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连续性血液净化治疗多器官功能障碍综合征的临床研究

【关键词】  持续血液净化疗法 多器官功能障碍综合征

   多器官功能障碍综合征(MODS)是严重创伤、休克、感染及大手术等原发病24 h后,机体同时或序贯发生两个或两个以上器官或系统功能障碍的临床综合征,是重危患者首要的死亡原因之一[1]。MODS的早期阶段为全身炎性反应综合征(SIRS),后者是机体对感染性或非感染性致病因素的过度反应状态,是一种常见的临床综合征[2]。以单纯中和或阻断某些炎性介质为目标的临床试验均未达到理想的治疗效果[3]。连续性血液净化(CBP)是近年来发展起来的新的血液净化技术,由于治疗的连续性,血流动力学的稳定性,特别是能够有效的清除循环中的炎性介质,使得这种治疗方法不但应用在急慢性肾功能衰竭而且已广泛的被应用于非肾脏疾患,特别是MODS。我院ICU近3年来应用CBP抢救危重患者33例,取得了较好的效果,现总结如下。

    1  资料与方法

    1.1  临床资料  2005年5月—2008年5月,入住我院ICU的MODS患者33例进行CBP治疗,男19例,女14例,平均年龄46.8岁(23~79岁)。其中腹部手术后9例,复合外伤5例,重症急性胰腺炎5例,药物中毒4例,重度妊高征、剖宫产术后 4例,心脏骤停复苏后6例。

    1.2  方法  所有患者均进行呼吸机辅助呼吸并常规给予控制感染、营养支持以及呼吸、循环等器官功能支持治疗。由于病情危重,只能在ICU内接受持续血液净化疗法。采用美国Baxter公司生产的BM25型床旁血滤系统,血滤器为FH1 200血滤器,股静脉留置双腔导管。置换液基本配方:生理盐水3 000 ml,注射用水830 ml,25%硫酸镁3.2 ml,10%葡萄糖酸钙40 ml,10%氯化钾12 ml,5%碳酸氢钠250 ml,每2 h一次。根据血气分析及生化检查结果调整置换液中电解质含量和碳酸氢钠用量。CBP前,100 mg/L肝素盐水预充滤器和管路30 min,再用生理盐水1 000 ml冲洗。CBP开始时,抗凝剂采用最小肝素化法,肝素首量0.3~0.5 mg/kg,追加量2~10 mg/h,保持活化凝血酶原时间(APTT)延长1.5~2.0倍,由输液泵持续输入。DIC有明显出血倾向的患者行无肝素血滤,定期用生理盐水冲洗滤器。24 h超滤量根据治疗量和病人容量负荷情况设定。患者每次治疗14~72 h,平均治疗25.6 h,根据病情治疗1~4次不等;血流量为180~250 ml/min,采用连续静静脉血滤(CVVH),均以前稀释法补充置换液,置换液流速为2 000~4 000 ml/h。

    1.3  观察项目  监测患者CBP治疗前后心率、血压,测定肌酐、尿素氮、胆红素和电解质变化,计算氧合指数。

    1.4  统计学方法  全部数据应用SPSS 14.0软件进行统计分析,数据以x±s表示,治疗前后比较用配对t检验。

    2  结果

    26例病情好转,5例因严重肺部感染死亡,2例因脑血管意外死亡。所有患者在治疗过程中,平均动脉压(MAP)、心率(HR)和氧合指数均有所改善(P<0.05);治疗前后血清肌酐、尿素氮下降(P<0.05 ),但胆红素无明显变化(P>0.05),见表1。由于在治疗时根据血气分析及生化检查结果随时调整置换液中电解质含量,故CBP结束时电解质均在正常范围。治疗中未发现明显的副作用。表1  CBP治疗前后生化及临床指标变化时间心率(次/分)平均动脉压(mm Hg)游离胆红素(μmol/l)肌酐(μmol/L)尿素氮(mmol/L)氧合指数治疗前注:与CBP治疗前比较,*P<0.05

    3  讨论

    CBP防治MODS的主要机制在于:①有效清除血液中炎症介质和内毒素;②通过清除间质水肿改善微循环,增强实质细胞摄氧力,从而改善组织氧利用,降低MODS的死亡率;③持续稳定的调控氮质血症和水电解质酸碱失衡;④为营养和代谢支持治疗创造条件,这是作为普通血透难以达到的[4] 。

    CBP治疗后平均动脉压、心率和氧合指数均有所改善,考虑血流动力学的改善与血液滤过后循环中某些影响血管舒缩功能及损伤血管内皮细胞的毒素及炎症介质,如一氧化氮(NO)、肿瘤坏死因子α(TNFα)等的清除有关[5],同时CBP可有效清除循环中心肌抑制因子[6],患者心血管功能得到改善;而氧合指数的改善还与经过缓慢超滤脱水,清除了体内毒素,减轻了肺间质水肿有关。CBP高超滤率可以清除全肠外营养(TPN)以及肠内营养中用做营养物质载体的多余水分,允许给予大量的液体,从而可以对体液负荷过大的患者更好地进行营养支持,为患者行TPN和某些药物的治疗提供条件。本研究采用连续静静脉血滤,33例患者经治疗后,肾功能均有所恢复,全身水肿改善;但胆红素下降不明显,可能系静静脉血滤对胆红素等大分子物质清除率低所致;这可能也是影响预后的因素,故需考虑与血浆置换或血液灌流相结合,以提高治疗效果,提高生存率。

    我们认为早期应用CBP,对MODS患者有改善预后的作用,提高抢救成功率,患者耐受性较好。

【参考文献】
  [1] Walsh CR.Multiple organ dysfunction syndrome after multiple trauma[J].Orthopaedic Nurs,2005,24:324333.

[2] Dremsizov T,Clermont G,Kellum JA,et al.Severe sepsis in communityacquired pneumonia:when does it happen,and systemic inflammatory response syndrome criteria help predict course[J]?Chest,2006,129:968978.

[3] Cariou A,Vinsonneau C,Dhainaut JF.Adjunctive therapies in sepsis;an evidencebased review[J].Crit Care Med,2004,32:562570.

[4] 顾勇,林善锬.连续性肾脏替代疗法在治疗多器官功能障碍综合征中的应用[J].中华肾脏病杂志,2001,17(5):350352.

[5] Klouche K,Cavadore P,Portales P,et al.Continuous venovenous hemofiltration improves hemodynamics in septic shock with acute renalfailure without modifying TNF alpha and IL6 plasma concentrations[J].J Nephrol,2002,15(2):150157.

[6] GarciaFernandez N,Lavilla FJ,Rocha E,et al.Haemostatic changes in systemic inflammatory response syndrome during continuous renal replacement therapy[J].J Nephrol,2000,13(4):282289.


作者单位:滨州医学院附属医院ICU 滨州市 256603

日期:2008年12月27日 - 来自[2008年第31卷第4期]栏目

急性肾衰竭患者使用连续性血液净化治疗的护理

【摘要】  血液净化技术(CBP)应用临床治疗急性肾衰竭(ARF)已有近半个世纪的历史。随着血液净化技术的不断革新,ARF的死亡率逐渐下降。尽管CBP在临床上使用已经比较广泛也仍然存在一些问题,比如在护理方面仍可以进行深入的讨论。比如:血管通路的护理、患者在治疗期间病情变化的处理、患者进行治疗时出现低温的护理以及患者的心理护理。CBP技术不仅为血液净化专业人员所必须掌握,而且由于其广泛应用于重症监护病房,因此要求专科护士特别是ICU护士亦能了解并掌握其一般操作,以充分发挥CBP在救治ARF中的作用。

【关键词】  连续性血液净化治疗;深静脉导管的护理;低温护理;心理护理

血液净化技术(CBP)应用临床治疗急性肾衰竭(ARF)已有近半个世纪的历史。随着血液净化技术的不断革新,ARF的死亡率逐渐下降,但是近十年来ARF死亡率却略有上升趋势,合并三个脏器功能衰竭者死亡率可达85%以上[1]。主要原因是ARF 的病谱发生重大变化,单纯性ARF比例下降,而并发多脏器功能障碍综合征(multiple organ dysfunction syndrome,MODS)、老年人比例上升以及传统血液净化技术仍有不可避免的缺陷,为了解决这一问题,Scrbner等在1960年提出了连续性血液净化治疗的概念[2]。所谓连续性血液净化治疗(CBP)是指所有缓慢、连续清除水和溶质的治疗方式。包括AVSCUF、CAVH、CAVHD等技术。正是由于CBP缓慢、连续、等渗的特点使得它在重症患者治疗时更显示其优越性。

    急性肾衰竭(acute renal failure,ARF)是指各种原因导致的肾小球急骤减退以肾小球滤过率明显降低以及肾小管功能障碍所致的氮质血症、水电解质、酸碱平衡紊乱为临床表现的一组综合征。

    1  病历摘要

    患者,男,66岁,2008年7月17日因“阑尾炎”于外院行阑尾切除术,7月18日出现全腹剧痛转入我院,当日在全麻下行“开腹探查右半结肠根治术,空肠造瘘术”,术中见腹腔内约有2 000 ml粪便性腹腔积液,距回盲瓣5 cm处升结肠可见长约3~4 cm破溃边缘坏死,有粪便流出。术后转入外科重症监护室(SICU)监护。转入后患者无尿。7月22日血液实验室检查尿素氮83.9 mg/dl、肌酐6.1 mg/dl、血钾5.3 mmol/L,随之给予患者CBP治疗。7月23日尿素氮36.5 mg/dl、肌酐2.5 mg/dl、血钾5.0 mmol/L;7月24日尿素氮20.5 mg/dl、肌酐1.5 mg/dl、血钾4.8 mmol/L;7月25日尿素氮19.4 mg/dl、肌酐1.0 mg/dl、血钾4.5 mmol/l。实验室检查恢复正常停止CBP治疗。由此可见,患者在使用CBP治疗期间,CBP对于纠正氮质血症及电解质紊乱起到了明显的作用。

    2  护理体会

    2.1  制定合理可行的护理计划   CBP开始治疗之前仔细了解病情及各项化验指标以便合理的制定治疗和护理计划,了解患者病情,根据患者的生命体征及病情来选择深静脉导管的置入部位和治疗初期血流速度、每小时置换液的入量和滤出液量、电解质的补充、碳酸氢钠的泵入等。尽可能地避免CBP开始时因血容量短时间内减少致血压下降,患者因不能耐受而发生其他不良反应。

    2.2  监测血压   CBP运转初期血流速度应逐渐加速,同时监测血压,如无明显变化可逐渐调至理想流速,当血压有所下降时除仔细观察症状外,应减慢流速,分析原因待血压平稳后再将流速调至最佳状态。

    2.3  血管通路的护理  对于CBP患者来说血管通路可谓“生命线”,监护室首选深静脉双腔留置导管,此管具有插管迅速、血流量充足、稳妥安全、留置时间长、不影响患者活动等优点。深静脉留置导管最常见的并发症是感染,因此插管处的无菌技术操作及细致护理尤为重要。应保持插管处敷料干燥清洁,观察穿刺处有无渗血、血肿及插管侧肢有无肿胀,深静脉穿刺处有渗出时敷料应该及时更换,若没有渗出72 h给予更换一次,更换敷料时要严格无菌操作,深静脉导管应7天更换一次穿刺部位,原导管拔除后应送管尖培养。连接管路时应注意无菌操作,管路要妥善固定防止管路打折、脱出从而影响CBP的顺利进行。

    2.4  CBP治疗期间的加热  患者在进行CBP治疗时大量置换液输入体内(2 L/min),易致患者体温骤降引起寒战,告知患者不要紧张,及时给患者保暖,可以加盖棉被,也可以使用血滤机自身的加温装置为患者保暖(将血滤机加温装置调节至37 ℃~38 ℃),必要时可以使用温毯机为患者保暖。

    2.5  做好患者的心理护理  为患者进行CBP治疗时,许多患者对病情不了解而感到恐惧,对于神志清楚能够配合的患者,应该充分安慰患者,简单告知患者进行此项操作是将患者体内毒素排除,减轻患者目前的症状。为患者摆好舒适体位,并告知管路的重要性,尽量插管侧肢体制动,以取得患者的配合,保证CBP的顺利进行。

    2.6  监测出入量  保持出入量平衡,量出为入。若滤出量过多,血容量在短时间内减少,可导致患者低血压性休克。反之,若输入过多,患者可发生心衰、肺水肿。应尽可能均匀的分配每日置换总量。ARF患者因治疗需要每天不得不接受大量液体输入,因此就体液平衡而言CBP有很大的优势,可以根据病情需要随时控制或调节水、电解质及酸碱平衡,而护理中详细正确的观察将为治疗提供最有力的依据。

    2.7  置换液的配置  利用无菌蒸馏水和0.9%生理盐水及电解质配置置换液,其pH值与血浆pH值近似,可以避免置换液进入患者体内后引起的pH值及电解质紊乱,一改过去瓶装置换液串联的方法,5 000 ml为一袋(内含4 000 ml 0.9%生理盐水+1 000 ml无菌蒸馏水+电解质),5%碳酸氢钠另建一条通路与一袋置换液同步输入,如此循环往复(5%碳酸氢钠根据患者的血气结果调节输入速度,电解质的补充可视生化结果而定)。

    2.8  CBP治疗中抗凝方法的使用  由于CBP是连续性体外循环而每位患者的凝血状况又有很大不同,因此抗凝剂的使用十分关键[1]。凝血通常最先发生在静脉小壶和滤器中。凝血最常见的原因有:(1)患者病情不允许使用抗凝剂;(2)使用小剂量抗凝剂;(3)患者处于高凝状态;(4)血滤机频繁停机,造成血细胞附着于滤器纤维素上;(5)血流量不足。长时间CBP治疗SICU尝试用“肝素吸附法”预冲管路及滤器即以肝素盐水2 000 ml+肝素钠100 mg预冲管路再以3 000 ml 0.9%盐水冲净未吸附的肝素连接血液循环开始CBP可有效抗凝。另外,CBP的患者在治疗期间应给予定时监测部分凝血活酶原时间(APTT)常规给予2 h测一次。APTT正常值为25~35 s。作为CBP患者APTT值结果应为正常的2.5倍约为70~100 s,这样可以减少血细胞较少的吸附在滤器上,提高滤过效果防止滤器发生凝集,延长滤器的使用寿命。

    ARF合并多器官脏器功能衰竭(MODS)是重症疾患,若抢救不及时可危及生命。重症ARF患者接受CBP治疗的时机目前尚无统一认识,通常当患者尿素氮>30 mmol/L,或者肌酐>5.6 mg/dl就开始进行CBP的治疗。尿毒素的蓄积可能导致更为严重的后果,而CBP可以有效地清除体内的毒素,而且CBP对纠正电解质紊乱以及出现的精神症状、消化道症状等有着明显的效果。从治疗过程中不难看出,患者经过4天的CBP治疗,患者的尿素氮、肌酐以及电解质均恢复正常水平,及时有效的控制患者ARF的发展,为抢救患者生命赢得了大量宝贵的时间。另外,正是由于CBP缓慢、连续、等渗的特点使得它在重症患者治疗时更显示其优越的特性[3]。另外,CBP技术不仅为血液净化专业人员所必须掌握,而且由于其广泛应用于重症监护病房,因此要求专科护士尤其是ICU护士亦能掌握其操作,以充分发挥CBP在救治ARF中的作用。CBP对护理要求较高,在CBP的治疗过程中护理人员必须具备高度责任心、熟练、专业化的技术操作,治疗过程专人护理,严密观察,护理人员只有掌握专科技能,通过专业细致的护理才能保证CBP顺利进行,预防并发症发生,提高治疗效果。

【参考文献】
  1 王一山.实用重症监护治疗学.上海:上海科技文献出版社,2000,763,437,79.

2 季大玺,谢红浪,黎磊石,等.连续性血液净化疗法在重症急性肾功能衰竭治疗中的应用.中华内科杂志,1999,38:802.

3 龚德华,季大玺.透析膜的吸附特征.肾脏病透析与肾移植杂志,1997,6(3):272.


作者单位:北京,首都医科大学附属北京朝阳医院

日期:2008年12月27日 - 来自[2008年第5卷第21期]栏目

急性肾损伤合并多器官功能障碍综合征经连续性血液净化治疗的预后分析

【摘要】  目的 探讨急性肾损伤(AKI)合并急性肾损伤合并多器官功能障碍综合征(multiple organ dysfunction syndrome,MODS)患者经连续性血液净化治疗(CBP)后的存活率及影响因素。方法 对2006年6月-2008年7月收治的153例AKI伴MODS经CBP治疗患者的年龄、治疗前APACHEIII评分,需要通气和升压情况及治疗前肾功能进行分析。结果 AKI伴 MODS病人经CBP治疗病死率为37.9%,较资料报道低。年龄较低、治疗前APACHEIII评分低、需要机械通气和升压药比例低的病人存活率较高。治疗前尿素氮、血清肌酐越低,存活率越高。结论 CBP是治疗AKI伴MODS病人的首选方案,对于APACHE III分值低、年龄较轻、肾功能受损较轻病人的预后有明显改善。

【关键词】  肾损伤;急性;血液净化疗法;多脏器功能障碍


    急性肾损伤(acute kidney injury, AKI)合并急性肾损伤合并多器官功能障碍综合征(multiple  organ  dysfunction  syndrome,MODS), 病死率可达50%~70%,传统血液透析方法对病死率并无明显改善且多有禁忌。近年来随着连续性肾脏替代治疗(continous  renal  replacement therapy,CRRT),现多称为连续性血液净化(continous blood purification,CBP)治疗技术的应用,使AKI伴MODS病人的预后明显改善。本文对我院2006年6月-2008年7月收治的153例AKI伴MODS经CBP治疗的病人预后进行分析,报告如下。

    1  资料与方法

    1.1  一般资料  153例中男81例,女72例;年龄13~82(48.1±16.7)岁,CBP前尿素氮(30.8±27.1)mmol/L,血清肌酐(Scr)(580.3±435.7)μmol/L。肾实质性疾病71例,药物中毒15例,外伤10例,肺心病8例,肿瘤2例,感染18例,毒蛛咬伤8例,急性出血坏死性胰腺炎13例,肝硬化2例,产后DIC 7例,急性心衰11例。153例病人中89例(58.2%)在发病3个月前健康状况良好,其他病人都存在一种或多种不同严重程度的疾病。

    1.2  MODS病情评价方法及分组  按照Knaus法收集每一患者第1个24h所需的17项急性生理参数的最差值、年龄和慢性健康状况,三者的分值相加,计算每一患者APACHEIII的分值,根据分值分三组。A组:APACHEIII的分值<10;B组:APACHEIII的分值10~20。C组:APACHEIII的分值>20。

    1.3  CBP指征  153例患者均符合Bellomo 2001年提出的ICU患者CBP治疗指征:(1)少尿(12h尿量<200ml)。(2)无尿(12h尿量<50ml)。(3)由于代谢性酸中毒导致的严重酸血症(pH<7.11)。(4)氮质血症(尿素氮>30mmol/L)。(5)高钾血症(血K+>6.5 mmol/L或血K+快速上升)。(6)怀疑有与尿毒症相关的疾病(心包炎、脑病、神经病、肌病)。(7)严重的钠离子紊乱(血Na+>160mmol/L或<115mmol/L)。(8)高热(体温>39.5℃)。(9)临床上明显的器官水肿(尤其是肺)。(10)可透析毒物导致的中毒或药物过量。(11)有肺水肿或急性呼吸窘迫综合征(ARDS)危险性,需大量输入血液制品又有凝血机制障碍者。达到其中1项标准的基线时可以开始CBP治疗,达到2项标准时必须进行CBP治疗,未达到标准但出现精神错乱也须行CBP治疗。

    1.4  血管通路  所有患者均使用颈内静脉或锁骨下静脉、股静脉单针双腔导管建立临时血管通路,留置时间7~30(18.5±11.5)天。

    1.5  CBP装置

    1.5.1  机器  采用日本ACH-10持续徐缓式血液净化装置。

    1.5.2  血滤器  使用日产APS-650滤器,OP-08W膜型血浆分离器,均为一次性使用。爱尔Aier一次性使用炭肾。

    1.5.3  置换液  采用改进Port等的配方,每一循环包括两组液体:第1组生理盐水3000ml+灭菌注射用水750ml+5%葡萄糖溶液200ml+25%MgSO4 3.2ml+15%KCl 8ml+10%葡萄糖酸钙40ml;第2组5%NaHCO3250ml。离子浓度分别为:Na+143.6mmol/L,Cl-112.4mmol/L,HCO-335.0mmol/L,Ca2+2.09 mmol/L,Mg2+1.57mmol/L,葡萄糖13.6mmol/L,每一循环根据患者血电解质情况可酌情调整离子浓度。

    1.6  CBP方法

    1.6.1  CBP治疗时间和方法  根据患者病情,每天床边连续替代治疗12~48h,血流量180~200ml/min。置换液流量2000~4000ml/h。

    1.6.2  CBP抗凝剂的应用  滤器和管路使用前先以50mg/L的肝素生理盐水预充。126例有出血倾向者用低分子肝素抗凝,首量2000~5000U,追加量400~800U/h,27例严重出血倾向或手术后的患者未使用抗凝剂。

    1.7  统计学方法  两样本均数的比较采用t检验,两样本率的比较采用U检验,P<0.05为差异有显著性。

    2  结果

    2.1  两组患者临床资料比较  见表1。153例病人95例存活(62.1%),存活组患者年龄明显较死亡组低且差异有显著性(P<0.01)。APACHEIII评分死亡组较存活组分数明显高,且差异有显著性(P<0.01)。死亡组需要机械通气和使用升压药的患者比例明显高于存活组。

    2.2  各组APACHEIII评分、肾功能、病死率比较  治疗前BUN、Scr A组较B组较低,B组较C组低,差异均有显著性;病死率A组较B组低,B组较C组低。见表2。表1  存活组与治疗组的临床资料比较注:与存活组比较,*P<0.01表2  APACHE Ⅲ评分、肾功能、病死率比较注:与A组比较,*P<0.01;与B组比较,△P<0.01

    3  讨论

    随着急救医学和肾脏替代治疗技术的发展,近30年来单纯ARF的存活率已有显著提高,但AKI合并MODS患者的病死率仍高达50%~70%。传统血液净化疗法未能明显降低AKI的病死率。近年来国内外采用CBP显示出较传统血液净化不可比拟的优势[1]。

    MODS合并AKI采用CBP比间歇性血液透析更能改善肺通气功能、血液动力学、氮质血症、液体过剩和营养支持。血液动力学状态不稳定,高分解代谢和超容量负荷的重症AKI患者应当首选CBP。CBP时,溶质和液体的清除持续缓慢进行,渗透压变化小,血流动力学状态稳定,溶质清除满意。连续性静脉-静脉血液滤过1~2L/h,尿素的清除率可达17~33L/min,患者不需要严格限制蛋白摄入量,重症者可辅助肠内、肠外营养,以利于脏器功能的恢复。CBP使用成物膜滤器,生物相容性好,血-膜反应小,而且通透性好,可持续清除各种中、大分子的炎症介质,如肿瘤坏死因子、白细胞介素-1、心肌抑制因子、前列腺素、血栓素等,使血流动力学状态更为稳定,而MODS时启动体内炎性反应,使机体细胞因子、炎性物质增多,目前认为CBP能重建机体免疫系统内稳状态,且CBP的疗效与患者接受治疗时机体免疫系统所处的状态有关,早期治疗效果远比晚期显著,这一研究不再锁定血液净化的清除作用,而是从免疫学机制的角度进行更深层的内涵探讨[2]。本研究对我院行CBP的病人生存率和病死率进行了系统的分析,发现病死率为37.9%,较资料报道略低。APACHEⅢ评分系统是目前国际上广泛用于危重患者病情分类评定及预测预后的一种评分方法,由年龄、急性生理学评分和患病前的慢性健康状况评价3部分组成,MODS患者采用APACHEⅢ评分系统能较准确地反应预后。BRIVET等报道,MODS患者的年龄、既往健康状况等因素与预后相关。SCHAEFER等发现低血压和需要机械通气是预示患者死亡的危险因素。本研究以APACHEⅢ评分估计患者病情发现死亡组年龄明显增大,需要升压药维持及机械通气者更多。在进行CBP治疗时,肾功能受损较轻组比肾功能受损较重组存活率明显增高。对MODS患者APACHEⅢ评分值<10分,医院死亡的可能性小;分值10~20分,病死率约50%;分值>20分,病死率约80%~100%。从本研究看APACHE值越低,存活率越高,存活率与资料报道基本一致。同时我们发现APACHE评分低的组肾功能受损明显较轻。曾有人认为危重患者的存活率与多脏器功能障碍的发生与进展密切相关,而不仅仅取决于是否出现肾功能损伤,即AKI的出现仅是作为多脏器功能障碍的症状之一,而不是造成患者死亡的原因。但新近临床研究认为及早地应用肾脏替代治疗能够最大限度地改善预后,本研究认为肾功能受损越轻,CBP治疗越早存活率越高。

    综上所述,CBP 是治疗AKI 伴 MODS病人的首选方案,对于APACHEⅢ分值低、年龄较轻、肾功能受损不重的病人的预后有明显改善,值得广泛用于临床。

  

【参考文献】
  1 沈波,李艳,张薇.连续性血液净化治疗急性肾损伤合并多器官功能障碍综合征12例临床分析.实用医学杂志,2005,21(13):1433-1435.

2 季大玺.连续性血液净化与重症感染.肾脏病与透析肾移植杂志,2004,13(3):235-236.


作者单位:841000 新疆库尔勒,新疆巴州人民医院ICU室

日期:2008年12月27日 - 来自[2008年第9卷第10期]栏目

第四军医大学西京医院研究表明:CBP技术可减轻MODS患者的氧化损伤


  
  中国医药报陕西讯 日前,第四军医大学西京医院放射科刘宏宝主治医师等,根据其开展的“危重病症救治中连续性血液净化的疗效评估和机制探讨”研究及相关临床实践,得出了连续性血液净化(CBP)技术可减轻多脏器功能障碍综合征(MODS)患者的氧化损伤的结论。
  据介绍,CBP已不再仅仅局限于替代肾脏功能,其在严重感染、创伤、中毒及MODS等危重病症的救治中也发挥了传统的药物治疗方法所无法比拟的效应。如何根据患者不同病情把握CBP的治疗时机、治疗方式和治疗剂量,以及探寻其疗效机制,始终是临床研究的热点。对此,在科主任陈威教授的指导下,刘宏宝等对CBP临床应用中的疗效和机制进行了深入研究。研究中,他们首次提出CBP技术可以改善MODS患者的氧化应激状态、减轻氧化损伤的观点;提出对MODS患者早期进行预防性CBP治疗的理念,发现提高MODS患者存活率的关键在于早期诊断和及时CBP治疗。研究者还较早地在国内将低糖配方置换液和血浆透析联合高容量血液滤过(HVHF)技术应用于临床,较早地系统证实了在CBP治疗过程中进行动态APACHE评分、MODS评分和细胞因子的检测可以综合判断患者的预后和指导CBP方案的调整,并证实了增加置换率更有利于清除炎性介质和改善机体免疫状态。
  据悉,到目前为止,研究人员已应用CBP技术有效地治疗了各种危重病症达411例,其方法在西安市中心医院、河南省三门峡黄河医院、解放军323医院等9家医院推广后,获得了较好的社会效益和经济效益。不久前,该研究成果荣获军队医疗成果二等奖。
   (张中桥)
日期:2007年11月21日 - 来自[血液]栏目

Evidence That the Coactivator CBP/p300 Is Important for Phenobarbital-Induced but Not Basal Expression of the CYP2H1 Gene

Department of Molecular Biosciences, Discipline of Biochemistry, the University of Adelaide, Adelaide, Australia (S.C.D., B.K.M.); and Chromatin and Transcriptional Regulation Group, the John Curtin School of Medical Research, Australian National University, Canberra, Australia (D.T.)

 

  Abstract

Top
Astract
Introduction
Materials and Methods
Results
Discussion
References

We have previously identified an upstream 556-bp enhancer domain for the chicken CYP2H1 gene that responds to phenobarbital and binds several transcription factors, including the orphan chicken xenobiotic receptor (CXR). By contrast, the promoter lacks a CXR site and is not inducible by phenobarbital. Although it has been established that CXR can interact with the coactivator SRC-1, there are no reports as to whether other coactivators may be important for phenobarbital-mediated inducibility. Our studies using the adenovirus E1A wild-type protein, which inhibits the coactivators cAMP response element binding protein (CBP) and CBP associated factor (p/CAF), provide evidence for the involvement of one or both of these coactivators at the enhancer but not at the promoter of the CYP2H1 gene. The observations that mutant E1A proteins did not affect the enhancer activity and that inhibition by wild-type E1A was reversed by CBP and p/CAF confirmed the involvement of these coactivators in the induction process. We propose that the intrinsic histone acetyl transferase activity of one or both of these coactivators participates in chromatin remodeling thereby stimulating drug induction of the promoter. This proposal was supported by experiments with the histone deacetylase inhibitor, trichostatin A, which resulted in the superinduction of the drug response but had little effect on basal expression of the CYP2H1 gene. The work provides evidence for the first time for the involvement of the coactivators CBP and p/CAF in the phenobarbital-mediated induction of the CYP2H1 gene.

 

  Introduction

Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The cytochrome P450 proteins comprise a superfamily of heme-containing enzymes that play an important role in the metabolism of diverse lipophilic compounds, including foreign chemicals, such as pharmaceutical drugs and other xenobiotics (Gonzalez, 1990; Dogra et al., 1998; Waxman, 1999). The synthesis of specific cytochrome P450 enzymes can be selectively induced by their own substrates after the interaction of ligand-receptor complexes with upstream enhancer sequences in these genes (Kliewer et al., 1999; Waxman, 1999; Honkakoski and Negishi, 2000). Phenobarbital is a prototype inducer that markedly increases the expression of CYP2 genes in mammals (Waxman and Azaroff, 1992; Waxman, 1999; Honkakoski and Negishi, 2000) and chicken (Mattschoss et al., 1986; Hansen et al., 1989; Dogra et al., 1998). In recent years, a great deal of information has been obtained regarding the molecular mechanism of phenobarbital-mediated induction of cyp2b10, CYP2B1, and CYP2H1 genes (Dogra et al., 1999; Honkakoski and Negishi, 2000; Handschin et al., 2001; Kim et al., 2001). These studies have revealed that a nuclear orphan receptor constitutive androstane receptor (CAR) plays a central role in the phenobarbital-mediated induction mechanism. Furthermore, an essential role for CAR in this induction mechanism has been confirmed by the loss of phenobarbital-mediated inducibility of the cyp2b10 gene in CAR knockout mice (Wei et al., 2000). Interestingly, it has been recently shown that only 50% of phenobarbital responsive genes are affected in CAR-null mice, indicating that CAR has diverse roles (Ueda et al., 2002). Studies in the mouse have shown that in response to phenobarbital, CAR is translocated to the nucleus, where it forms a heterodimer with retinoid X receptor and activates drug response elements in the 5'-flanking region of the cyp2b10 gene (Honkakoski and Negishi, 2000).

Activation of gene expression involves direct recruitment of coactivator complexes to the enhancer and promoter regions of target genes. Studies have suggested a strong link between histone acetylation, chromatin remodeling, and gene regulation (Grunstein, 1997; Wade and Wolffe, 1997; Kadonaga, 1998). A number of transcriptional coactivators, including the ubiquitous cAMP response element-binding protein (CBP) and its structural homolog p300 (Bannister and Kouzarides, 1996), CBP-associated factor (p/CAF) (Yang et al., 1996) and steroid receptor coactivator, SRC-1 (Spencer et al., 1997) have been found to possess intrinsic histone acetyltransferase activity, which can modulate chromatin structure and gene transcription (Wang et al., 1998). In addition, CBP/p300 and p/CAF have been postulated to link transcription activators to the basal transcriptional machinery (Eckner, 1996). Human p300 was initially identified by its ability to bind the adenovirus E1A oncoprotein (Moran, 1993). E1A binding to CBP/p300 and p/CAF probably perturbs chromatin structure, which accounts for the transcription modulating effects of these proteins (Goodman and Smolik, 2000). Because E1A interacts with the transcriptional coactivators CBP/p300, its exogenous expression has been used as a tool to study the role of coactivators in specific gene transactivation.

We are studying the molecular mechanism of CYP2H1 gene induction by phenobarbital in chick embryo livers (Hansen et al., 1989; Dogra et al., 1998, 1999). We previously identified in this gene an upstream enhancer domain (located between 5900 and 1100) that responds to drug and, within this, a 556-bp enhancer region has been analyzed in detail (Dogra et al., 1999). It has been observed that DNA-binding sites for a number of transcription factors including a nuclear receptor are required for the phenobarbital response (Dogra et al., 1999; Handschin and Meyer, 2000). The nuclear receptor binding site in the 556-bp enhancer of CYP2H1 gene is similar in sequence to that identified as a CAR binding site in CYP2B genes and mutagenesis of this motif in the CYP2H1 enhancer abrogates the phenobarbital response (Handschin et al., 2001). Recently, a nuclear receptor CXR, the chicken homolog of CAR, was cloned and identified as an activator of the chicken CYP2H1 gene and has activation properties similar to CAR and pregnane X receptor (Handschin et al., 2000). However, there is no information on whether coactivators such as CBP/p300, p/CAF, and SRC-1 are part of the enhancer/promoter complex that drives the phenobarbital-induced activation of the CYP2H1 gene.

In this study, we show that expression of wild-type E1A in chick embryo primary hepatocytes inhibited only the phenobarbital-induced level of CYP2H1 mRNA but did not alter the constitutive expression of this mRNA in the absence of drug. Consistent with this, E1A also repressed the phenobarbital-induced activity of the 556-bp CYP2H1 enhancer but had no effect on the promoter activity of this gene. Trichostatin A (TSA), a selective inhibitor of histone deacetylase activity, further increased the phenobarbital-induced level of CYP2H1 mRNA. This suggests that drug action involves acetylation, probably mediated through the histone acetyltransferase activity of the coactivator complex recruited to the 556-bp CYP2H1 enhancer.

 

  Materials and Methods

Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

RT-PCR Analysis of Endogenous CYP2H1 Gene Expression. Primary hepatocytes were prepared from 17-day-old chick embryos by a method described previously (Dogra and May, 1997). Hepatocytes (2 × 107) were cotransfected with 40 µg of expression clone for E1A (E1A12S) and 5 µg of pEGFP-C1 (BD Clontech, Palo Alto, CA). After transfection, each sample was split so that approximately 1 × 107 cells were plated onto 60-mm dishes and cultured in Williams' E medium plus 10% Serum Supreme (Edward Keller Ltd., Hallam, Victoria, Australia). Hepatocytes were incubated at 37°C over night, after which medium was changed and hepatocytes were treated with either phenobarbital (final concentration, 500 µM in PBS) or solvent. Cells were harvested after 24 h and cells expressing enhanced green fluorescence protein (EGFP) were isolated by FACS (2-3 × 105 cells). Total RNA was isolated from these cells by a method described previously (Chomczynski and Sacchi, 1987). cDNA was synthesized from 50 ng of total RNA in 20 µl using Oligo (dT)23 primer and a two-step enhanced avian RT-PCR kit (Sigma, St. Louis, MO) according to the manufacturer's instructions. An increasing amount of cDNA was used in the semiquantitative PCR reaction containing 400 nM of each primer in a final volume of 50 µl, as described in the two-step enhanced avian RT-PCR kit (Sigma). The reaction mix was spiked with [32P]dCTP to quantify CYP2H1 mRNA using -actin as an internal control. Primer sequences employed were CYP2H1, 5'-CACTGCAGGGAAAGCGGTCAAT-3'; 5'-TGCTGGACTGTACTTACTGGACC-3', and -actin, 5'-CCTGAACCCCAAAGCCAACAGA-3'; 5'-GGACTCCATACCCAAGAAAGATG-3'.

Northern Blot Analysis. Primary hepatocytes were plated at 6 to 8 × 106 in Williams's E medium supplemented with 10% serum supreme. Medium was changed after overnight culture and cells were treated with TSA at 1 or 2 µM concentrations for 1 h before phenobarbital was added at a 500 µM final concentration. Hepatocytes were further incubated for 6 h and used to prepare total RNA (Chomczynski and Sacchi, 1987). Total RNA (15 µg) was electrophoresed on a 1% agarose gel containing 1.1 M formaldehyde. The fractionated RNA was blotted onto NYTRAN membrane (Schleicher and Schuell, Keene, NH) and UV cross-linked using a UV Stratalinker 1800 (Stratagene, La Jolla, CA). The filters were prehybridized for 16 h in 50% formamide, 5× standard saline citrate (0.75 M NaCl, 75 mM sodium citrate, pH 7.0), 5× Denhardt's (0.1% Ficoll, 0.1% polyvinylpyrrolidine, and 0.1% bovine serum albumin), 0.05% sodium pyrophosphate, 0.1% SDS, and 200 µg/ml salmon sperm DNA, and then hybridized with cDNA probes labeled with [32P]dATP by random priming using a DNA labeling kit (Amersham Biosciences, Piscataway, NJ). The specific DNA probes were as follows: pCHPB15 for CYP2H1 mRNA, p105B1 was used to detect ALAS1 (5-aminolevulinate synthase1) mRNA, and a full-length cDNA clone was used for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (Dogra and May, 1996). Each probe was added at an activity of 0.5 to 1.0 × 106 cpm/ml. Filters were washed and quantified as described previously (Dogra and May, 1996).

Cell Culture and Transfection. For transfection experiments, plasmid DNA was prepared by CsCl/ethidium bromide equilibrium density gradients and quantified by spectrophotometry. The RSV-driven adenovirus E1A12S and mutant clones were a gift from Prof. T. Kouzarides (Cambridge, United Kindom) and Dr Y. Tsuji (Wake Forest University, NC), respectively. TSA was purchased from Wako Pure Chemical Industries, Japan. For transient transfection assays, primary hepatocytes (2 × 107 cells) in 0.8 ml of electroporation buffer (20 mM HEPES, pH 7.05, containing 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM glucose, and 400 µg of sonicated Salmon sperm DNA as carrier) were electroporated at 960 µF and 250 V using a Gene Pulser and Capacitance Extender (Bio-Rad, Hercules, CA). Electroporation efficiency, as observed in cell-sorting experiments using EGFP, was about 8 to 10%. Various CAT reporter constructs used in transient transfection assays to analyze the effect of E1A on the enhancer or promoter regions of the CYP2H1 gene included p4.8-SVCAT [4.8-kb BamH1 enhancer domain (5900/1100) SVCAT], p556-SVCAT [556-bp enhancer region (1956/1400)-SVCAT], pCYP-205CAT [205-bp promoter (205/1)CAT], and enhancerless SVCAT (Dogra et al., 1999). After transfection, each sample was split so that approximately 1 × 107 cells were plated onto 60-mm dishes and cultured in Williams' E medium plus 10% serum supreme. Hepatocytes were incubated at 37°C overnight, after which the medium was changed and hepatocytes were treated with either phenobarbital (final concentration, 500 µM in PBS) or solvent. The cells were further cultured for 48 h and then CAT activities were determined. Leghorn male hepatoma (LMH) cells were obtained from the American Type Culture Collection (Manassas, VA). LMH cells were cultured in Williams' E medium supplemented with 10% serum supreme and 50 µg of gentamicin/ml of medium. Cells were seeded on gelatin-coated 24-well plates at the density of 6 × 104 per well 24 h before transfection. On the next day, the medium was replaced with serum free Williams' E medium and transfections were performed using p556-SV-luciferase, pRL-SV40 (Promega, Madison, WI), CXR expression vector kindly provided by Prof. Urs Meyer (Handschin et al., 2000), E1A12S expression clone (Bannister and Kouzarides, 1996), expression clones for CBP (Chrivia et al., 1993); p/CAF, SRC-1, and hSRC-1A (a kind gift from Dr. B. W. O'Malley, Baylor College of Medicine, Houston, TX) and FuGENE6 transfection agent (Roche Molecular Biochemicals, Mannheim, Germany). The medium was replaced after 6 h with Williams' E containing 10% serum supreme with or without phenobarbital (final concentration, 500 µM in PBS). After 24 h, cells were lysed and luciferase assays carried out using the Promega dual-luciferase kit.

CAT Assay. Transfected cells were harvested in 40 mM Tris-HCl, pH 7.5, containing 1 mM EDTA and 150 mM NaCl, by scraping with a rubber policeman. The cells were pelleted and resuspended in 50 to 100 µl of 250 mM Tris-HCl, pH 7.6, lysed by three cycles of freezing and thawing, and centrifuged for 5 min to remove cell debris. The protein concentration of each cell was determined by protein microassay (Bio-Rad). For CAT assays, the cell supernatant was heated at 68°C for 6 to 8 min to remove deacetylase activity and CAT activity was then determined (Gorman et al., 1982). The acetylated products of [14C]chloramphenicol were separated by thin-layer chromatography. After autoradiography, CAT activity was quantified by cutting out the spots from the plate and measuring the radioactivity in a scintillation counter. The results were expressed as percentage of acetylated chloramphenicol or calculated as fold-induction over control values.

 

  Results

Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

E1A Represses Phenobarbital-Induced Expression of Endogenous CYP2H1 mRNA. The orphan receptor CAR underlies phenobarbital-induced activation of the CYP2B2, cyp2b10, and CYP2H1 genes (Honkakoski and Negishi, 2000; Handschin et al., 2001; Kim et al., 2001). Activation of genes by receptors generally involves coregulator proteins such as SRC-1, CBP/p300, and p/CAF (Perlmann and Evans, 1997; Torchia et al., 1998). Although there is evidence that SRC-1 is required for CAR activity (Sueyoshi and Negishi, 2001), the participation of other coactivators in the phenobarbital induction process has not been reported. Adenovirus E1A has been shown to abrogate the transcriptional activity of coactivators, including CBP/p300 and p/CAF (Eckner et al., 1994; Reid et al., 1998). Therefore, in this study, we examined the effect of E1A on the phenobarbital-mediated induction of the endogenous CYP2H1 gene in chick embryo hepatocytes to determine whether CBP or p/CAF participates in the phenobarbital-mediated induction process. Hepatocytes were cotransfected with expression plasmids for E1A (E1A12S) and EGFP (pEGFP-C1), and treated with or without phenobarbital at a final concentration of 500 µM for 24 h. Transfected cells expressing EGFP were sorted by FACS and the expression of endogenous CYP2H1 mRNA was determined by semiquantitative RT-PCR. RNA from control hepatocytes treated with or without phenobarbital was also analyzed to compare the effect of E1A on the basal and phenobarbital-induced levels of CYP2H1 mRNA. As shown in , expression of E1A reduced the steady-state level of drug-induced CYP2H1 mRNA to that of basal without affecting -actin mRNA used as a control. The average decrease in phenobarbital-induced levels of CYP2H1 mRNA by E1A from two independent experiments was from 4.3- to 1.4-fold . However, basal expression of the CYP2H1 mRNA was not altered by E1A . These results show that there is an E1A-sensitive step in the phenobarbital-mediated induction mechanism that is likely to involve CBP/p300 and/or p/CAF coactivators.

fig.ommitted

  Effect of E1A expression on the phenobarbital-mediated induction of endogenous CYP2H1 mRNA. Chick primary hepatocytes were cotransfected with expression clone for E1A and enhanced green fluorescence (pEGFP-C1) by electroporation and treated with and without phenobarbital (PB) for 24h. Transfected hepatocytes expressing EGFP were selected by FACS. Total RNA was isolated from 2 × 105 EGFP expressing cells, reversed transcribed and increasing amounts of cDNA used in the semiquantitative PCR reaction to quantify CYP2H1 mRNA with -actin mRNA as an internal control. A reaction was also run with no cDNA to serve as a negative control. A, RT-PCR reactions, spiked with [32P]dCTP, were resolved on a 1.5% agarose gel and a representative ethidium bromide stained gel is shown. B, PCR reaction products from the agarose gel were cut and quantified in the scintillation counter and the ratio of CYP2H1 to -actin as standardized against control is shown.

 

E1A Represses Expression of the Phenobarbital-Responsive Enhancer Region. We have previously identified an upstream 4.8-kb enhancer at 5900/1100 in the chicken CYP2H1 gene that responds to phenobarbital and have subsequently defined a 556-bp region at 1956/1400 within this domain that retains drug responsiveness when tested in both orientations (Dogra et al., 1999). Maximum induction of this enhancer is dependent on the presence of a number of transcription factor binding sites (Dogra et al., 1999) including a binding site for CXR, the chicken homolog of CAR (Handschin et al., 2000). Basal expression of the CYP2H1 gene is driven by a number of liver-specific transcription binding sites in the first 205 bp of promoter sequence. This region is not drug-inducible and does not contain a CXR binding site (Dogra and May, 1997).

We investigated the effect of exogenously expressed E1A on the expression of two CAT reporter gene constructs, p4.8-SVCAT and pCYP-205CAT, in chick embryo hepatocytes. pCYP-205CAT is driven by the CYP2H1 proximal promoter (Dogra and May, 1997) and p4.8-SVCAT by the 4.8-kb upstream drug responsive domain fused to the enhancerless SV40 promoter (Dogra et al., 1999). E1A expressing plasmid at 400 ng markedly repressed phenobarbital-mediated induction of p4.8-SVCAT, but had no effect on the basal expression of this construct. When the 4.8-kb enhancer region was replaced with the drug responsive 556-bp enhancer sequence (p556-SVCAT), induction by phenobarbital was again inhibited by E1A . A similar inhibition by E1A on the phenobarbital-induced 556-bp enhancer activity was observed when the 556-bp enhancer sequence was tested in the opposite orientation (5'- 3') (result not shown). A control vector lacking E1A had no effect on the expression of these constructs (data not shown). Note that E1A did not affect the transcriptional activity of the 205-bp proximal promoter. From these experiments, we conclude that the phenobarbital inducible 556-bp enhancer region is a target for E1A inhibitory action.

fig.ommitted

  Effect of E1A on the phenobarbital-mediated induction of p4.8-SVCAT, p556-SVCAT, and pCYP-205CAT. The p4.8-SVCAT, p556-SVCAT, and pCYP-205CAT constructs were cotransfected with 0.4 µg of the expression vector for E1A (p4.8-SVCAT also was transfected with 0.1 µg of E1A) into chick embryo primary hepatocytes. Each sample was halved (but not for the pCYP-205CAT construct), and 500 µM of PB was added to one dish and solvent to the control dish. A, the average of three independent experiments, repeated in duplicate and represented as fold-induction over the activity of control value of the p4.8-SVCAT enhancer construct is shown. The CAT activity of the pCYP-205CAT construct is 12- to 15-fold higher than p4.8-SVCAT and did not change with E1A expression. Therefore, results of the CAT assay only are shown . Control vector containing no E1A insert had no effect on phenobarbital induction of either enhancer construct (p4.8-SVCAT and p556-SVCAT). B, a typical CAT assay of one such experiment is shown.

 

Repression by E1A Requires an Intact p300/CBP Binding Domain. The presence of the CR1 domain of E1A (Offringa et al., 1990) is essential for repression of CBP/p300-mediated transcriptional activity. Such repression could involve titration by E1A of CBP/p300 away from the promoter or displacement of p/CAF by E1A (Bannister and Kouzarides, 1996; Reid et al., 1998), because residues in the CR1 domain can directly bind p/CAF independent of CBP (Reid et al., 1998. To test whether E1A repressed the 556-bp CYP2H1 enhancer by interacting with CBP/p300, mutants of E1A with deletions in the CR1 domain (15-35 and 23-107) were examined. The inhibitory action of the E1A mutants on the phenobarbital-induced 556-bp enhancer activity in transfected chick embryo hepatocytes, was considerably abrogated compared with that observed by wild-type E1A . Similarly, an E1A mutant that contained deletion 23-150, thereby abrogating interaction with both CBP/p300 and the retinoblastoma family members, was weakly inhibitory. These results show that the domain of the E1A protein that interacts with CBP/p300 and p/CAF is required for E1A repression of drug-induced enhancer activity.

fig.ommitted

   E1A mutations defective in CBP/p300 binding lose the ability to repress phenobarbital-mediated induction of CYP2H1 enhancer. Chick embryo primary hepatocytes were transiently cotransfected with the p556-SVCAT enhancer construct and either the control vector, containing no E1A insert; the expression vector for E1A; or mutants of E1A 23-107, 23-150, and 15-35. Each sample was halved and 500 µM of PB was added to one dish and solvent to the control dish. The average of three independent experiments repeated in duplicate and represented as relative CAT activity of the p556-SVCAT enhancer construct is shown. Gray and black bars represent control and phenobarbital-induced activity, respectively, of the p556-SVCAT enhancer construct

 

If E1A represses drug induction by targeting CBP/p300, excess CBP should reverse the inhibition. To explore this possibility, E1A expression plasmid (200 ng and 400 ng) was cotransfected with a CBP expression vector (2.5 and 5.0 µg) and p556-SVCAT, a CAT reporter construct containing the 556-bp enhancer. As shown in , CAT activity in response to phenobarbital was repressed by E1A, whereas CBP at 5.0 µg restored this to a maximal level of about 60%. No further reversal was seen with CBP at 10 µg (data not shown). Thus, full restoration of enhancer activity by CBP was not observed. This could reflect an interaction of E1A, not only with CBP but also with other regulatory proteins acting on the 556-bp enhancer region. This possibility was assessed by examining the effect of E1A on enhancer activity when transcription factor binding sites within the 556-bp enhancer were individually mutated. As reported previously (Dogra et al., 1999) mutagenesis of individual binding sites for the factors HNF-1, CCAAT, E-box like protein, and an unknown factor lowered the level of phenobarbital induction. However, E1A repression was still observed in the presence of these mutations, demonstrating that E1A does not directly inhibit the activity of the transcription factors that interact with the 556-bp enhancer. A functional binding site for CXR is located in the 556-bp enhancer region at 1637/1622 (Handschin and Meyer, 2000). However, the possibility that E1A inhibits CXR activity could not be tested by this approach because we found that inactivation of the CXR binding site resulted in a very low level of phenobarbital-mediated induction.

fig.ommitted

  CBP partially reverses the E1A-mediated repression of phenobarbital-induced CYP2H1 enhancer. Chick embryo primary hepatocytes were transiently cotransfected with the p556-SVCAT enhancer construct and either the control vector, containing no E1A insert, or expression vector for E1A and the expression vector for CBP. Each sample was halved and 500 µM of PB was added to one dish and solvent to the control dish. The average of three independent experiments repeated in duplicate and represented as percentage conversion of chloroamphenicol to acetylated form is shown. Gray and black bars represent control and phenobarbital-induced activity, respectively, of p556-SVCAT.


fig.ommitted

  Identification of CYP2H1 enhancer element(s) involved in E1A-mediated repression. The p556-SVCAT enhancer construct containing mutations in either of the four functional transcriptional binding sites, denoted as A (E box like), B (HNF-1), C (Unknown), and D (CCAAT), was cotransfected with either 0.4 µg of the expression vector for E1A or control vector containing no insert into chick embryo primary hepatocytes. Each sample was halved and 500 µM of PB was added to one dish and solvent to the control dish. The average of three independent experiments repeated in duplicate and represented as fold-induction over the activity of control value of the p556-SVCAT enhancer construct is shown. Black and gray bars represent phenobarbital-induced activity of various enhancer constructs cotransfected with E1A expression vector or control vector containing no insert, respectively.

 

E1A Inhibits CXR Activation of the 556-bp Enhancer in LMH Cells. We next examined the effect of exogenous CBP, p/CAF, and E1A on CXR-activation of the CYP2H1 556-bp enhancer construct in chicken hepatoma LMH cells. CXR increased expression of the transfected construct by about 5-fold. Cotransfection with increasing concentrations of CBP (50-400 ng) further increased CXR-induced activation by about 2.6-fold at 400 ng. At these concentrations, a similar result was seen for p/CAF. In the absence of exogenous CXR, neither CBP nor p/CAF altered expression of the 556-bp enhancer construct (data not shown). CXR-driven activation of the 556-bp enhancer construct was inhibited by E1A , and this was totally reversed by CBP at 100 ng concentration. Interestingly, at this concentration, p/CAF also reversed the E1A inhibition, suggesting that this coactivator can replace CBP in the enhancer complex. Our data provide evidence that CXR activation of the enhancer involves CBP and/or p/CAF. Experiments were also carried out to determine whether phenobarbital-induced enhancer activity in primary chick embryo hepatocytes was altered by exogenous CBP. Only a weak response to CBP expression (about 1.2-fold) was seen in repeated experiments (data not shown), most probably reflecting a high endogenous level of this coactivator in the hepatocytes.

fig.ommitted

  The E1A inhibition of CXR-induced activity of 556-bp enhancer is reversed by expression of CBP/pCAF. LMH cells were cotransfected with an enhancer-luciferase construct (p556SV-luciferase), CXR, with control vector containing no E1A insert or an E1A expression clone, various concentrations of either CBP or p/CAF expression clones, and pRL-SV40 as an internal transfection control. A, CXR-induced activity of the p556SV-luciferase construct is further induced with the increasing concentrations (50-400 ng) of CBP and p/CAF coactivators. B, the CXR-induced activity of the p556SV-luciferase construct is inhibited by E1A, and this inhibition is abrogated by coexpressing CBP and p/CAF. Cell extracts were assayed for luciferase activity. The average of three independent experiments repeated in duplicate and represented as fold-induction in activity is expressed as a ratio of test to the control.

 

Trichostatin A Stimulates Phenobarbital-induced Expression of CYP2H1 mRNA but not Basal Expression. It is now well documented that CBP/p300 and p/CAF possess intrinsic histone acetyltransferase activity that can modify chromatin structure through acetylation events (Ogryzko et al., 1996). We therefore examined whether histone acetylation is important in the enhancer-dependent phenobarbital response using TSA, an inhibitor of histone deacetylase activity. Chick embryo primary hepatocytes were pretreated with TSA at a concentration of 1 or 2 µM for 1 h before the addition of phenobarbital and incubation of hepatocytes was continued for a further 6 h. Total RNA was isolated and mRNA amounts determined by Northern blot analysis. Quantification of the results showed that treatment with phenobarbital induced the levels of mRNA for both CYP2H1 (15.2-fold) and ALAS1 (9.5), another phenobarbital-inducible gene (Dogra and May, 1996). TSA alone at 1 µM did not significantly alter the levels of either of the mRNAs, but in the presence of phenobarbital, promoted a super induction of both CYP2H1 (2.8-fold) and ALAS1 mRNAs (12.6-fold). In this study, phenobarbital, TSA, or a combination of both did not affect mRNA levels for GAPDH. Super induction of CYP2H1 and ALAS1 mRNAs with 1 µM TSA treatment was maximum, and no further increase with 2 µM TSA was observed. The data suggest that one action of phenobarbital induction involves acetylation and chromatin remodeling. The fact that neither basal expression nor the control GAPDH gene was altered by TSA shows that the potentiating response reflects a specificity for the inducing agent and not a global change in chromatin structure.

fig.ommitted

   Effect of TSA on the phenobarbital-induced levels of CYP2H1 mRNA. Representative Northern blot analyses of steady-state levels of mRNAs is shown. Hepatocytes were pretreated with TSA (1 or 2 µM) for 1 h and then with or without PB at 500 µM for a further 6 h. Cells without TSA pretreatment were treated with or without PB for 6 h and were included as controls. Total RNA was isolated and 15 µg analyzed by Northern blotting. The filter was hybridized in turn with 32P-labeled probes specific for CYP2H1, ALAS1, and GAPDH (as a control). Radiolabeled filters were quantified using a PhosphorImager (model 300A; Amersham Biosciences), the level of CYP2H1 and ALAS1 mRNA was standardized to that of GAPDH mRNA. This experiment was repeated and similar results were obtained.

 

 

  Discussion

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Abstract
Introduction
Materials and Methods
Results
Discussion
References

Phenobarbital-induced activation of the mouse cyp2b10, rat CYP2B1, and chicken CYP2H1 genes is mediated by complex phenobarbital-responsive enhancer regions, with CAR playing a central role (Dogra et al., 1999; Honkakoski and Negishi, 2000; Handschin et al., 2001; Kim et al., 2001). A 163-bp enhancer region that confers phenobarbital responsiveness to the CYP2B1 gene contains more than three transcription binding sites that, together with a CAR binding site, are required for maximal phenobarbital responsiveness (Stoltz et al., 1998). A 51-bp enhancer region that independently responds to phenobarbital and induces the cyp2b10 gene was shown to contain a nuclear factor 1 binding site flanked by CAR sites (Honkakoski et al., 1998). Similarly, the phenobarbital responsive chicken CYP2H1 gene enhancer region binds at least four transcription factors in addition to CXR (chicken homolog of CAR), with each contributing to enhancer activity (Dogra et al., 1999; Handschin and Meyer, 2000). These studies suggest that phenobarbital-mediated induction requires interactions among multiple regulatory proteins on the enhancer region to constitute a phenobarbital response unit. However, although the coactivator SRC-1 is known to bind CAR (Forman et al., 1998), there is no information as to whether other coactivators are recruited to the phenobarbital-inducible enhancer regions.

In this study, we provide evidence that CBP/p300 and p/CAF are required for CYP2H1 enhancer activity and that E1A as an inhibitor of these components strongly reduces phenobarbital-mediated expression. It was observed that in chick embryo primary hepatocytes, expression of E1A strongly reduced the phenobarbital-induced steady-state level of endogenous CYP2H1 transcript without influencing basal expression of the mRNA in the absence of phenobarbital. Similarly, in transient expression assays, phenobarbital-induced activity of the 556-bp CYP2H1 enhancer was inhibited by expression of wild-type E1A but not by E1A mutants defective in CBP/p300 binding. Hence, E1A specifically interferes with phenobarbital-mediated induction of the gene through altering the enhancer activity.

Overexpression of CBP did not completely reverse E1A-mediated inhibition of the 556-bp CYP2H1 enhancer induced by phenobarbital. We speculated that E1A, in addition to interacting with CBP and pCAF, may also directly target other transcription factors bound on the enhancer, but our mutagenesis data did not support this possibility. Perhaps insufficient CBP is produced in transiently transfected primary hepatocytes to completely abrogate the effect of E1A. This may also be the reason that transient overexpression of CBP only marginally (about 1.2-fold) potentiated the effect of phenobarbital exposure in primary hepatocytes (result not shown).

To further examine whether overexpression of CBP and p/CAF can activate the 556-bp enhancer, chicken hepatoma LMH cells were employed. We reasoned that LMH cells might have low endogenous levels of these cofactors. The basal level of expression of the 556-bp enhancer construct in LMH cells was substantially induced in the presence of exogenously expressed CXR, whereas E1A repressed this activation. In these cells, coexpression of either CBP or p/CAF was able to further activate CXR-mediated transactivation and also completely reverse the inhibitory action of E1A on the 556-bp enhancer. These results strongly indicate that in LMH cells, CBP and p/CAF are involved in the CXR-mediated induction process and that the inhibitory action of E1A is dependent on an interaction with these coactivators. Because E1A expression in LMH cells did not affect the basal activity of the 556-bp enhancer, it can be reasoned that in these cells, coactivator assembly and activation of the 556-bp enhancer is dependent on the presence of CXR. Therefore, our studies with LMH cells and primary hepatocytes demonstrate that CXR plays a critical role in the activation of the 556-bp CYP2H1 enhancer through the recruitment and assembly of the coactivators CBP and p/CAF.

The steady-state level of histone acetylation at a promoter is a balance between the action of histone acetyl transferases and histone deacetylases. It is now well-documented that CBP/p300 and p/CAF possess intrinsic histone acetyl transferase activities that can modify chromatin structure at enhancer/promoter sites to facilitate transcriptional activation (Wade and Wolffe, 1997; Grunstein, 1997; Kadonaga, 1998). In keeping with an acetylation role of CBP/p300 and p/CAF at the enhancer, we have shown that there is a substantial increase in endogenous CYP2H1 mRNA in the presence of phenobarbital, but not in its absence, when histone deacetylases are inhibited with TSA. Moreover, in a preliminary study using the chromatin immunoprecipitation assay, we have observed that exposure of chick embryo primary hepatocytes to phenobarbital leads to enhanced acetylation of histone H3 on the 556-bp enhancer region (data not shown).

In summary, our E1A inhibitor and coactivator overexpression studies demonstrate the involvement of CBP/p300 and p/CAF in the phenobarbital-mediated induction mechanism, with histone acetylation being a likely step in the phenobarbital response. A speculative model can be proposed for phenobarbital-mediated induction of this gene. As described previously (Dogra et al., 1998), we suggest that the intrinsically strong CYP2H1 gene promoter in the native chromatin situation is repressed by a nucleosome. After phenobarbital exposure, CBP and p/CAF form a higher order complex with transcription factors assembled on the enhancer in response to CXR (see . These coactivators lead to hyperacetylation of the nucleosome at the promoter, with subsequent binding of transcription factors and activation of the promoter. In this model, TSA could superinduce CYP2H1 promoter activity by inhibiting deacetylases, responsible for removing acetyl groups on the promoter nucleosome, thereby shifting the equilibrium to a higher acetylation state. Further detailed studies using the chromatin immunoprecipitation assay to analyze the acetylation status of the enhancer and promoter regions after phenobarbital exposure in the presence of activators and inhibitors will be of great interest.

fig.ommitted
 

  Speculative model for phenobarbital-induced activation of CYP2H1 gene. In the absence of phenobarbital, the promoter of the CYP2H1 gene (200 bp) is repressed by a nucleosome. After exposure to drug, CXR (chicken homolog of CAR) translocates to the nucleus and, together with retinoid X receptor (RXR) binds to its site on the enhancer. CXR binding nucleates binding of nearby transcription factors, HNF-1, CCAAT, E-box factor, and unknown factor (?), with recruitment of coactivators including CBP/p300 and p/CAF. This leads to the acetylation of the nucleosome on the promoter, subsequent binding of liver specific factors, and activation of the promoter.

 

 

  Abbreviations

CAR, constitutive androstane receptor; CBP, cAMP response element binding protein; p/CAF, CBP associated factor; CXR, chicken xenobiotic receptor; TSA, trichostatin A; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGFP, enhanced green fluorescent protein; LMH, leghorn male hepatoma; SV, simian virus 40; FACS, fluorescence-activated cell sorting; HNF-1, hepatocyte nuclear factor-1; SRC-1, steroid receptor coactivator-1; bp, base pair(s); kb, kilobase; PB, phenobarbital; CAT, chloramphenicol acetyltransferase.

 

  References

Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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日期:2007年5月15日 - 来自[2003年第63卷第1期]栏目

连续性血液净化治疗热射病并发多脏器功能不全综合征的临床分析

     [摘要]   目的   分析连续性血液净化(CBP)技术在热射病并发多脏器功能不全综合征(MODS)患者救治中的作用。方法   对2例热射病合并MODS患者采用Baxter BM25机器行CBP治疗24次,在每次CBP治疗前及治疗后2 h、4 h、8 h分别取血样,采用ELISA法检测肿瘤坏死因子(TNF-α)、白细胞介素-1(IL-1)、白细胞介素-8(IL-8)的含量,并分别于治疗前与治疗后即时取血化验肌酐(SCr)、尿素氮(BUN)、总胆红素(TBIL)、血肌红蛋白(bMb)浓度。结果   2例患者24次CBP后血浆TNF-α、IL-1、IL-8水平均明显下降(P<0.05),血清BUN、SCr、bMb、TBIL均有不同程度降低(P<0.05或<0.01)。 结论   CBP能清除热射病并发MODS患者血浆中的细胞因子,并可以改善肝、肾功能指标,有效阻止MODS的进展。

    [关键词]   持续性血液净化;热射病;多脏器功能不全综合征;细胞因子

      Clinic analysis of heartstroke associated with multiple organ dysfunction syndrome treated by continuous blood purification

    HE Junfeng,GAO Juping,LI Bing.Department of ICU,the Affiliated Hospital of Medical College,Qingdao University,Qingdao 266071,China

    [Abstract]   Objective   To analyze the role of continuous blood purification(CBP) in treatment of heartstroke associated with multiple organ dysfunction syndrome(MODS). Methods    2 patients met heartstroke associated with MODS were treated 24 times with Baxter BM25 CBP.The plasma TNF-α,IL-1,IL-8 were measured by ELISA at 0,2,4,8h following CBP,and the levels of Bun,SCr,totle Bil,blood Mb.  Results   The plasma concentrations of TNF-α,IL-1,IL-8 decreased actually(P<0.05).The levels of Bun,SCr,totle Bil,blood Mb descended at the different extent(P<0.05 or <0.01).Conclusion   CBP can remove many inflammatory cytokines in plasma,and improve the function markers of liver and kidney in the patients met heartstroke associated with MODS.

    [Key words]   continuous blood purification;heartstroke;multiple organ dysfunction syndrome;cytokine

    热射病是因高温引起体温调节中枢功能障碍,热平衡失调致使体内热蓄积,是中暑最严重的一种类型。它可对机体造成广泛的损伤,导致多脏器功能不全综合征(multiple organ dysfunction syndrome,MODS)。近年来发现,连续性血液净化(continuous blood purification,CBP)技术在救治各种原因导致的MODS患者有较好的疗效,但应用于热射病并发MODS方面的研究很少。我们对2例热射病并发MODS患者进行了24次CBP治疗,临床分析如下。

    1   资料与方法

    1.1   一般资料   病例选自2005年8月我院收治的军事训练时患热射病并发MODS患者2例,均为男性,19岁。热射病的诊断标准依据我国《职业性中暑诊断标准》(GB11508-89)。MODS的诊断标准参照我国1995年庐山全国危重病学术会议通过的MODS诊断评分标准,均发生了包括脑、肾、肝、消化道、胰腺在内的重要器官功能障碍。入院时2例患者均处于休克状态,同时并发了横纹肌溶解症、弥漫性血管内凝血(DIC),在治疗过程中均出现菌血症和二次横纹肌溶解。

    1.2   治疗方法   在抗休克、水化治疗、碱化尿液、药物保护各大脏器功能的基础上,2例患者每日1次,共行24次CBP治疗。采用Baxter BM25机器治疗,具体方法见表1。2例均采用股静脉插管建立血管通路,采用瑞典金宝公司生产的AN69膜透析器,肝素使用普通肝素,剂量25 mg,追加12.5 mg/4 h。血流量100~150 ml/min,连续性静脉—静脉血液滤过(CVVH)全部采用后稀释法,透析液流量30~50 ml/min,为达到降温效果,透析液温度控制在4 ℃。置换液为新鲜冰冻血浆,流量为30 ml/min,每次置换2 L。透析液配方参考季大玺等配方[1]。

    1.3   监测指标   治疗过程中持续体温、心率、血压、中心静脉压监测。在每次CBP治疗前、治疗后2 h、4 h、8 h时取血样,采用ELISA法检测TNF-α、IL-1、IL-8的浓度,试剂盒由美国Bioscience公司提供,检测灵敏度为30 pg/ml,批内、批间误差均<9.5%,操作步骤按其说明书进行。另分别于治疗前与治疗后即时取血,应用全自动生化分析仪(日本Roche 7600型)检测肌酐(SCr)、尿素氮(BUN)、总胆红素(TBIL)、血肌红蛋白(bMb)。 表1   2例MODS患者24次CBP方法(略) 注:CVVH为连续性静脉-静脉血液滤过;PE为血浆置换;HP为血液灌流

    1.4   统计学方法   应用SPSS 10.0软件包进行统计分析,各统计指标以均数±标准差(x±s)表示。计量资料的组间比较采用方差分析和q检验,P<0.05具有统计学意义。     

    2   结果

    2.1   CBP对心血管系统的影响   2例患者均能耐受CBP治疗,生命体征平稳,血流动力学指标良好,全身水肿逐渐减轻。治疗中体温下降至正常范围,血压平均为(122.8±23.4)/(74.2±18.6)mmHg,中心静脉压波动在9~14 cmH2O。

    2.2   出血情况   无因治疗出现出血倾向或加重出血倾向者。     

    2.3   肾功能、胆红素指标的变化   CBP治疗前后,血清BUN、SCr、TBIL、bMb变化差异有显著性(P<0.05)。见表2。表2   CBP治疗前后肾功能及胆红素指标变化比较   (略)

    2.4   外周血细胞因子的变化   CBP治疗后2h及4h,血浆TNF-α、IL-1、IL-8水平明显低于CBP治疗前,差异有统计学意义。见表3。表3   CBP治疗前后细胞因子的变化   (略)注:治疗后各时段与治疗前相比,P<0.05

    2.5   转归   2例患者经抗休克、CBP、纠正DIC、抗感染等综合治疗后,均康复出院,且未留任何后遗症。

    3   讨论

    中暑指体温由于失控或调节障碍而被动地升高,超过了体温调定点水平的一种病理性体温升高过程。热射病是重症中暑的一种类型。一般认为中暑的发病机制是由于身体内部或外部的热负荷超过了机体的散热能力,使身体过度蓄热导致过热。高热可直接损伤细胞膜和细胞内结构,使蛋白质热变性,改变脂膜的流动性,损伤线粒体,从而造成组织细胞的广泛损伤,引起MODS。近年的研究表明,感染、创伤、高热等均可诱发初期的炎症反应及大量的炎性介质的释放,成为发生全身性炎症反应综合征(SIRS)、代偿性抗炎反应综合征(CARS)和MODS的重要环节,而早期控制炎症反应,阻断其发展或有效清除循环中的炎性介质可能是防治SIRS、CARS和MODS的关键[2]。CBP是近年来在间歇性血液透析基础上发展起来的血液净化技术,它可以非选择性地清除血液中的炎症介质,有利于重建内环境的稳态[3]。炎症介质中的细胞因子TNF-α、IL-1、IL-8是目前被公认的能较好地反映SIRS的指标[4],本研究观察到,CBP治疗后血浆TNF-α、IL-1、IL-8水平较治疗前明显降低,差异有统计学意义(P<0.05),证明了CBP具有清除体内炎症介质的作用。因此,重症中暑后早期应用CBP治疗,可减轻SIRS,防止MODS出现,或改善已有的MODS,从而改善患者的预后。本文所观察的BUN、SCr、TBIL、bMb等指标,在CBP治疗前后,差异有显著性(P<0.05),这也从功能指标上说明该2例患者能够救治成功,在很大程度上得益于早期使用了CBP治疗。另外,与间歇性血液透析相比,CBP更符合生理状态,血流动力学更稳定,机体液体入量控制更良好[5],其中血浆置换(PE)与血液灌流(HP)可清除体内有害的大分子物质,这与我们观察的结果是一致的。就本研究中2例患者而言,需要积极有效的降温治疗,采取物理或药物降温有时难以起到预期的效果,在行CBP治疗时,使透析液温度保持在4 ℃,可起到相当好的降温效果。在治疗过程中,2例患者均并发了横纹肌溶解及菌血症,前者所产生的肌红蛋白可导致急性肾衰竭,后者所产生的内毒素可诱导巨噬细胞产生TNF-α等细胞因子,加重MODS。应用CBP治疗既有效清除血肌红蛋白,又能清除炎症细胞因子,很好地保护了各大脏器功能。故不论是对热射病本身,还是对其所发生的并发症所导致的MODS,早期应用CBP治疗是正确而有效的。

    [参考文献]

    1   季大玺.连续性肾脏替代治疗技术的现状.肾脏病与透析肾移植杂志,2000,9(2):75-79.

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    4   Endo S,Kasai T,Inana K.Evaluation of procalcition in levels in patients with systemic inflammatory response syndrome as the diagnosis of infection and the severity of illness.Kansenshogaku Zasshi,1999,73:197-204.

    5   许元文,余学清,梁艳冰,等.连续性肾替代治疗与间歇性血液透析治疗多脏器功能障碍综合征的疗效比较.中华肾脏病杂志,2001,17(11):290-293.

   作者单位: 266071 山东青岛,青岛大学医学院附属医院综合ICU

   (编辑:夏   琳)

日期:2007年4月26日 - 来自[2007年第4卷第1期]栏目

慢性前列腺炎的治疗现状

  关键词:慢性前列腺炎;治疗;药物;手术;非手术非药物

  慢性前列腺炎(CBP)是青壮年男性的一种常见病,发病率约5%~8%\[1\]。现就目前治疗情况综述如下。

  1 一般治疗

  消除病人心理因素,树立能治愈信念,建立良好的生活习惯。如不易过度疲劳、长时间骑车或久坐;注意参加体育活动;少食剌激性食物,少喝烈性酒、咖啡、可可和茶等。多饮水增加尿量冲洗尿道,保持大便通畅;性生活规律,不可纵欲或手淫。注意治疗尿路感染等。

  2 物理疗法

  因前列腺液排泄不畅,积聚液内脓细胞较多,感染持续存在,按摩可排出脓性腺液,增加血液循环促进炎症吸收。同时注射丙酸睾丸酮,增加腺液的分泌量,以利引流。TRM多功能前列腺治疗仪是集热疗、旋磁、振动按摩于一体的物理治疗,可解痉镇痛、化瘀消肿、促进引流。金化民等报道有效率86.2%。

  3  热水坐浴

  45~50℃热水坐浴,对缓解症状有效。有人介绍恒温坐浴装置,使病人臀及会阴坐入盆内,启动加热电源,经反复调试,盆内水温45.4℃为最适水温。每日2次,每次20min,20d为1疗程,治疗过程中病人全身温热微汗,有舒适感。林培生等报告治疗有效率为88.4%。

  4 药物治疗

  4.1 抗生素                                              

  首选复方新诺明,1g每日2次,连续治疗12周,治愈率可达30%~40%。其他如呋喃坦啶、氟哌酸、吡哌酸、红霉素、四环素、强力霉素、庆大霉素、卡那霉素、先锋霉素Ⅳ、头孢唑啉,青、链霉素等均有较好疗效。泰利必妥(Dfloxacin),抗菌谱广,组织内穿透力强,不良反应少。那彦群等报告有效率为76.7%。美满霉素(Minocin),主要通过抑制细菌的蛋白质合成而起抑菌作用,具有高度亲脂性。刘定益等报告治疗4周后细菌培养转阴率达85.7%。

  三种或四种药联合应用屡有报道。袁孔华等介绍3组,A组口服复方新诺明(smzTmp 2片,每日2次)、泌尿灵(0.2g每日3次);B组口服氟嗪酸(0.2g每日3次)、四环素(0.5g每日4次)、消炎痛(25mg每日3次);C组在B组基础上加先锋霉素Ⅵ(针剂,4g溶于注射用水50mL,尿道灌注膀胱保留半小时以上,每周2次)。均4周为1疗程,结果C组有效率为94.4%。陶然等\[2\]报告采用:①左克100mg 3次/d、美满霉素100mg 2次/d。②α1受体阻滞剂马沙泥片2mg/晚。③选择性环氧化酶2(COX2)抑制剂莫必可片7.5mg/白天。④前列舒乐颗粒3g 3次/d。连用4周后,后三药联用平均2.8个月,根据美国国立卫生研究所(NIH)制定的CBP症状评分标准(CPSI)和前列腺按摩液(EPS)指标变化及细菌学检查结果评估疗效,总有效率为83.6%。

  4.2  植物类药 
                                            
  锯叶棕属花粉提出物舍尼通(Prostat)每天2次,每次1片,持续治疗8周后有效率为82.1%\[3\]。相似药护前列片(Urgenin) 主要成分干锯叶棕浸出物(Extra Srbalae serrulatae) 和干紫锥花叶浸出物(Extra Echinangustifoliae siccum)。干锯叶棕果实含有挥发油,能消除膀胱和前列腺黏膜的充血,而干紫锥花叶浸出物能增强机体的抗感染能力,使机体吞噬作用加强,细胞崩解产物及致病菌加速去除。因此,护前列片对CBP有治疗作用。孟荟等报告用护前列片治疗,分A、B两组,结果B组显效率62%,有效率为38%。

  4.3  中医中药                                              

  应用辩证论治,采用活血化瘀和清热解毒药物,如黄芹、黄柏、连翘、车前子、红花、赤芍、滑石、茴香、橘核、荔枝核、王不留行、桃仁等。中成药如清热通淋胶囊、东泰清淋颗粒、炎立消、炎可宁、伏特灵、三金片、宁泌泰、前列腺丸、六味地黄丸、肾气丸、癃闭舒胶囊、癃清片、前列欣胶囊、前列舒乐等,以及单方、验方、民间偏方、中药灌肠、坐浴等有一定疗效。

  4.4  药物对症疗法                                              

  包括镇痛药阿米替林、消炎痛;抗炎药酮洛芬、双氯芬酸、泌尿灵、尿多灵、膀胱灵等;解痉剂普鲁本辛或颠茄合剂等可改善症状。抗雄激素药、肾上腺皮质激素。四季青、毛冬青、黄连素、大蒜等药物的离子透入有缓解症状的作用。有人提出吲哚美辛是最强的PG合成酶即氧化酶(COX)抑制药之一,抗炎作用较氢化可的松大2倍。除抑制PG合成酶外,还能抑制白细胞的运动,减少其在炎症部位浸润和释放致痛物质,减少组织损伤。阻止炎症刺激物引起的细胞炎症反应。另外还有减少缓激肽生成,抑制红细胞和血小板凝集而发挥抗炎作用\[4\]。根据这一理论,张前等\[5\]用吲哚美辛治疗174例,3周时疗效判定,与对照组相比差异均有显著性意义。

  4.5 自由基清除剂                                              

  近年发现CBP患者前列腺液中铜锌超氧化物歧化酶(CuZnSOD)含量明显减少,提示氧自由基在CBP发病中的重要作用\[6\]。有人为探讨自由基清除剂在CBP中的治疗作用,按治疗方法不同随机分为A、B、C、D四组,分别采用自由基清除剂、抗生素、α1受体阻滞剂和安慰剂治疗,结果A组有效率47.3%,并提出自由基清除剂可能成为治疗CBP的一种新方法\[7\]。

  4.6  镇静及解除焦虑药                                              

  紧张和焦虑一直是公认的导致或加重CBP的重要因素之一,因此,关注并缓解CBP患者精神紧张和焦虑的技术方法和药物已成为治疗CBP的辅助手段。对盆底肌紧张痛者应用地西泮(安定)1.25~5mg,1~3次/d,可以对α肾上腺素能受体阻滞剂治疗完全无反应的患者收到一定疗效,它可以减轻外括约肌的痉挛程度降低后尿道压力。地西泮对缓解患者的情绪紧张、焦虑也有一定作用,对心理负担较重的患者有较好的治疗效果。另外可辅以抗抑郁、抗焦虑药物如帕罗西汀20~40mg/d,5羟色胺抑制剂曲唑酮等。

  4.7  综合疗法
                                             
  有人\[8\]对600例CBP,应用喹诺酮类抗生素4~12周,根据病情适当应用α受体阻滞剂、热疗或前列腺按摩,治疗6周后CPSI症状评分判定疗效,结果Ⅱ型有效率13.3%,ⅢA有效率55.8%,ⅢB有效率30.9%。中波或超短波、针灸、气功疗法等对CBP均有一定的疗效。

  5  生物反馈技术

  生物反馈技术是利用自然反射原理进行组织器官的功能训练,最终达到改善和协调局部肌肉和器官功能状态的一种自然疗法。包括:①坚持提肛练习,反复收缩上提,然后放松肛门和睾丸,可以改善局部的血液循环。②一手伸入患者的肛门内嘱咐患者不用腹压而用排便动作将手指推出。③在温暖松弛的环境下用手指反复牵拉阴囊壁20~30次,可以使阴囊内膜和提睾肌松弛,部分患者可在2~3周内缓解或使疼痛消失。④指导患者认识并纠正排尿过程中的盆底收缩状态,进行收缩/舒张锻炼,使肌肉活动恢复到正常的动力学范围,同时进行逐渐增加排尿间隔时间的排尿训练,从而打破痉挛和疼痛的恶性循环状态。⑤低频电刺激治疗也获得了良好的临床效果\[9\]。

  6 灌注疗法

  6.1 双囊四腔硅橡胶导管注药                                              
  程怀瑾等报道68例,均参照Meare氏法明确CBP的诊断,每3~4日治疗1次,6~8次为1疗程,每次治疗时导管留置尿道30~40min,治愈及好转率为80.8%。刘昌荣等采用自制的单囊双腔前列腺灌注管加压灌注药物治疗有效率达94.3%。

  6.2 活力碘膀胱灌注                                              
  活力磺即碘诺酮,亦名聚乙烯吡咯酮碘,其浓度药典规定为6%~12%,市售活力碘为10%。李朝钦介绍此法,经尿道插入导尿管,用1%活力碘150~200mL膀胱灌注,取坐位或站立位,保留2h自行排出。每3d灌洗1次,15~21d为1疗程。治疗98例,临床症状明显减轻,前列腺液镜检正常84例(86%),未出现不良反应。

  6.3 直肠栓剂 
                                            
  前列安栓作为直肠栓剂的代表药物,对前列腺炎的治疗作用已得到初步证实\[10\]。有人作随机双盲安慰剂对照试验,治疗组总有效率71.9%,对照组总有效率41.67%\[11\]。

  7 注射疗法

  7.1  经会阴穿剌注药法                                              

  食指插入直肠内作引导,每次注入1侧叶,也可两侧叶,超声波引导可克服注药的盲目性。注入药物多为丁胺卡那霉素、庆大霉素、先锋霉素或妥布霉素等。但对抗生素的选择应以前列腺液细菌培养和药敏试验为依据。作者采用2%普鲁卡因6mL,0.5%甲硝唑溶液6mL前列腺内注射,每周1次,4次为1疗程,辅以热水坐浴,总有效率达80%。

  7.2 经直肠注入法                                              

  长针头尖端紧贴于左手食指腹侧一并进入直肠内,食指触及前列腺后穿过直肠黏膜,进入前列腺包膜内,回抽无血再注药,此法安全,无痛苦,少数人有轻度不适,有效率75.0%。

  7.3 耻骨上穿剌注入法                                              

  耻骨联合上1.5cm,长针剌入,针身倾斜与腹壁成45~50度进针,达耻骨后间隙有落空感,进针7cm左右遇阻力,再向前推进0.5cm,回抽无血液注入药液。操作注意进针的角度和深度,以免剌入膀胱或耻骨后静脉丛。此法简便,成功率亦较高,无并发症。闵立贵等报告有效率为63.0%。

  7.4 前列腺周围注射法                                              

  与经会阴穿剌前列腺注入法相似,唯一区别在于针尖不进入前列腺包膜内。

  7.5 经输精管注药                                              

  用输精管固定钳将输精管固定于阴囊皮下,以7号针头剌破皮肤及输精管后,再用4号钝针头顺势进入输精管腔内,注入庆大霉素8万单位,0.5%地塞米松1mL,透明质酸酶1500单位,稀释成5mL。每周注射1次,5次为1疗程。王浩然等报告治愈率85%。

  7.6  经附睾头给药                                              

  将睾丸提起握于手内,以拇指及食指固定附睾头,再以8号针头剌附睾头部,穿入后即可注药。王浩然等报告治愈率86.7%。

  7.7 骶管注射法                                              
  有人采用骶管注药阻滞骶神经治疗CBP 489例,有效率83%\[12\],通过阻滞骶神经,可以降低α受体及神经肌肉的兴奋性,降低尿道压力,改善排尿困难症状,减少尿液返流。通过阻断痛觉向中枢的传导,缓解疼痛。

  8 紫外线腔内照射治疗

  赵学兰等采用GRSⅡ型脉冲式低压紫外线治疗机。该机辐射的紫外线主要为短波2537A,生物剂量为<1s。机内设有脉冲时间控制装置和手制动器。可根据需要调节紫外线强度及照射时间。通过弓型石英导子,经直肠进行前列腺照射。病人取膝胸卧位,石英导子涂润滑剂,轻轻插入肛门6~7cm,直达前列腺部位。首次6~7个生物剂量,5日1次,6~10次为1个疗程。用此法治疗CBP 62例,结果总有效率为96.8%。

  9 射频治疗

  桂西青等报告采用体外射频热疗法(ERFH)治疗有效率为87.5%。方法为治疗床上及患者体表应无一滴水及金属异物,以患者耻骨联合下缘为中心(相当于前列腺垂直部位),在臀侧和下腹各置圆形电极板一块,距皮肤约5~7cm,中间以空气为递质。治疗全过程由计算机控制,射频输出功率在65%~74%,局部体表温度达42.4~43.3℃,连续2~2.5h为1疗程,1周后进行第2及第3个疗程治疗。

  经尿道射频热疗,治疗前口服氟哌酸0.2g,每日3次,连服1周。射频治疗时,经尿道置入F16电极尿管于膀胱内,气囊内注蒸馏水10mL后向外轻拉尿管,使电极固定于前列腺部位,连接电极板导线并接通前列腺射频热疗仪。设定温度42℃,时间60min。李宗来等报告治疗1次,有细菌性症状改善者占21.7%;无菌性症状改善者占61.8%。

  10 微波经直肠治疗

  治疗前嘱排空膀胱及大便,先将微波治疗仪接通电源预热5min后,患者露出臀部,微波辐射器套避孕套,套外及肛门涂以石蜡油,辐射器经肛门放置至前列腺部位,开通微波机,调节电源强度,使直肠温度保持在41.5~42.5℃,患者有明显热感,保持50min,10~12次为1疗程,酌情行第2~3个疗程。经直肠微波与药物离子导入同步治疗治愈率为72%,结果表明微波与药物离子导入同步化方法能明显提高转入前列腺内的药物含量\[13\]。直肠内直流电药物导入法治疗CBP治愈率达80.6%\[14\]。

  11 激光治疗

  有人曾创用氦-氖激光治疗CBP,方法为用氦-氖激光治疗仪,病人显露会阴部,用4毫伏功率激光在会阴穴(肛门与会阴部中点)照射,每周照5次,每次10min,20次为1个疗程。此法对减轻症状,促进肿胀腺体的消退有一定效果。

  12  前列腺尿道局部表面用药

  5%弱蛋白银每次10mL尿道灌入,每周1次,对伴有后尿道炎明显者有一定作用。
对伴精阜充血肿胀明显者可经尿道镜直接涂10%硝酸银。闵立贵等\[15\]对4例CBP行膀胱三角区和后尿道灌注1%硝酸银声称效果颇佳。

  13 锌离子体外导入

  正常前列腺液或精液的锌含量大约相当于血浆的100倍,说明前列腺的组织细胞可从血浆或组织中主动摄取锌离子。锌在前列腺液或精液中具有直接杀菌和活化提高组织细胞抗菌能力的作用,是局部免疫防御机制的重要因子。故康新立等采用锌离子体外导入治疗,主观症状好转65.0%,前列腺液镜检改善52.5%,92.5%前列腺液锌含量提高,有望成为CBP的综合治疗措施之一。

  14  电化学(电解)治疗

  电化学(TEP Tansurerhral electrolysis of the prostate)是1987年Nordenstrom教授首次把‘生物闭合电路学说’理论介绍给我国学者。1992年姜其钧研制的电化学治疗仪成功地用于前列腺疾病的治疗。电解治疗CBP的治疗原理是尿道周围约3~5mm内的组织被液化,起到了切除前列腺体导管闭塞和狭窄开口的作用,治疗后有利于前列腺液的排出和引流;直流电将抗菌药直接导入腺体和腺管内起到了其他给药方法无法达到的局部给药效果;电解消除了屏障作用,药物容易进入前列腺组织发挥消炎作用;电解激活了前列腺组织局部的免疫反应,大量的免疫吞噬细胞进入前列腺组织吞噬致病菌,有利于消除前列腺炎症。

  15 手术治疗

  15.1 尿道扩张术                                              
  对伴有尿道狭窄或不通畅者可以进行尿道扩张,以利尿液排泄,降低尿流阻力,减轻症状,有利于炎症吸收。

  15.2 经尿道电切术  
                                           
  经尿道切除CBP之病灶,尤其炎症伴有膀胱颈挛缩者可行经尿道电切术,以解除梗阻缓解症状。保守治疗无效者也可作选择性经尿道电切治疗\[16\]。李黔生等报告4例CBP,经电切术后随访1年效果良好。

  15.3 开放手术   
                                          
  保守治疗不能控制,且并发前列腺结石,可行前列腺摘除术。但手术对一个炎性腺体组织不易切除尽,又有使感染播散的风险。因术后性功能等问题,对于较年轻的病人是不适宜的,病人也难以接受,故较少应用。王振声等\[16\]报告采用前列腺被膜十字切开、膀胱颈成型术治疗CBP,结果非细菌性有效率达85.3%,梗阻型92.6%,疼痛型70.6%。手术机理主要是因被膜十字切开,病灶充分引流,减轻了对血管、神经的压迫,硬化的膀胱颈和受压的尿道得以减压。前列腺被膜动脉-勃起神经束的分支分布在前列腺被膜上,故被膜切开、松解、减压,血循环可恢复正常,性功能得到改善\[17\]。

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  \[15\]闵立贵,段鲁永,马哈苏提. CBP 200例治疗总结 \[J\]. 中华泌尿外科杂志, 2001, 22:180-180.

  \[16\]彭光平主编. 泌尿生殖疾病的临床治疗 \[M\]. 第1版. 北京:北京科技出版社, 2003.21-27.

  \[17\]王振声,赵庆利,白墙,等. 慢性非细菌性前列腺炎的手术治疗 \[J\]. 中华泌尿外科杂志, 2000, 21:746-748.
                                                                  
  作者简介:彭光平(1952),男,山东诸城人,主任医师,从事泌尿外科男科专业. Tel:(0532)7265198

  (青岛市胶州中心医院泌尿外科,山东青岛266300)

  (编辑 邱芬)

  收稿日期:20041208  

 

日期:2006年12月20日 - 来自[2005年第9卷第6期]栏目

连续性血液净化在2例多器官功能不全综合征中的应用

  连续性血液净化(CBP)疗法是缓慢、连续地排除机体内过多的水分及有害毒素(包括细胞因子及炎症介质),比间断的血液透析更接近肾脏的作用,更符合生理状态,从而改善急危重症患者的预后。

    CBP技术是近年来国际国内在危重症治疗技术中的重大进展,与传统的血液透析技术相比,CBP具有以下特点:(1)血流动力学稳定;(2)溶质清除率高;(3)清除炎症介质;(4)营养改善好;(5)改善组织氧代谢;(6)保持水电解质酸碱平衡;(7)生物相容性好。因此,该治疗技术临床适应证包括:(1)各种原因所致的重症急性肾功能衰竭;(2)多器官功能不全综合征(MODS);(3)非急性肾功能衰竭疾病:重症感染、ARDS、药物或毒物中毒、挤压综合症、乳酸酸中毒、体外循环手术、充血性心力衰竭、肝功能衰竭等。

    我科自2003年至今借助移动式血滤机行CBP先后治疗近十余例危重病人,均取得了令人满意的疗效,本文就其中2例因急性坏死性胰腺炎致MODS病人的治疗,浅谈CBP技术在此类病人中的应用。

    1  病例资料

    本组病例中男1例,72岁;女1例,44岁。均因上腹不适于我院外科就诊,经常规检查确诊为急性胰腺炎。入院后病情进行性加重,病程中先后出现烦躁等精神症状加重、少尿急性肾衰、ARDS、心律失常或心衰、胰腺超声及CT均可见胰腺肿胀并可见胰腺坏死区及胰周积液。生化检查可见白细胞(WBC)均超过30×109/L,明显核左移;肝肾功生化异常进行性加重;面罩吸氧时血氧饱和度(SpO2)低于95%;C反应蛋白(CRP)均超过300mg/L。故急性坏死性胰腺炎、MODS诊断明确。

    经外科系统治疗后,2例病人病情仍无好转,于是开始行CBP治疗。采用连续性静静脉透析滤过方式,以滤过为主,置换液每小时2000~4000ml不等,观察主要指标为神志变化;生命体征中的心率、血压、呼吸频率;理化检查WBC、SpO2、CRP的变化。

    2例病人经CBP治疗24h后,烦躁明显减轻, 问话回答正确,能主动配合治疗。心率及血压未经特殊药物(如升压药、西地兰等)应用已稳定于正常范围内。自觉气短症状缓解,呼吸频率不超过25次/min,鼻塞吸氧时SpO2不低于98%。WBC及CRP较入院时明显下降。因我院无法检测细胞因子等炎症介质,故该项目无法监测。

    CBP治疗48~72h后,2例经常规药物治疗均能稳定病情,未有病情加重趋势,故CBP治疗结束。CBP治疗前后观察项目比较见表1。 表1  CBP治疗前后观察项目比较

    2  讨论

    2.1  MODS的发病过程

    2.1.1  MODS相关的几个概念  ①全身炎性反应综合征(SIRS):指机体对不同的严重损伤所产生的全身性炎性反应。包括由细菌等微生物感染导致者(Sepsis)以及由胰腺炎、烧伤、外伤等其他因素所致。②多器官功能不全综合征(MODS):指急性疾病时出现两个或两个以上器官功能的改变,机体的内环境必须靠临床干预才能够维持。③多器官功能衰竭(MOF):指在损伤因素的作用下,机体序贯性发生两个或两个以上器官或系统的功能衰竭,且受损程度进行性发展。

    2.2.2  发病过程  MODS可以相对地分为原发性和继发性。原发性MODS是由明确的损伤因素引起,器官功能的损伤出现早,并与病因的损伤因素直接相关,或者说是由损伤因素本身直接导致的器官功能损伤。例如,子弹的贯通伤导致的肺、肝、肾的功能损伤。继发性MODS中器官功能损伤不是由病因的损伤因素直接引起,而是机体炎性反应的结果。SIRS始终存在于MODS的发生发展过程中,从而导致了似乎与原发损伤无关的远隔器官的功能损伤。有时损伤因素作用于机体后,经过一段时间的潜伏期之后才出现继发性MODS。继发性MODS是ICU病人发生MODS的主要类型。目前已有证据表明,在SIRS过程中大量细胞因子的释放是导致MODS的基础[1]。机体在受到损伤后不同的组织细胞释放出众多的细胞因子是机体对损伤的正常反应,细胞因子的功能具有高度的多向性,可以产生多种不同性质、不同程度的生理效应。机体对细胞因子通过受体、拮抗及其他方式进行复杂调控,连续地控制细胞因子的释放和作用。如果这种平衡范围较广或者开始失衡,则引起SIRS,继而引发MODS。

    2.2.3  MODS发病原因

    损伤 机体应激反应 炎性介质    SIRS  范围扩大或失衡     范围缩小或平衡    MODS   治疗无效  MOF可见,各种细胞因子等炎性介质的作用不可忽视,而其失衡更是MODS发病的主要原因。

    2.3  CBP在MODS中应用的理论依据  人们已经认识到各种炎性介质在SIRS及MODS中的地位,但是目前尚无有效药物控制如此多样的炎性介质,所以人们将注意力转向应用体外疗法清除细胞因子等炎性介质和循环中的内毒素。这就是CBP治疗MODS的初步理论依据。Gomez的研究表明,脓毒血症动物在血液滤过时可清除许多炎性介质,如果把这些炎性介质再注入动物体内则可产生脓毒血症[2]。因此,应用CBP治疗MODS有合理的治疗思路。

    2.4  CBP在MODS中的作用

    2.4.1  炎性介质的清除  CBP中应用高通量透析器可清除相对分子量大于30000的溶质,这些炎症介质是导致SIRS的危险因素,通过CBP的清除,改善MODS的预后。Bellomo等证实,CBP可减轻SIRS,从而显著减少全身性感染及MODS患者的血管活性物质的用量[1]。近几年研究表明透析膜吸附也参与炎性介质的清除。部分细胞因子在通过透析膜时被膜吸附直接清除,无需膜的物质转运。谢红浪等[3]发现,CBP时应用聚酰胺膜能清除肿瘤坏死因子(TNF),并使血浆中TNF水平下降,但超滤液中未检出,提示它是以吸附清除为主。

    2.4.2  内毒素的清除  在MODS的发病中,内毒素同样起着关键性的作用。内毒素含量增高将使机体产生大量细胞因子等炎性介质,造成严重的组织损伤,与血流动力学紊乱、代谢异常、感染性休克、MODS和死亡等密切相关,如能将体内内毒素清除将减轻或防止炎症反应。Kodama等[4]的试验表明,应用聚苯乙烯纤维膜治疗42例脓毒血症患者,治疗前后血中内毒素降低超过60%,提示CBP对内毒素的清除确实有效。但应强调要在脓毒血症的早期进行,否则疗效无明显改善。

    2.4.3  稳定内环境  CBP具有血液透析相同的优点,对于清除间质中多余的水分,改善离子紊乱及酸碱平衡效果确切。对于保证危重患者大量静脉用药时的体液平衡及营养支持的作用不可替代。Reeves等[5]证实,在使用CBP过程中,心血管功能稳定,患者的平均动脉压、心脏指数、心输出量均会升高,平均肺动脉压降低,PaO2上升,动脉氧分压得到改善。

    2.4.4  CBP疗法分类  ①SCUF—连续缓慢超滤;②CAVH—连续动静脉血液滤过;③CVVH—连续静静脉血液滤过;④CAVHD—连续动静脉血液透析;⑤CVVHD—连续静静脉血液透析;⑥CAVHDF—连续动静脉血液透析滤过;⑦CVVHDF—连续静静脉血液透析滤过。

    2.5  结论

    (1)MODS是一个连续发展的动态过程,临床医师应关注从SIRS到MODS整个过程的动态变化,不应局限于某个阶段,力求早期诊断,提倡多学科的相互协作,理想的治疗是早期干预SIRS的发展。(2)炎性介质的大量且过度释放及机体对抗炎调控的失衡是导致MODS的主要矛盾,CBP可以清除这些炎症介质,从而控制MODS的进展,为临床治疗争取时间。(3)强调CBP应尽早开始。对于预计将出现SIRS的病人,在器官发生不可逆的功能损害(MOF)之前,应用CBP清除炎症介质越早越好,一旦发生MOF,则疗效极差。

     【参考文献】

    1  Bone R.Immunologic dissonance : A continuing evolution in our understanding of the systemic inflammatory response syndrome (SIRS) and the multiple organ dysfunction syndrome (MODS). Ann Intern Med,1996,125:68.

    2  Gomez A, Wang R, Unruh H et al. Hemofiltration reverses left ventricular dysfunction during sepsis in dogs. Anesthesiology, 1990,8:161.

    3  谢红浪.连续性肾脏替代治疗对外周血细胞因子的影响.肾脏病与透析肾移植杂志,1999,3:217.

    4  Kodama M, Aoki H, Tani T. Hemoperfusion using a polymyxin B immobilized fiber column for the removal of endotoxin. In: Levin J, Alving CR, Munford RS, eds: Bacterial Endotoxin: Recognition and Effector Mechanisms. Amsterdam. The Netherlands Elsevier Science Publishers, 1993.

    5  Reeves JH, Butt WW, Shann F,et al. Continuous plasma in sepsis syndrome. Crit Care Med, 1999,27:2096.

  作者单位: 118002 辽宁丹东,丹东市中心医院肾内科   

   (编辑:若  木)

日期:2006年12月18日 - 来自[2006年第6卷第15期]栏目
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