【摘要】 目的 探讨CD147与基质金属蛋白酶2（MMP2）在子宫内膜样腺癌组织中的表达及其与临床病理参数的关系。方法 采用免疫组化SP法检测31例正常子宫内膜、73例子宫内膜样腺癌组织中CD147与MMP2的表达情况。结果 CD147、MMP2在子宫内膜样腺癌组织中的阳性表达率分别为76.71%、89.04%，均高于其在正常子宫内膜组织中的表达（χ2=29.734、38.176，P＜0.01）。子宫内膜样腺癌CD147、MMP2表达与手术病理分期、肌层浸润深度、淋巴结转移有关（χ2=4.002～6.627，P＜0.05；P=0.047、0.040）。子宫内膜样腺癌组织中CD147与MMP2的表达一致率为61.64%，两者表达呈正相关（r=0.752,P＜0.01）。结论 子宫内膜样腺癌组织中CD147、MMP2的高表达可能作为子宫内膜样腺癌生物学行为的客观指标。
【关键词】 癌,子宫内膜样；抗原,CD147；明胶酶A；免疫组织化学
THE EXPRESSIONS AND CLINICAL SIGNIFICANCE OF CD147 AND MMP2 IN ENDOMETRIOID ADENOCARCINOMAYANG YANDONG, ZHANG YINGZI, LIANG KUIXIANG, et al (Department of Obsterics and Gynecology, The Affiliated Hospital of Binzhou Medical College, Binzhou 256603, China); ［ABSTRACT］ Objective To investigate the expressions of MMP2 and CD147 in endometrioid adenocarcinoma (EA),and their relationship with clinicopathologic parameters. Methods The expressions of CD147 and MMP2 were detected in normal endometrium and EA tissues in 31 and 73 cases, respectively, by using immunohistochemical technique. Results The positive expressions of CD147 and MMP2 in EA were 76.71% and 89.04%, respectively, which were significantly higher than those in normal endometrium tissue (χ2=29.734,38.176;P<0.01), these two expressions were associated with pathologic staging, depth of muscular invasion and lymphatic metastasis (χ2=4.002-6.627,P<0.05;P=0.047,0.040). The concordance rate of expressions of CD147 and MMP2 in EA was 61.64%, a positive correlation of expressions existed between the two groups (r=0.752,P<0.01). Conclusion The high expressions of CD147 and MMP2 in EA may be used as an objective marker of biological behavior of this maliglancy.
［KEY WORDS］ Carcinoma, endometrioid; Antigens, CD147; Gelatinase A; Imnunohistochemistry
子宫内膜癌是常见的妇科恶性肿瘤之一，多见于围绝经期及绝经后的妇女，但在近年来，该病的发病年龄呈年轻化趋势，且发病率明显上升，尤其是与雌激素相关的Ⅰ型子宫内膜样腺癌，发病人数已占到子宫内膜癌病人的80%～90%,严重威胁着广大妇女的生命和健康。恶性肿瘤的侵袭和转移是多步骤过程的结果，基质金属蛋白酶(MMPs)在多种肿瘤的浸润和转移过程中起了重要作用。肿瘤细胞分泌的CD147可刺激MMPs的大量分泌，从而影响肿瘤的生物学行为和临床进展过程。本研究应用免疫组织化学SP法检测CD147和基质金属蛋白酶2（MMP2）在子宫内膜样腺癌组织中的表达,探讨二者与子宫内膜样腺癌侵袭、转移的相关性。
1 材料和方法
1.1 标本来源
收集2000年01月～2006年12月滨州医学院附属医院妇科子宫内膜样腺癌术后标本73例，其中高分化腺癌（G1）27例，中分化腺癌（G2）26例，未分化腺癌（G3）20例；浸润肌层深度≤1/2者51例，＞1/2者22例；淋巴结有转移者23例，无转移者50例；按2000年FIGO分期，Ⅰ期25例，Ⅱ期23例，Ⅲ期25例。同时，收集诊断性刮宫所得的正常增生期子宫内膜31例作对照。所有病例术前均未行放、化疗和雌、孕激素治疗。
1.2 检测方法
采用免疫组化SP法，操作严格按SP试剂盒说明进行。兔抗人CD147分子多克隆抗体、三步法染色试剂盒（SP 9001）、DAB试剂盒均购自北京中杉金桥生物技术有限公司，MMP2单克隆抗体试剂盒购自福州迈新生物技术公司。每批均用已知阳性切片（试剂公司提供）作阳性对照，用磷酸盐缓冲液（PBS）代替一抗作阴性对照。
1.3 结果判定标准
以细胞出现棕黄色颗粒为阳性，其结果根据SHIMIZU等[1]的方法分析。着色范围以阳性细胞数占总细胞数的百分数计分：无着色为0分，≤20%为1分，21%～50%为2分，>50%为3分；着色强度依据着色深浅计分:无着色为0分，浅着色为1分，深着色为2分，上述两项相加为着色程度积分，结果0、1分为阴性(-),2分为弱阳性(+),3分为阳性(),≥4分为强阳性()。
1.4 统计学处理
采用SPSS 11.5和PPMS 1.51[2]统计软件进行数据处理，组间比较采用χ2检验、Fisher精确概率法，两者的相关性用Spearman等级相关分析。
2 结 果
2.1 CD147、MMP2着色部位
CD147阳性染色主要定位于细胞膜；MMP2阳性染色主要定位于癌细胞胞浆中，胞膜上亦有少许表达。
2.2 CD147、MMP2在正常子宫内膜组织和子宫内膜样腺癌组织中的表达
CD147在正常子宫内膜与子宫内膜样腺癌组织中的阳性表达率分别为19.35%、76.71%，差异有极显著性（χ2=29.734,P<0.01）。MMP2在正常子宫内膜与子宫内膜样腺癌组织中的阳性表达率分别为29.03%、89.04%，差异有统计学意义（χ2=38.176,P<0.01）。
2.3 CD147、MMP2与子宫内膜样腺癌临床病理特征的关系
子宫内膜样腺癌CD147、MMP2表达与手术病理分期、肌层浸润深度、淋巴结转移有关（χ2=4.002～6.627，P<0.05；P=0.047、0.040）。MMP2表达与子宫内膜样腺癌病理分级有关(χ2=5.571，P<0.05)，而CD147表达与病理分级无关(χ2=2.420，P>0.05)。见表1。
2.4 子宫内膜样腺癌组织中CD147与MMP2表达的关系
CD147与MMP2在子宫内膜样腺癌组织中同时阴性表达和同时阳性表达，且程度相同的分别为7和38例，两者表达的一致率为0.616 4, CD147与MMP2的表达呈正相关（r=0.752，P<0.01）。见表2。
3 讨 论
肿瘤的浸润与转移是恶性肿瘤的主要生物学特征,也是恶性肿瘤病人的主要死亡原因。肿瘤细胞对细胞外基质成分及基底膜的降解是肿瘤浸润转移表1 子宫内膜样腺癌组织中CD147、MMP2表达与临床病理特征的关系临床病理特征nCD147表达表2 子宫内膜样腺癌组织中CD147与MMP2表达的关键步骤。MMPs是一组重要的细胞外基质降解酶,它通过对细胞外基质中不同成分的降解,在肿瘤浸润转移中起重要作用。作为一个有着23个成员的MMPs大家族,MMP2在肿瘤细胞介导的细胞外基质降解中起关键作用。MMP2是一种锌离子依赖的蛋白水解酶,又称Ⅳ型胶原酶,由肿瘤细胞和间质细胞以酶原形式分泌,经水解后激活。在病理情况下,一方面通过降解基底膜和包绕肿瘤的基质,突破基质屏障,促进肿瘤浸润转移；另一方面则通过毛细血管增生、新生血管生成等,促进肿瘤生长和扩散[3]。而多项研究表明,MMP2是降解Ⅳ型胶原最主要的酶,活化的MMP2定位于细胞穿透基质的突出部位,具有“钻头”样作用,通过转染MMP2激活因子即膜结合的MMPs,能增强肿瘤细胞穿过基底膜基质的功能[4]。本文结果显示，MMP2在子宫内膜样腺癌组织中高表达，其阳性表达率为89.04%，显著高于正常子宫内膜；随着子宫内膜癌淋巴结的转移、肌层浸润深度、病理分级、手术病理分期的升高,MMP2的阳性表达率也随之增高。由此推测，在子宫内膜样腺癌组织中，由于机体免疫监视功能降低，使肿瘤细胞分泌MMP2增加，导致MMP2高水平表达，降解细胞外基质及基膜能力增强，促进肿瘤新生血管的形成，从而促使肿瘤细胞侵袭周围正常组织并向远处转移。
CD147是相对分子质量为5万～6万的单次跨膜糖蛋白,属免疫球蛋白超家族成员,在人体内分布广泛,具有多种不同功能。恶性肿瘤细胞侵袭生长时需穿过基膜和间质成分构成的细胞外基质,基膜的主要成分是Ⅳ型胶原蛋白,间质的主要成分是Ⅰ型胶原蛋白,大部分肿瘤细胞可产生降解Ⅳ型胶原蛋白的酶(如MMP2、MMP9),但很少有肿瘤细胞能产生降解Ⅰ型胶原蛋白的酶(如MMP1)来降解间质成分。KANDKURA等[5]对皮肤黑色素瘤进行的研究结果显示，表达CD147的肿瘤细胞能刺激纤维母细胞产生MMP1、MMP2、MMP3和MT1MMP,从而导致胶原酶的溶解活性增加。研究表明,CD147在胃癌、结直肠腺癌等肿瘤中有阳性表达,而且也相应发现在这些肿瘤组织中有MMPs的高表达[6,7]。在部分肿瘤中还发现随着肿瘤恶性程度的增高，CD147的表达也增高，且与肿瘤的浸润和转移相关[8,9]。本研究结果显示，CD147在子宫内膜样腺癌组织中的阳性表达率为76.71%，与其在正常子宫内膜组织表达比较，差异具有显著性，提示CD147在子宫内膜样腺癌的发生发展中发挥了重大作用，这与公莉等[10]研究结果相符。本研究结果还显示，CD147的阳性表达与子宫内膜样腺癌的手术病理分期、肌层浸润深度及淋巴结转移有关，提示临床分期越晚、分化越差的子宫内膜样腺癌，其表达CD147的能力越强。
本文结果亦显示，CD147与MMP2在子宫内膜样腺癌细胞中多同时表达,二者联合表达率为61.64%,二者表达呈显著正相关。这一结果与目前多数学者认为的CD147能刺激肿瘤周围成纤维细胞产生MMPs的理论相吻合。CD147不仅能刺激肿瘤周围成纤维细胞产生MMP2,而且还能刺激成纤维细胞产生MT1MMP、MT2MMP,而后两者又是MMP2的激活物[11]。同时,CD147还可与MMP2在肿瘤细胞表面形成复合物,这种复合物的形成将MMP2浓缩在肿瘤细胞周围,使得肿瘤细胞周围由多种蛋白酶修饰重塑的微环境发生了破坏,肿瘤细胞周围的基质降解,使肿瘤细胞易于扩散和转移[12]。鉴于CD147和MMP2的阳性表达与子宫内膜样腺癌浸润和转移的密切关系,CD147和MMP2抑制剂治疗肿瘤已进入临床试验阶段，因此进一步探索CD147、MMP2在子宫内膜样腺癌组织中的表达及其适宜的抑制剂,可为抑制子宫内膜样腺癌的浸润转移提供一条新途径,对于改善子宫内膜癌病人的预后具有重要意义。
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作者单位：滨州医学院附属医院妇产科，山东 滨州 256603
Department of Cell Biology, the Fourth Military Medical University
Department of Clinical Immunology, Xijing Hospital, the Fourth Military Medical University, Xi'an
Institute of Basic Medical Sciences, Academy of Military Medical Sciences
Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
Department of Molecular Medicine, University of California, San Diego, San Diego
To identify the function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome (SARS) coronavirus (CoV), we analyzed the protein-protein interaction among HAb18G/CD147, cyclophilin A (CyPA), and SARS-CoV structural proteins by coimmunoprecipitation and surface plasmon resonance analysis. Although none of the SARS-CoV proteins was found to be directly bound to HAb18G/CD147, the nucleocapsid (N) protein of SARS-CoV was bound to CyPA, which interacted with HAb18G/CD147. Further research showed that HAb18G/CD147, a transmembrane molecule, was highly expressed on 293 cells and that CyPA was integrated with SARS-CoV. HAb18G/CD147antagonistic peptide (AP)9, an AP of HAb18G/CD147, had a high rate of binding to 293 cells and an inhibitory effect on SARS-CoV. These results show that HAb18G/CD147, mediated by CyPA bound to SARS-CoV N protein, plays a functional role in facilitating invasion of host cells by SARS-CoV. Our findings provide some evidence for the cytologic mechanism of invasion by SARS-CoV and provide a molecular basis for screening anti-SARS drugs.
Severe acute respiratory syndrome (SARS) coronavirus (CoV), the pathogen ascertained to be responsible for SARS, caused disastrous results around the world at the end of 2002 [1, 2]. How SARS-CoV infects host cells still remains unclear, and the results of studies of the other CoVs suggest that CoVs infect host cells by direct or indirect interaction between viral proteins and cellular proteins expressed on the unit membrane [25]. Cyclophilin A (CyPA) is a ubiquitously distributed cellular protein and is thought to assist protein folding and to function as a chaperone. It can be integrated with virions of some kinds of viruses, such as HIV-1 [6] and vaccinia virus [7]. By interacting with cellular receptors or through other pathways, integrated CyPA plays a role in viral invasion or replication. CD147 (called "EMMPRIN" in [8] and "basigin" in [9]) is a transmembrane glycoprotein and belongs to the immunoglobulin superfamily. It is a receptor for CyPA and contributes to viral infection or inflammation [6, 10]. HAb18G/CD147, which was developed and was identified to be a member of the CD147 family in our laboratory, is involved in tumor metastasis, inflammation, and virus infection [1119]. Inspired by the known relationship between CD147 and HIV-1, we conducted the present study to investigate the possible function of HAb18G/CD147 in invasion of host cells by SARS-CoV.
MATERIALS AND METHODS
Viruses and cells.
SARS-CoV (BJ04) and 293 cells (ATCC CRL-1573) were supplied by the Chinese Center for Disease Control and Prevention (China CDC). The cells were cultured in Dulbecco's MEM (Gibco BRL) containing 10% fetal calf serum (Gibco BRL).
Peptides.
HAb18G/CD147antagonistic peptide (AP)9 (an AP of HAb18G/CD147 obtained in our laboratory, composed of 12 aa residues) [2024], CB-2 (a randomly synthesized peptide also containing 12 aa residues, used as negative control), and biotinAP-9 were synthesized by CL Bio-Scientific. Their purity, assayed by high-pressure liquid chromatography, was >95%, and their amino-acid sequences and molecular weights, respectively, were as follows: AP-9, YKLPGHHHHYRP and 1541.09; CB-2, LHRHSHGHSYTS and 1389.50; and biotinAP-9, biotin-YKLPGHHHHYRP and 1767.69.
Coimmunoprecipitation (Co-IP) analysis.
The ProFound mammalian Co-IP kit (Pierce) and the ECL Plus Western blotting detection system (Amersham Pharmacia) were used in accordance with the manufacturers' instructions. To analyze the interaction between HAb18G/CD147 and CyPA, 100 g of HAb18G/CD147 (a recombinant extracellular fragment of HAb18G/CD147, prepared in our lab) was incubated with an equivalent amount of CyPA (Alexis Biochemical) overnight at room temperature (RT). The mixture was then added to the gel, which was coupled with 200 g of mouse antihuman HAb18G/CD147 monoclonal antibody (MAb) HAb18 (prepared in our lab) [2533], for incubation with gentle end-over-end mixing for 4 h. After the bound proteins were eluted, aliquots of the eluent were analyzed by a Western blot developed with rabbit antihuman CyPA antibody (diluted 1 : 2000; Calbiochem-Novabiochem) and horseradish peroxidase (HRP)conjugated goat antirabbit IgG (diluted 1 : 5000; Amersham Pharmacia). Purchased CyPA (2.5 g) was used as the positive control.
To analyze the interaction between CyPA and SARS-CoV structural proteins (spike [S], membrane [M], envelope [E], and nucleocapsid [N]; provided by China CDC), the gel was coupled with 200 g of CyPA, which was used as the bait protein. Mouse antibodies to the 4 proteins (diluted 1 : 1000) and HRP-conjugated goat antimouse IgG (diluted 1 : 5000) were used in the subsequent Western blot analyses. Five micrograms of the respective protein was used as the positive control. Blank controls were established in accordance with the manufacturer's instructions.
BIAcore spectroscopy.
Surface plasmon resonance analysis was performed, by use of a BIAcore X biosensor system (BIAcore), as described elsewhere [34]. HAb18G/CD147 or CyPA was immobilized on research-grade CM5 sensor chips (BIAcore), with a concentration of 100 g/mL in 10 mmol/L sodium acetate (pH 6.0), by use of the amine coupling kit supplied by the manufacturer. Approximately 2000 resonance units of HAb18G/CD147 or CyPA was immobilized under these conditions. Unreacted moieties on the surface were blocked with ethanolamine, and the chips were measured in HEPES-buffered saline containing 10 mmol/L HEPES (pH 7.4).
Analyses were performed at 25°C, at a flow rate of 5 L/min for determination of on-rates and equilibrium binding and 50 L/min for determination of off-rates. To determine which protein of SARS-CoV could be captured by CyPA or HAb18G/CD147, the S, M, E, and N proteins of SARS-CoV were diluted to 1 g/mL in HEPES-buffered saline for capture by the CyPA or HAb18G/CD147 surface. Surfaces were typically regenerated with 100 mmol/L hydrochloric acid and 0.2 mol/L Tris buffer.
To assess the affinity of N protein to CyPA, N protein was diluted to 40, 32, 24, 16, and 8 nmol/L for capture by the CyPA surface. Biosensor data were prepared for kinetic analysis, by zeroing the time and response before the first injection. To correct refractive index changes and nonspecific binding, the binding responses generated in the control experiments were subtracted from the responses generated in the CyPAN protein interaction. The binding data were then globally fitted to the following reaction mechanism, in which CyPA and N protein are represented by A and B, respectively: A + B AB. An equilibrium dissociation rate constant (Kd) was calculated from the kinetic rate constants by nonlinear fitting of the primary sensorgram data by use of BIAevaluation software (version 3.1; BIAcore).
Flow-cytometric analysis.
293 cells, at a density of 106 cells/mL, were incubated with either 10 L of fluorescein isothiocyanate (FITC)conjugated anti-CD147 antibody (Pharmingin) or 10 L of FITC-conjugated mouse IgG1 (control; Pharmingin) in the dark for 30 min at 4°C. After being washed once with PBS, cells were fixed with 1% paraformaldehyde and analyzed by use of a FACSCalibur flow cytometer (Becton Dickinson) and CellQuest software (Becton Dickinson).
For analysis of AP-9 binding, the cells, blocked by goat serum for 30 min and incubated with biotinAP-9 (final concentration, 160 g/mL) for 60 min, were washed and treated with 5 L of avidin-FITC (Pharmingin) in the dark for 60 min at 4°C. Fixation and data analysis were performed as described above.
Confocal microscopy.
293 cells infected with SARS-CoV (BJ04) were cultured overnight on the sterilized cover slips and then washed in PBS and fixed with cold acetone for 30 min. For analysis of AP-9 binding, the cells were incubated with 25 g of biotinAP-9 and avidin-Cy3 (diluted 1 : 100; Sigma) in the dark overnight at 4°C. For analysis of blocking of AP-9, 25 g of biotinAP-9 was incubated with 2.5 g of HAb18G/CD147 for 2 h at RT. Then, the mixture (biotinAp-9 and HAb18G/CD147) and avidin-Cy3 were added to the cells and incubated in the dark overnight at 4°C. The negative control, which was treated only with avidin-Cy3, was established at the same time. By use of the immuofluorescence double-staining method, the cells were incubated with 25 g of HAb18 and an equivalent amount of biotinAP-9 for 2 h at RT. After being washed with PBS, the cells were incubated with avidin-Cy3 (diluted 1 : 100) and FITC-labeled goat antimouse IgG (diluted 1 : 100; Wuhan BOSTER Bioengneer) in the dark overnight at 4°C. Careful observations were made by use of a confocal microscope (IX-7; Olympus) with an object lens (UPLAPO; ×20 or ×40) and image-acquisition software (FLUOVIEW FV 300; Olympus).
Immunoelectronic microscopy.
293 cells infected with SARS-CoV (BJ04) were harvested and pelleted. The cells were treated with 4% glutaral for 30 min at 4°C and then were washed with phosphate buffer twice and treated with 1% osmium tetroxide (Polysciences) for 1.5 h at RT. After they had been dehydrated and embedded, the ultrathin sections of the cells were prepared.
The indirect gold colloidlabeling method was used to detect the localization of CyPA and HAb18G/CD147. After being washed in distilled water for 3 min, the ultrathin sections were treated with 1% H2O2 for 5 min and incubated with normal goat serum (diluted 1 : 75) for 10 min at RT and with rabbit antihuman CyPA antibody (diluted 1 : 25; Calbiochem-Novabiochem) or mouse antihuman CD147 antibody (diluted 1 : 50; Abcam) for 16 h at 4°C and for 1 h at RT. After being washed in PBS 3 times and in distilled water once, the sections were treated with PBS (containing 1% bovine serum albumin [BSA] [pH 8.2]) for 5 min and then were incubated with gold colloidlabeled protein A (diluted 1 : 50; diameter of the gold particle, 10 nm; prepared by the Academy of Military Medical Sciences [China]) for 1 h at RT and consecutively stained with 5% uranium acetate and lead acetate.
Then, the gold colloid double-labeling method was used to determine the colocalization of HAb18G/CD147 and AP-9. After being washed, the sections were treated with 1% H2O2 and normal goat serum (diluted 1 : 75), as described above. After being incubated with PBS (containing 1% BSA [pH 8.2]) for 5 min, the cells were incubated with the mixture of gold colloidlabeled AP-9 (diluted 1 : 400; diameter of the gold particle, 20 nm; prepared by the Academy of Military Medical Sciences [China]) and mouse antihuman CD147 antibody (diluted 1 : 50) and then were incubated with the gold colloidlabeled protein A (diluted 1 : 50) for 1 h at RT and were consecutively stained with 5% uranium acetate and lead acetate. Careful electronic microscopic observations were made by use of a JEM-100SX microscope (JEDL).
Analysis of cytopathic effect.
293 cells (4 × 105 cells/mL) were planted into 96-well plates (Costar) and kept in 5% CO2 for 24 h at 37°C. For analysis of the toxicity of AP-9 to 293 cells, 2-fold dilution series of AP-9 (range, 3893.47.6 mol/L), CB-2 (negative control; range, 4318.18.4 mol/L), and gancyclovir (an antivirus chemical drug purchased from Hubei Keyi Pharmacy) injection (positive control; range, 23,508.245.8 mol/L) were added to the cells. The TD0 and TD50 were determined. For analysis of the virulence of SARS-CoV (BJ04) to 293 cells, viruses in 8 dilutions (10-110-8) were added to the cells, to determine the TCID50 of SARS-CoV (BJ04). For the inhibitory-effect analysis of AP-9, the cells were incubated with 100 TCID50 of SARS-CoV (BJ04) for 2 h. After the supernatant was discarded, 2-fold dilution series of TD0 of AP-9 (range, 973.31.9 mol/L), CB-2 (range, 359.80.7 mol/L), and gancyclovir (range, 23,508.245.8 mol/L) injection were added to the appropriate wells. Normal cell controls and virus controls were established. Cytopathic effects on the above cells were observed daily by use of an inverted microscope (XSZ-D; Nanjing Optical Instrument Factory of China) with an object lens (×10) and a camera (Ricon-5). Morphological changes observed in <25% of total cells were marked as "+", in 25%50% were marked as "++", in 51%75% were marked as "+++", and in 76%100% were marked as "++++". TD50, TD0, TCID50, IC50, MIC, and treatment index (TI) were calculated by use of the Reed-Muench method. (For the inhibitory-effect analysis, when "+++" or "++++" was marked in virus control, the test was stopped.)
RESULTS
Interaction among N protein, CyPA, and HAb18G/CD147.
Surface plasmon resonance analysis showed that none of the SARS-CoV S, M, E, or N proteins directly bound to HAb18G/CD147. However, N protein was found to interact with CyPA. In Co-IP analysis, when the mixture of HAb18G/CD147 and CyPA was added, CyPA was found to be indirectly bound to the gel, mediated by the specific interaction of HAb18G/CD147 to its MAb, HAb18, which was used as the capture antibody. Thus, the HAb18-HAb18G/CD147-CyPA complex was formed. After the coimmunoprecipitated CyPA was eluted, it was visible by Western blot analysis performed with anti-CyPA antibody, which confirmed the interaction between CyPA and HAb18G/CD147 (figure 1A). Co-IP analysis further confirmed the interaction between N protein and CyPA. Since CyPA was used as the bait protein, the coimmunoprecipitated N protein was visible by Western blot analysis performed with antiN protein antibody (figure 1B). The binding kinetics of N protein (in different concentrations) to CyPA was determined by surface plasmon resonance analysis, with a Kd of 0.04 mol/L (figure 1C).
Quantitative analysis of the expression of HAb18G/CD147 on SARS-CoVpermissive 293 cells and the binding of AP-9 to them.
HAb18G/CD147 was found to be highly expressed on 293 cells, at the expression rate of 98.56% (figure 2A). AP-9 was shown to be bound to the detected cells, at the binding rate of 98.15% (figure 2B), which was very close to the expression rate of HAb18G/CD147 on the same kind of cells. AP-9 also had a median fluorescence intensity that was similar to that of HAb18G/CD147.
Cellular localization of HAb18G/CD147 and AP-9 on SARS-CoVinfected 293 cells.
After being traced by fluoresceins, by use of laser scanning confocal microscopy, AP-9 was found to have high affinity to the detected cells. After incubation with HAb18G/CD147, the binding of AP-9 to the cells obviously decreased (figure 3A). By use of the immunofluorescence double-staining method, both HAb18G/CD147 and AP-9 were seen on the same binding sites (the cell membrane and the cytoplasm) of SARS-CoVinfected 293 cells (figure 3C).
Subcellular localization of CyPA, HAb18G/CD147, and AP-9 on SARS-CoVinfected 293 cells.
After being traced by gold colloidlabeled antibody, by use of electronic microscopy, CyPA was present on or around the surface of SARS-CoV (figure 4A), indicating that CyPA was integrated with SARS-CoV. HAb18G/CD147 was located on the detected cell surface and the unit membrane, especially on the membrane of endoplasmic reticulum (ER) in cytoplasm (figure 4B). The gold colloid double-labeling method was used, and the results showed that AP-9 was present on the same sites as was HAb18G/CD147 (figure 4C).
Inhibitory effect of AP-9 on SARS-CoV.
The results of analysis of cytopathic effect showed that AP-9, at a concentration of 973.3 mol/L, was nontoxic to 293 cellsthe TD50 and TD0 for AP-9 were 1946.7 and 973.3 mol/L, respectively. The TCID50 of SARS-CoV to 293 cells was 10-3. When 100 TCID50 of SARS-CoV was added to the normally cultured 293 cells, the cells became infected and necrotic. After an MIC of AP-9 was added, the infected cells gradually recovered, whereas the AP-9untreated virus control cells remained necrotic. The IC50, MIC, and TI of AP-9 were 60.8 mol/L, 30.4 mol/L, and 32, respectively, whereas those of the positive control (with gancyclovir injection) were 91.8 mol/L, 45.8 mol/L, and 256, respectively; the TI of the negative control (with CB-2) was only 4.
DISCUSSION
It has been reported that CyPA can be specifically incorporated into the virions of HIV-1 and can significantly enhance an early step of cellular HIV-1 infection. CD147 can facilitate HIV-1 infection by interacting with virus-associated CyPA [6]. The present study has shown that HAb18G/CD147, a member of the CD147 family, can interact with CyPA, which may be associated with the SARS viruses and play a functional role in invasion of host cells by SARS-CoV. AP-9 has an inhibitory effect on SARS-CoVs.
SARS-CoV is known to have 4 structural proteins: S, M, E, and N. S, M, and E proteins are envelope proteins of SARS-CoV, and N protein is bound to viral RNA in the core. S protein of the other known CoVs is regarded to be responsible for both binding to receptors on host cells and membrane fusion [2]. In the present study, we have found that N protein may play a role in invasion of host cells by SARS-CoV. N protein was bound to CyPA, but S, M, and E proteins were not. However, CyPA was present only on the surface of mature SARS-CoVs, as observed by electronic microscopy. This finding seems to be contradictory to the finding that N protein locates in the core of the virus. This difference can be partly explained by the finding of a previous report that CyPA bound to viral proteins in the core can be relocated from the core to the viral surface during maturation of the virus [35]. The results of the present study also confirm the interaction between CyPA and HAb18G/CD147, as determined by Co-IP analysis, indicating that CyPA may act as a mediator between SARS-CoV N protein and HAb18G/CD147 in the process of invasion of host cells by SARS-CoV. By flow-cytometric analysis, confocal microscopy, and immunoelectronic microscopy, we found that HAb18G/CD147 was highly expressed in the SARS-CoVpermissive 293 cells and that it was located on the cytomembrane and the unit membrane in the cytoplasm (especially on the membrane of ER) as a transmembrane molecule. In the present study, the binding site of AP-9 was confirmed to be on the HAb18G/CD147 molecule in the infected 293 cells, and AP-9 could efficiently block the binding sites of HAb18G/CD147 on the cytomembrane and the unit membrane in the cytoplasm of the 293 cells and had an inhibitory effect on SARS-CoV in vitro. From this finding, we can conclude that HAb18G/CD147 is a functional molecule in SARS-CoV infection of host cells.
The mechanism of HAb18G/CD147 as a functional molecule can be inferred as follows: (1) CyPA is bound to N protein after invasion of host cells by SARS-CoV, and CyPA relocates to the virus surface during the maturation of the virus [35]; (2) the exposed CyPA molecules interact with HAb18G/CD147 on the cell membrane, which leads to the infection of other host cells; and (3) AP-9 blocks HAb18G/CD147, to prevent virus infection after those viruses complete their life cycle.
It also has been reported that the life cycle of CoVs that invade host cells includes N protein assembling with the full-length replicated RNA to form the RNA protein complex, which is associated with the M protein embedded in the membrane of ER and virus particles, which are formed as the nucleocapsid complex buds into ER [2, 36]. SARS-CoV is believed to have a similar process in the life cycle [36]. Therefore, we also inferred that SARS-CoV N protein associated with CyPA could interact with HAb18G/CD147 located on the membrane of ER and that the interaction could facilitate the virus particles in forming or budding into ER. AP-9, the small peptide, could enter the cells to prevent the virus particles from forming or budding into ER, by blocking HAb18G/CD147 located on ER. Thus, HAb18G/CD147 plays an important role in the process of invasion of host cells by SARS-CoV.
Both the function of HAb18G/CD147 in invasion of host cells by SARS-CoV and a clearer understanding of the mechanism responsible for the process need further confirmation. However, the present study may provide some evidence for the cytologic mechanism of invasion by SARS-CoV and may provide an important molecular basis for screening some anti-SARS drugs, such as antibody, polypeptide, immunocomplex, and small-molecule compounds.
Acknowledgments
We thank Dexing Li, Mifang Liang, Shengli Bi, Yinghua Chen, Hongwei Yang, Jian Liu, Bingfeng Wang, and Mei Huang, for their help in our experiments, and Yumei Zhou, Wenli Yan, and Fan Peng, for their careful reading of the manuscript.
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以下是引用片段：
【摘要】 基质金属蛋白酶（MMPs）是一组结构和功能相关的锌离子依赖性内肽酶，MMP-2是其中的一种，活化后可分解基底膜的主要成分—Ⅳ型胶原，从而在肿瘤的侵袭转移中具有重要意义。MMP-2已被证实在侵袭性垂体腺瘤中表达增强，可以作为肿瘤侵袭性一项有效的指标。CD147分子是一种广泛存在于人体各个组织器官的属于免疫球蛋白超家族的糖蛋白，参与机体多种生理过程。目前认为CD147可刺激肿瘤周围的纤维母细胞产生MMP，从而促进肿瘤的浸润与转移。
目前的研究认为，肿瘤的侵袭生长是一个多因素参与、多步骤完成的过程:肿瘤细胞通过细胞膜表面受体粘附到细胞基质（extracellular matrix，ECM）层粘连蛋白上;蛋白酶水解前体的释放与活化;蛋白水解酶对ECM（主要是Ⅳ型胶原蛋白）的水解;以及肿瘤细胞通过基底膜的迁移。其中，ECM的降解是肿瘤侵袭的标志。而在此级蛋白水解过程中涉及两类酶系统:纤溶酶原系统和基质金属蛋白酶（Maˉtrix Metalloproteinase.MMP）系统。MMP是锌离子依赖性内肽酶，具有降解各种基底膜的能力。MMP-2已被证实在侵袭性垂体腺瘤中表达增强，可以作为肿瘤侵袭性一项有效的指标。目前认为CD147能刺激肿瘤周围的纤维母细胞产生基质金属蛋白酶，从而促进肿瘤的侵袭与转移。CD147也已被证实在多种体内肿瘤细胞中表达增强。人们正从不同水平，不同途径对垂体腺瘤浸润与转移的发生机制进行研究，无疑MMP-2、CD147是目前的一个研究热点。
1 MMP及MMP-2的结构与功能
基质金属蛋白酶（MMPs）是一组结构和功能相关的锌离子依赖性内肽酶，至少由20种能降解细胞外基质的酶类组成。依据其基本结构和底物不同，可分为4类:胶原酶、明胶酶、间质溶素和膜型MMP（membrane type MMP，MT-MMP）。MMP由信号肽、前肽和催化域等组成:N末端的前肽区大约有80个氨基酸组成。在所有的MMP中相同，前肽区有一半胱氨酸残基。催化域高度保守，结合有锌原子，明胶酶的催化域内有纤连蛋白二型样序列的插入。C端的血色素结合蛋白样结构域与血色素结合蛋白具有很强的同源性。C末端与N末端以绞链区相连。在正常组织中，MMPs可由内皮细胞、结缔组织细胞、巨噬细胞、淋巴细胞等产生，在肿瘤组织中，多有间质细胞产生，部分可由肿瘤细胞产生。大多数MMP的前体以酶原的形式分泌至细胞外，然后被活化。MMP-2是MMPs家族中的一员，国外文献报道 ［1，2］ MMP-2在正常的胚胎发育，器官发生，血管形成及子宫复旧等多种生理过程中参与细胞外基质的调节。MMP-2以无活性的前体形式产生的，激活后可以分解基底膜的主要成分—Ⅳ型胶原蛋白，这说明MMP-2在肿瘤的侵袭转移中具有相当重要的意义。
2 CD147的结构、功能、调节及其对MMP的调节
CD147 ［3］ 是一类属于免疫球蛋白超家族（Immunoglobulin superfamily，IGSF）的跨膜糖蛋白，分子量约为50～60KD，是一种单链跨膜糖蛋白，其胞膜外区有2个IGSF结构区域，这种IGSF结构域与免疫球蛋白可变区和Ⅱ类主要组织相容性复合物的β链非常相似。存在于不同体系中的CD147同源分子名称不同:人体内的称其为:细胞外基质金属蛋白酶诱导者（extracellular matrix metalloproteinase induer EMMˉPRIN）、基础免疫球蛋白（Basic immunoglobulin，basigin）或M6。人类的EMMPRIN的cDNA序列分析发现，cDNA编码269个氨基酸残基，包括21个氨基酸残基组成的信号肽，185个氨基酸残基组成的膜外结构域，24个氨基酸残基组成的跨膜结构域和39个氨基酸残基组成的胞浆内结构域。CD147在体内分布非常广泛，使之能参与体内多种生理过程并具有多种不同的生理功能。Chen等 ［4］ 对人胚胎、幼儿及成人皮肤中CD147表达的研究发现CD147分子与角质形成细胞分化有关的特点，并证实了CD147分子主要位于角质形成细胞的微绒毛。这充分说明，CD147的表达与细胞分化状态密切相关。Renno T等 ［5］ 对胸腺细胞的研究发现，表达于胸腺细胞表面的CD147与不成熟的胸腺细胞成正比例关系，推测CD147与胸腺细胞的结合可能会抑制其发展成为成熟的T细胞。Papousi M等 ［6］研究证明，CD147还广泛存在于中枢神经系统，其表达与中枢神经系统内皮细胞的成熟相一致，是血脑屏障形成的标志之一。Kanekura等 ［3］ 对黑色素瘤细胞研究结果证明，CD147在黑色素瘤细胞呈高表达并可能在促进其浸润转移过程中刺激其周围的纤维母细胞产生MMP起着重要的作用。CD147在人体内的广泛分布，不仅参与机体多种不同的生理功能，还与肿瘤的增殖、浸润、转移密切相关。
CD147的调节及其对MMP-2的调节，国外有一些研究报道。Menashi等 ［7］ 在对NS2T2A1乳腺癌肿瘤细胞实验中检测到双调蛋白和表皮生长因子能影响CD147的表达和活性。表皮生长因子不仅影响CD147的mRNA和蛋白表达，还影响MMP-2和MMP-9的活性。双调蛋白对CD147的诱导是通过表皮生长因子受体酪氨酸激活作用所介导，其可以被ZD1839所抑制。双调蛋白和表皮生长因子的反cDNAs能抑制CD147表达和MMP活性。实验证明双调蛋白和表皮生长因子可以调节CD147和其介导的MMP的表达。Sun等 ［8］ 报道CD147存在着亲同种受体反应，并发现CD147单克隆抗体8G6不仅可抑制其亲同种受体反应，还可以通过乳腺癌细胞系MDA-435抑制MMp-2的产生以及可通过重建的基底膜Matrigel抑制MMP-2依赖的MDA-435细胞的侵袭。提出亲同种CD147受体反应可能会在肿瘤的侵袭和MMP-2的生成中起到关键作用，并认为对其干预可能会在MMP-2、MMP-1依赖的恶性肿瘤转移中起到治疗的作用。
3 MMP-2、CD147与肿瘤
Dallberg等 ［9］ 对18例浸润性乳腺癌患者临床研究结果发现MMP-2mRNA主要在中度至重度浸润性生长的肿瘤细胞周围的基质细胞中表达，也可以在原位性损害和一些正常腺细胞中呈低水平表达，而MT1-MMP和CD147在所有的肿瘤细胞中表达，且主要位于肿瘤细胞。由于MT1-MMP可能是通过活化前MMP-2而参与肿瘤浸润与转移的，提示CD147是MMP-2活化中的重要因子。KandKura等 ［3］ 对皮肤黑色素瘤进行的研究结果说明了表达CD147的肿瘤细胞能刺激纤维母细胞产生MMP-1、MMP-2、MMP-3和MT1-MMP，从而导致胶原酶的溶解活性增加。抗CD147抗体能抑制与黑色素瘤共同培养下纤维母细胞MMP的产生，则进一步说明了CD147的作用。许多研究报道在骨巨细胞瘤、泌尿系肿瘤、肺癌、成釉细胞癌、乳腺癌、子宫内膜癌、结直肠癌、神经胶质瘤、骨髓癌、皮肤鳞状细胞癌、黑色素瘤等肿瘤细胞均有CD147表达增强的出现 ［5］ 。在部分肿瘤中还发现随着肿瘤恶性程度的增高，CD147表达也随之增多 ［3］ ，且与肿瘤的浸润和转移相关 ［9］ 。更多的体内与体外研究证明，肿瘤的侵袭性与其MMP-2的表达与活化有着密切关系。而CD147作为细胞外基质金属蛋白酶刺激物（EMMPRIN），具有刺激肿瘤细胞周围间质的成纤维母细胞产生基质金属蛋白酶，降解Ⅳ型胶原，从而促进肿瘤细胞的浸润和转移 ［10］ 。
4 MMP-2、CD147与垂体腺瘤的侵袭性
垂体腺瘤占颅内肿瘤的9.43%，仅次于胶质瘤与脑膜瘤。垂体腺瘤一般上来讲是良性肿瘤，大多数不会发生远处转移，但是仍有大约30%左右的垂体腺瘤，可侵袭邻近组织，如海绵窦、蝶窦、下丘脑以及斜坡。垂体腺瘤侵袭性生长的发生率，由于判断标准不一，其结果存在差异，过去手术中肉眼见侵袭者约5%～35%不等。而现今报道垂体腺瘤经蝶窦切除，术中见侵袭者为40%左右，取鞍底硬脑膜行组织学的切片检查侵袭率可达80%。并发现肿瘤越大侵袭发生率越多，垂体腺瘤的侵袭性生长方式为手术切除带来了一定的困难，这是其治疗后预后欠佳及复发的重要原因。肿瘤细胞的侵袭性生长浸润时主要是通过对Ⅳ型胶原酶的降解，而降解基底膜，从而得以浸润转移。大量研究实验说明在肿瘤浸润性生长过程中MMP-2是非常重要的因子，而CD147则是MMP-2活化过程中的重要因子。Liu WP等 ［11］ 用免疫组化的方法对54例垂体瘤患者组织标本中MMP-2的表达进行检测，并对其中16例患者采用逆转录-聚合酶链反应（RT-PCR）的方法检测了MMP-2m RNA的表达。结果表明，侵袭性垂体腺瘤MMP-2的表达明显高于非侵袭性垂体腺瘤，侵袭性垂体腺瘤MMP-2m RNA的表达明显高于非侵袭性垂体腺瘤。MMP-2的高表达与垂体腺瘤的侵袭性密切相关，但与肿瘤的大小及分泌功能无明显关系。有学者 ［12］ 用ELISA和Norther印迹分析垂体腺瘤中MMP-2、MMP-9、MT-MMP以及组织蛋白酶抑制物（tissue inhibitors of metalloproteinas，TIMPs）-1、TIMP-2，发现MMPs活性显著增高，且TIMPs呈低表达，但这些改变在侵袭性与非侵袭性垂体腺瘤间未见显著相关性。而 Beaulieu等 ［13］ 用Western印迹方法对垂体腺瘤的研究表明MMPs（MMP-1、MMP-2、MMP-3）在大多数中均有表达，其表达水平与肿瘤级别和其侵袭性无关，而TIMP-2、TIMP-3表达水平与肿瘤级别之间呈较好的负相关。认为检测TIMP-2和TIMP-3表达水平具有预后判断价值。在上述不同的实验MMPs的表达有着不尽相同的相关性结果，可能与其选择不同的实验方法，不同的MMPs抗体以及不同的侵袭性与非侵袭性界定标准有一定的关系。
Sameshima等 ［14］ 于2000年在对胶质瘤脑组织中纤维母细胞共同培养实验中首次报道CD147不仅刺激MMP-2前体的生成，而且能将其激活，还发现作为MMP-2前体激活剂的MT1-MMP和MT2-MMP同时也被刺激呈高表达。而应用抗-CD147单克隆抗体后MT1-MMP，MT2-MMP及MMP-2表达被抑制。同年Sameshima等 ［15］ 对正常脑组织和颅内胶质瘤研究中发现，作为MMP诱导因子的CD147在正常脑组织和胶质瘤中有着不同的表达并与星形细胞瘤进展有一定的相关性，提出CD147影响胶质瘤进展可能的机制将值得讨论。目前国内外尚未见CD147在侵袭性垂体腺瘤中表达的相关报道，但作为MMP上游因子的CD147也可能与垂体腺瘤的侵袭性有着一定的联系。进一步研究垂体腺瘤中MMP-2与CD147的作用及作用机制，有助于我们对垂体腺瘤侵袭性的认识，同时也为其治疗提供了新的方向。
5 展望
作为一种广泛分布于体内的跨膜蛋白CD147以及MMP-2与侵袭性垂体腺瘤之间，人们对其感兴趣还是在于它们在肿瘤浸润和转移中所发挥的作用。多数学者认为MMP-2在肿瘤浸润、转移过程中起到重要作用，这样如何阻断MMP的表达已成为今后治疗垂体腺瘤侵袭性的热点之一。近来，MMP抑制剂已进入Ⅲ期临床试验阶段，而作为其诱导因子的CD147，相信能阻断其表达或功能也将更有效地控制垂体腺瘤侵袭性的发展。最近有学者 ［16］报道在巨噬细胞内，Sp1和Sp3能与CD147启动子功能性Sp1位点相结合，从而调节CD147基因表达，Sp1位点的变异将大大降低其启动活性，这为在基因水平调节CD147的表达提供了一条新方向。相信，肿瘤的治疗将进入一个新的阶段。
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（收稿日期:2004-08-03）
作者单位:400016重庆医科大学第一附属医院神经外科
（编辑罗 彬）
