结直肠癌相关抗原MC5-Ag的临床意义及抗原初步鉴定
摘要
【背景】
结直肠癌是高发病率和死亡率的恶性肿瘤之一，在中国人群消化系肿瘤发病率中位列第三。肿瘤标志物检测是临床上结直肠癌早期筛查和诊断的重要方式，但是，目前临床上常用的传统肿瘤标志物的敏感性和特异性均不高，不利于该肿瘤的筛查和早诊。在二十世纪八十年代，第四军医大学西京医院全军消化病研究所樊代明院士等利用淋巴细胞杂交瘤技术成功制备了一系列结直肠癌相关的单克隆抗体， MC5单抗是其中一株，其识别的抗原命名为MC5-Ag。前期研究显示MC5-Ag的结直肠癌组织特异性很高，有希望成为敏感性高、特异性强的新型结直肠癌标志物。但是MC5-Ag的蛋白质序列尚未鉴定，制约其深入研究和应用。本课题在前期研究基础上，对MC5-Ag的表达、分布和临床意义进行了系统全面的分析，并利用蛋白组学技术进行抗原鉴定。
【目的】
1．检测MC5-Ag在结直肠癌中的表达、分布及其与结肠癌患者各项临床病理参数之间的相关性。比较MC5-Ag和CEA在结肠癌组织中的表达情况。
2．研究MC5-Ag对结肠癌患者预后生存的影响。
3．系统全面地观察MC5-Ag在全身多器官肿瘤组织和正常组织中的表达。
4．利用蛋白组学研究技术分离和鉴定MC5-Ag。
【方法】
（1）利用免疫组织化学技术，在组织芯片和临床收集的组织标本中检测MC5-Ag在正常结肠组织、结肠腺瘤、腺瘤伴低级别上皮内瘤变、结肠癌和转移淋巴结等组织中的表达和分布；并在全身多器官肿瘤组织和正常组织中检测MC5-Ag的表达。同时，在大样本中比较了MC5-Ag和CEA在结肠癌组织中的表达情况。
（2）统计分析MC5-Ag在结肠癌中的表达水平与结肠癌患者的年龄、性别、肿瘤分化程度以及TNM分期等临床病理参数之间的相关性。根据患者术后随访资料，Kaplan–Meier法绘制生存曲线，Log Rank检验MC5-Ag表达水平与患者预后生存的相关性。
（3）利用细胞免疫荧光染色/激光共聚焦技术、免疫电镜、Western blot检测MC5-Ag在多种结肠癌细胞系和正常结肠上皮细胞系中的表达和定位。
（4）利用免疫沉淀（Immunoprecipitation, IP）、双向凝胶电泳（two-dimensional gelelectrophoresis,2-DE），辅以Western blot和硝酸银染色验证，富集和分离MC5-Ag，切胶、胶内酶解及基质辅助激光解吸电离飞行时间质谱（matrix-assisted laserdesorption/ionization time of flight mass spectrometry, MALDI-TOF MS）分析，对MC5-Ag进行初步鉴定。
【结果】
（1）MC5-Ag在40例结肠癌组织中的阳性表达率达到87.5%（35/40），显著高于其在癌旁正常组织中的表达（15/40，37.5%），在转移淋巴结中的表达显著高于结肠癌原发灶。随着结肠癌分化程度的降低，MC5-Ag的表达强度逐渐增强。MC5-Ag在正常结肠粘膜和单纯腺瘤中未见表达，在腺瘤伴低级别上皮内瘤变中表达增加，在结肠癌中表达水平显著提高。在26种不同器官的恶性肿瘤中，MC5-Ag除了在结肠癌特异性表达外，在消化系肿瘤胃癌、食管癌和胰腺癌中也有阳性表达。还可见于少部分恶性肿瘤如肺癌、甲状腺癌、乳腺癌、恶性黑素瘤。而在大多数脏器的恶性肿瘤中，该抗原均未见表达；在20种常见正常组织中，MC5-Ag仅表达于肺、脾和皮肤组织，而在睾丸、子宫、骨髓、淋巴结、卵巢、脑、肝脏、前列腺、乳腺、肾脏、食管、小肠、胸腺、胰腺、子宫内膜和胃等大多数正常组织中均不表达。MC5-Ag在248例结肠癌组织中的阳性率显著高于CEA，并且组织学诊断敏感性优于CEA。
（2）MC5-Ag在结肠癌中的表达与结肠癌患者的肿瘤分化程度、TNM分期以及淋巴结转移密切相关，结肠癌分化程度越差，临床分期越高以及发生淋巴结转移的结肠癌患者，MC5-Ag的表达越强，其表达与患者年龄和性别无关。MC5-Ag的表达强度与结肠癌患者的预后密切相关，其表达强度越强，患者预后越差，术后生存期越短；并且MC5-Ag的表达水平可作为影响结肠癌患者预后的独立风险因素。
（3）MC5-Ag在结肠癌细胞SW480、CRYPT以及LOVO的胞浆中广泛表达，而在正常结肠上皮细胞HIEC及结肠癌细胞SW620中则不表达。在SW480细胞和CRYPT细胞中也可见胞膜着色。免疫电镜的结果显示，MC5-Ag在SW480细胞中广泛表达于胞浆和胞膜附近的分泌小泡中，该抗原在SW620细胞中完全不表达。
（4）裂解结肠癌细胞系SW480和SW620，免疫沉淀结合双向凝胶电泳富集MC5-Ag，在50kDa、70kDa和100kDa位置可见清晰蛋白带；双向凝胶电泳进一步分离，在50kDa和100kDa可见特异蛋白点。蛋白带/点和对照进行质谱分析后，确定MC5-Ag的候选分子为eukaryotic translation initiation factor3, subunit B(EIF3B)和sodium/potassium-transporting ATPase subunit α1(ATP1A1)。
【结论】
本研究系统分析了MC5-Ag在结肠癌以及全身多器官肿瘤组织和正常组织中的表达和分布，发现该抗原在结肠癌以及胃癌、胰腺癌、食管癌中具有良好的特异性，在结肠癌组织中的表达显著高于癌旁正常组织，在结肠癌转移淋巴结中的表达显著高于肿瘤原发灶。MC5-Ag的表达与结肠癌患者的肿瘤分化程度、TNM分期以及淋巴结转移密切相关，同时MC5-Ag表达水平升高预示着结肠癌患者预后不良。蛋白组学技术初步鉴定MC5-Ag可能为EIF3B或ATP1A1。综上，MC5-Ag有希望成为新型的结直肠癌组织学诊断和预后标志物。
【Background】
Colorectal cancer (CRC) is one of the malignancies with relatively high morbidityand mortality. It is also the3rd leading tumor type among all gastrointestinal cancers inChina. The biomarkers are critical for the early screening and diagnosis of CRC, but thepractical values of current biomarkers are limited due to their poor sensitivity andspecificity. In the1980s, Fan and his colleagues generated series of monoclonal antibodiesagainst CRC-associated antigens. Of these, MC5which recognized MC5-Ag, was showedto be highly CRC-specific. However, the nature of MC5-Ag remains unclear. In this study,we carried out a systemic study to further clarify the distribution, expression and clinicalvalue of MC5-Ag in CRC, and meanwhile we also made preliminary identification ofMC5-Ag by means of proteomics.
【Objectives】
1. To analyze the distribution and expression pattern of MC5-Ag in CRC and to clarifytheir association with the clinicopathological parameters. And to compare the expressionof MC5-Ag and CEA in CRC.
2. To analyze the association between MC5-Ag and prognosis of CRC patients.
3. To study the expression pattern of MC5-Ag in tumors of different origins and adjacentnormal tissues.
4. To identify MC5-Ag by means of proteomics.
【Methods】
1. Immunohistochemistry(IHC) was employed to detect MC5-Ag in formalin fixed,paraffin embedded specimen from tissue microarray and tumor database. These specimeninclude tissues of normal colon tissues, adenoma with or without low-grade intraepithelialneoplasia, colon cancer with adjacent lymph node and tumors derived from multipleorgans. The expression of MC5-Ag and CEA in CRC were also compared by IHC.
2. The correlation of MC5-Ag and clinicopathological parameters were analyzed. Itsassociation with prognosis of CRC was studied by Kaplan–Meier curves.
3. The expression and subcellular localization was studied by immunostaining,immuno-electron microscope and western blot.
4. Immunoprecipitation, two-dimensional gel electrophoresis, western blot and silverstaining were used for the enrichment and isolation of MC5-Ag. Matrix-assisted laserdesorption/ionization time of flight mass spectrometry was used to identify MC5-Ag.
【Results】
1. The positive rate of MC5-Ag in40CRC cases was87.5%（35/40），a significantlyhigher rate than that in adjacent normal tissue（s15/40，37.5%）. MC5-Ag was up-regulatedin metastasizing lymph nodes than in primary cancers. MC5-Ag was not found in normalcolon tissues and adenomas, and its intensity gradually went higher among adenoma withlow-grade intraepithelial neoplasia and colon cancer. MC5-Ag was found in gastric,esophageal and pancreatic cancers. It was also observed in a minority of cancers withdifferent origins including lung cancer, thyroid cancer, breast cancer and melanoma. Innormal tissues, MC5-Ag was detected in lung, spleen and skins, with no positive staining in tissues including testis, uterus, bone marrow, lymph nodes, ovary, brain, liver, prostate,breast, kidney, esophagus, intestine, thymus, pancreas, endometrium and stomach. Thepositive rate of MC5-Ag was higher than CEA in248CRC cases with superior diagnosticsensitivity.
2. The expression level of MC5-Ag was significantly higher in gastric cancer in suchsituations including poor differentiation, advanced TNM staging and lymph nodemetastasis. MC5-Ag expression was not associate with gender and age of the patients. Theoverall survival of patients with stronger MC5-Ag expression was markedly reduced，andMC5-Ag was showed to be an independent risk factor for prognosis of CRC.
3. MC5-Ag was localized in the cytoplasma of multiple CRC cell lines including SW480,CRYPT and LOVO. It is not present in HIEC and SW620. A minority of MC5-Ag wasalso found on the cell membrane of SW480and CRYPT cells. Immuno-electronmicroscope showed that MC5-Ag was widely distributed in secretory vesicles which couldbe seen in cytoplasma and membrane of SW480cells.
4. Bands of50kDa,70kDa and100kDa were found with the lysis of SW480cells. Specificdots of50kDa and70kDa were confirmed by two-dimensional gel electrophoresis.MALDI-TOF-MS analysis indicated that eukaryotic translation initiation factor3, subunitB(EIF3B) and sodium/potassium-transporting ATPase subunit α1(ATP1A1) were thecandidate antigens for MC5-Ag.
【Conclusions】
MC5-Ag specifically recognized antigens in CRC, gastric cancer, pancreatic cancer andesophageal cancer. It was up-regulated in CRC than in the normal colon tissues. Theintensity of MC5-Ag was also higher in lymph nodes than in primary cancers. Theexpression of MC5-Ag was associated with differentiation, TNM staging, lymph nodemetastasis and overall survival of CRC patients. EIF3B and ATP1A1were the candidateantigens for MC5-Ag. Based on those data, MC5-Ag could serve as a novel biomarker forthe diagnosis and prognosis of CRC.
结直肠癌是高发病率和死亡率的恶性肿瘤之一，在中国人群消化系肿瘤发病率中位列第三。肿瘤标志物检测是临床上结直肠癌早期筛查和诊断的重要方式，但是，目前临床上常用的传统肿瘤标志物的敏感性和特异性均不高，不利于该肿瘤的筛查和早诊。在二十世纪八十年代，第四军医大学西京医院全军消化病研究所樊代明院士等利用淋巴细胞杂交瘤技术成功制备了一系列结直肠癌相关的单克隆抗体， MC5单抗是其中一株，其识别的抗原命名为MC5-Ag。前期研究显示MC5-Ag的结直肠癌组织特异性很高，有希望成为敏感性高、特异性强的新型结直肠癌标志物。但是MC5-Ag的蛋白质序列尚未鉴定，制约其深入研究和应用。本课题在前期研究基础上，对MC5-Ag的表达、分布和临床意义进行了系统全面的分析，并利用蛋白组学技术进行抗原鉴定。
【目的】
1．检测MC5-Ag在结直肠癌中的表达、分布及其与结肠癌患者各项临床病理参数之间的相关性。比较MC5-Ag和CEA在结肠癌组织中的表达情况。
2．研究MC5-Ag对结肠癌患者预后生存的影响。
3．系统全面地观察MC5-Ag在全身多器官肿瘤组织和正常组织中的表达。
4．利用蛋白组学研究技术分离和鉴定MC5-Ag。
【方法】
（1）利用免疫组织化学技术，在组织芯片和临床收集的组织标本中检测MC5-Ag在正常结肠组织、结肠腺瘤、腺瘤伴低级别上皮内瘤变、结肠癌和转移淋巴结等组织中的表达和分布；并在全身多器官肿瘤组织和正常组织中检测MC5-Ag的表达。同时，在大样本中比较了MC5-Ag和CEA在结肠癌组织中的表达情况。
（2）统计分析MC5-Ag在结肠癌中的表达水平与结肠癌患者的年龄、性别、肿瘤分化程度以及TNM分期等临床病理参数之间的相关性。根据患者术后随访资料，Kaplan–Meier法绘制生存曲线，Log Rank检验MC5-Ag表达水平与患者预后生存的相关性。
（3）利用细胞免疫荧光染色/激光共聚焦技术、免疫电镜、Western blot检测MC5-Ag在多种结肠癌细胞系和正常结肠上皮细胞系中的表达和定位。
（4）利用免疫沉淀（Immunoprecipitation, IP）、双向凝胶电泳（two-dimensional gelelectrophoresis,2-DE），辅以Western blot和硝酸银染色验证，富集和分离MC5-Ag，切胶、胶内酶解及基质辅助激光解吸电离飞行时间质谱（matrix-assisted laserdesorption/ionization time of flight mass spectrometry, MALDI-TOF MS）分析，对MC5-Ag进行初步鉴定。
【结果】
（1）MC5-Ag在40例结肠癌组织中的阳性表达率达到87.5%（35/40），显著高于其在癌旁正常组织中的表达（15/40，37.5%），在转移淋巴结中的表达显著高于结肠癌原发灶。随着结肠癌分化程度的降低，MC5-Ag的表达强度逐渐增强。MC5-Ag在正常结肠粘膜和单纯腺瘤中未见表达，在腺瘤伴低级别上皮内瘤变中表达增加，在结肠癌中表达水平显著提高。在26种不同器官的恶性肿瘤中，MC5-Ag除了在结肠癌特异性表达外，在消化系肿瘤胃癌、食管癌和胰腺癌中也有阳性表达。还可见于少部分恶性肿瘤如肺癌、甲状腺癌、乳腺癌、恶性黑素瘤。而在大多数脏器的恶性肿瘤中，该抗原均未见表达；在20种常见正常组织中，MC5-Ag仅表达于肺、脾和皮肤组织，而在睾丸、子宫、骨髓、淋巴结、卵巢、脑、肝脏、前列腺、乳腺、肾脏、食管、小肠、胸腺、胰腺、子宫内膜和胃等大多数正常组织中均不表达。MC5-Ag在248例结肠癌组织中的阳性率显著高于CEA，并且组织学诊断敏感性优于CEA。
（2）MC5-Ag在结肠癌中的表达与结肠癌患者的肿瘤分化程度、TNM分期以及淋巴结转移密切相关，结肠癌分化程度越差，临床分期越高以及发生淋巴结转移的结肠癌患者，MC5-Ag的表达越强，其表达与患者年龄和性别无关。MC5-Ag的表达强度与结肠癌患者的预后密切相关，其表达强度越强，患者预后越差，术后生存期越短；并且MC5-Ag的表达水平可作为影响结肠癌患者预后的独立风险因素。
（3）MC5-Ag在结肠癌细胞SW480、CRYPT以及LOVO的胞浆中广泛表达，而在正常结肠上皮细胞HIEC及结肠癌细胞SW620中则不表达。在SW480细胞和CRYPT细胞中也可见胞膜着色。免疫电镜的结果显示，MC5-Ag在SW480细胞中广泛表达于胞浆和胞膜附近的分泌小泡中，该抗原在SW620细胞中完全不表达。
（4）裂解结肠癌细胞系SW480和SW620，免疫沉淀结合双向凝胶电泳富集MC5-Ag，在50kDa、70kDa和100kDa位置可见清晰蛋白带；双向凝胶电泳进一步分离，在50kDa和100kDa可见特异蛋白点。蛋白带/点和对照进行质谱分析后，确定MC5-Ag的候选分子为eukaryotic translation initiation factor3, subunit B(EIF3B)和sodium/potassium-transporting ATPase subunit α1(ATP1A1)。
【结论】
本研究系统分析了MC5-Ag在结肠癌以及全身多器官肿瘤组织和正常组织中的表达和分布，发现该抗原在结肠癌以及胃癌、胰腺癌、食管癌中具有良好的特异性，在结肠癌组织中的表达显著高于癌旁正常组织，在结肠癌转移淋巴结中的表达显著高于肿瘤原发灶。MC5-Ag的表达与结肠癌患者的肿瘤分化程度、TNM分期以及淋巴结转移密切相关，同时MC5-Ag表达水平升高预示着结肠癌患者预后不良。蛋白组学技术初步鉴定MC5-Ag可能为EIF3B或ATP1A1。综上，MC5-Ag有希望成为新型的结直肠癌组织学诊断和预后标志物。
【Background】
Colorectal cancer (CRC) is one of the malignancies with relatively high morbidityand mortality. It is also the3rd leading tumor type among all gastrointestinal cancers inChina. The biomarkers are critical for the early screening and diagnosis of CRC, but thepractical values of current biomarkers are limited due to their poor sensitivity andspecificity. In the1980s, Fan and his colleagues generated series of monoclonal antibodiesagainst CRC-associated antigens. Of these, MC5which recognized MC5-Ag, was showedto be highly CRC-specific. However, the nature of MC5-Ag remains unclear. In this study,we carried out a systemic study to further clarify the distribution, expression and clinicalvalue of MC5-Ag in CRC, and meanwhile we also made preliminary identification ofMC5-Ag by means of proteomics.
【Objectives】
1. To analyze the distribution and expression pattern of MC5-Ag in CRC and to clarifytheir association with the clinicopathological parameters. And to compare the expressionof MC5-Ag and CEA in CRC.
2. To analyze the association between MC5-Ag and prognosis of CRC patients.
3. To study the expression pattern of MC5-Ag in tumors of different origins and adjacentnormal tissues.
4. To identify MC5-Ag by means of proteomics.
【Methods】
1. Immunohistochemistry(IHC) was employed to detect MC5-Ag in formalin fixed,paraffin embedded specimen from tissue microarray and tumor database. These specimeninclude tissues of normal colon tissues, adenoma with or without low-grade intraepithelialneoplasia, colon cancer with adjacent lymph node and tumors derived from multipleorgans. The expression of MC5-Ag and CEA in CRC were also compared by IHC.
2. The correlation of MC5-Ag and clinicopathological parameters were analyzed. Itsassociation with prognosis of CRC was studied by Kaplan–Meier curves.
3. The expression and subcellular localization was studied by immunostaining,immuno-electron microscope and western blot.
4. Immunoprecipitation, two-dimensional gel electrophoresis, western blot and silverstaining were used for the enrichment and isolation of MC5-Ag. Matrix-assisted laserdesorption/ionization time of flight mass spectrometry was used to identify MC5-Ag.
【Results】
1. The positive rate of MC5-Ag in40CRC cases was87.5%（35/40），a significantlyhigher rate than that in adjacent normal tissue（s15/40，37.5%）. MC5-Ag was up-regulatedin metastasizing lymph nodes than in primary cancers. MC5-Ag was not found in normalcolon tissues and adenomas, and its intensity gradually went higher among adenoma withlow-grade intraepithelial neoplasia and colon cancer. MC5-Ag was found in gastric,esophageal and pancreatic cancers. It was also observed in a minority of cancers withdifferent origins including lung cancer, thyroid cancer, breast cancer and melanoma. Innormal tissues, MC5-Ag was detected in lung, spleen and skins, with no positive staining in tissues including testis, uterus, bone marrow, lymph nodes, ovary, brain, liver, prostate,breast, kidney, esophagus, intestine, thymus, pancreas, endometrium and stomach. Thepositive rate of MC5-Ag was higher than CEA in248CRC cases with superior diagnosticsensitivity.
2. The expression level of MC5-Ag was significantly higher in gastric cancer in suchsituations including poor differentiation, advanced TNM staging and lymph nodemetastasis. MC5-Ag expression was not associate with gender and age of the patients. Theoverall survival of patients with stronger MC5-Ag expression was markedly reduced，andMC5-Ag was showed to be an independent risk factor for prognosis of CRC.
3. MC5-Ag was localized in the cytoplasma of multiple CRC cell lines including SW480,CRYPT and LOVO. It is not present in HIEC and SW620. A minority of MC5-Ag wasalso found on the cell membrane of SW480and CRYPT cells. Immuno-electronmicroscope showed that MC5-Ag was widely distributed in secretory vesicles which couldbe seen in cytoplasma and membrane of SW480cells.
4. Bands of50kDa,70kDa and100kDa were found with the lysis of SW480cells. Specificdots of50kDa and70kDa were confirmed by two-dimensional gel electrophoresis.MALDI-TOF-MS analysis indicated that eukaryotic translation initiation factor3, subunitB(EIF3B) and sodium/potassium-transporting ATPase subunit α1(ATP1A1) were thecandidate antigens for MC5-Ag.
【Conclusions】
MC5-Ag specifically recognized antigens in CRC, gastric cancer, pancreatic cancer andesophageal cancer. It was up-regulated in CRC than in the normal colon tissues. Theintensity of MC5-Ag was also higher in lymph nodes than in primary cancers. Theexpression of MC5-Ag was associated with differentiation, TNM staging, lymph nodemetastasis and overall survival of CRC patients. EIF3B and ATP1A1were the candidateantigens for MC5-Ag. Based on those data, MC5-Ag could serve as a novel biomarker forthe diagnosis and prognosis of CRC.
引文
1. Center MM, Jemal A, Smith RA, Ward E (2009) Worldwide variations in colorectalcancer. CA Cancer J Clin59:366-378.
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