新胃癌分子标志物GP128特性鉴定及在胃癌血管形成中的作用与分子机制

Gastric cancer (GC) is one of the most common human malignancies worldwide and is the leading cause of morbidity and mortality, especially in China. Although the clinical outcome of GC has gradually improved, the prognosis of patients at the advanced stage is still disappointing. The 5-year survival rate for GC patients is considerably lower than that of other common cancers. Moreover, there are no desirable tumor biomarkers with high specificity and sensitivity for early diagnosis of GC. While some works have been done on the mechanism, the pathogenesis of GC is not completely understood yet. Thus, the identification of novel biomarkers for diagnosis and novel targets with high specificity for treatment is spotlight and a major goal in basic research and clinical practice of GC. And these biomarkers may bring a better understanding of the pathogenesis and mechanism of GC..In the preliminary experiments, using the assembly of the hybridoma technology in combination with cell-surface-based FACS screening, we prepared 5 monoclonal antibodies (mAbs) against membrane proteins of GC with intact cancer cells instead of denatured proteins as the immunogen. Among these mAbs, mAb 38-2.1 was selected for further investigation and this novel mAb showed high specificity to the membrane antigen on malignant cells rather than normal cells by both FACS and Western Blotting. Under both reducing and non-reducing conditions, mAb 38-2.1 showed strict immunoreactivity to a protein from GC cells with an apparent molecular mass of 128 kDa. So the corresponding antigen to mAb 38-2.1 was named glycoprotein 128 (GP128). In the immunohistochemisty assay, GP128 showed strong expression in small and medium blood vessels, and considerable expression in cancer cells of GC tissues, rather than in the adjacent normal tissues. To identify further information of GP128, the antibody-based counter-screening was fabricated. Protein microarray comprising a large set of human proteins (>16, 000) showed mAb 38-2.1 recognized human SPRR3 (small proline rich protein 3). ELISA was further used to verify that GP128 shared the same epitope as SPRR3 expressing on the cell surface of a wide range of tumor and tumor-associated cells.. To date, our results demonstrate that GP128 may play a role in the development of malignant tumors. Now we are focusing on the gene and protein characteristic of GP128, the clinical significance of the GP128 expression in GC, the function and molecular mechanism of angiogenesis of GP128 in GC. The investigation will classify the possibility of this novel biomarker as the potential target for GC early diagnosis and antibody-based treatment. And the function of GP128 in GC angiogenesis may bring a better understanding of the tumorigenesis of this disease.

胃癌是我国最常见的恶性肿瘤之一，目前其早期诊断手段的特异性、敏感性并不令人满意。多数胃癌患者初诊时已发展至晚期，传统的治疗措施疗效差强人意，患者的五年生存率无明显改善。因此，寻找胃癌新的特异性分子标志物，建立胃癌敏感、特异的早期诊断方法以及探索胃癌发生的可能机制是胃癌基础和临床研究领域亟待解决的问题。本课题前期研究采用杂交瘤技术结合流式细胞术高通量筛选的创新方法，获得了胃癌特异性单克隆抗体38-2.1；经过反向鉴定技术，发现与该抗体结合的抗原GP128在胃癌中特异性表达；借助蛋白质组学策略，表明GP128与小脯氨酸富含蛋白3有共同的表位。本课题拟进一步鉴定GP128的基因与蛋白质特性，阐明其在胃癌表达的临床意义，探讨其在胃癌血管形成中的作用及分子机制。新胃癌分子标志物GP128的研究，可为胃癌的早期筛查诊断、胃癌新生血管形成机制的探索和胃癌治疗性抗体药物的研发奠定基础。

胃癌是我国最常见的恶性肿瘤之一，目前其早期诊断手段的特异性、敏感性并不令人满意。多数胃癌患者初诊时已发展至晚期，传统的治疗措施疗效差强人意。因此，寻找胃癌新的特异性分子标志物，建立胃癌敏感、特异的早期诊断方法以及探索胃癌发生的可能机制是胃癌基础和临床研究领域亟待解决的问题，对于胃癌的基础研究以及临床诊断治疗都具有深远意义。. 本课题“新胃癌分子标志物GP128 特性鉴定及在胃癌血管形成中的作用与分子机制”，采用完整的胃癌细胞免疫动物制备杂交瘤技术，结合流式细胞术高通量筛选的创新方法，在杂交瘤细胞分泌产生的多种抗体中鉴定可能用于胃癌诊断或治疗的单克隆抗体，目前已经制备获得5 株抗人胃癌特异性单克隆抗体。选取其中信号最强的一株单克隆抗体38-2.1进行进一步研究。经过“抗体-抗原”反向鉴定技术，发现与该抗体结合的抗原可能为一个新的分子标志物。Western Blotting 结果表明该标志物在胃癌细胞和胃癌组织中均特异性表达，分子量大小约为128 kDa，故将其命名为GP128。借助蛋白质组学策略，蛋白芯片结果表明GP128 与小脯氨酸富含蛋白3（small proline rich repeat protein 3, SPRR3）有共同的抗原表位。通过收集了830例胃癌及对照胃组织标本，制作成组织芯片，通过免疫组织化学染色显示，GP128 主要表达于胃癌肿瘤细胞和间质小血管，可能与胃癌的血管形成有一定关系。GP128蛋白在胃癌中阳性率65.83%，明显高于癌旁组织（38.20%），并且GP128的表达和分化，TNM分期，肿瘤大小，淋巴结转移，远处转移，幽门螺旋杆菌感染等相关，高表达GP128的患者呈现较差的生存预后情况。初步的机制研究表明，GP128可能与VEGR-VEGFR2信号通路有关，使用VEGR2抗体封闭或者下调VEGFR2表达从而阻断VEGR-VEGFR2信号通路后，GP128的表达量明显下降；上调VEGFR2表达后，GP128表达量明显上升。. 在本课题的资助下，申请者共发表SCI论文2篇，国际会议报告1次，中文综述1篇，培养博士研究生1人，七年制研究生1人。