B7-H4在胰腺癌中的表达及其临床意义

发布时间：2018-01-26 03:31

本文关键词： B7-H4 单克隆抗体胰腺癌免疫组化　出处：《浙江大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景与目的：胰腺癌是一种以起病隐匿、生长迅速并早期转移为特征的消化系统恶性肿瘤,是死亡率最高的恶性肿瘤之一,目前尚无有效的治疗措施,其发病率在世界范围内呈逐年上升趋势。由于胰腺组织的解剖位置关系,早期症状不明显且缺乏有效的检测手段,大多数患者就诊时已为中晚期,预后差,5年生存率不足5%。早期发现与早期诊断是改善胰腺癌患者预后的有效措施之一。因此,寻找早期诊断、特异性强、灵敏度高的肿瘤分子标记物对于改善胰腺癌患者的预后具有重要的临床意义,同时也能为胰腺癌新型靶向治疗技术提供依据。 B7-H4(也称为B7S1或B7x)是新近发现的B7家族成员,能抑制T细胞增殖、细胞因子产生和细胞周期的进行,从而抑制T细胞介导的免疫应答。B7-H4在肿瘤免疫应答中起重要作用。B7-H4mRNA的在脾、肺、胸腺、胎盘、肝、肾等正常组织中均有表达,然而,免疫组化显示,B7-H4蛋白在正常外周组织中几乎没有阳性表达,而在一些肿瘤组织中有丰富表达,例如乳腺癌、卵巢癌、肾癌、前列腺癌和非小细胞肺癌,并与肿瘤进展密切相关。鉴于目前尚无可用于多种方法检测B7-H4表达的抗体出售,本研究以稳定表达B7-H4的3T3-B7-H4细胞为免疫原,制备了能稳定分泌特异性单克隆抗体的杂交瘤细胞株,并对其进行了生物学特性分析。同时,应用免疫组织化学染色检测B7-H4在胰腺癌组织中的表达,探讨B7-H4的表达与胰腺癌临床病理特征之间的关系,分析B7-H4在胰腺癌诊疗及预后判断等方面的潜在价值。方法： 1.以稳定表达B7-H4胞外段的3T3-B7-H4细胞为免疫原,免疫Babl/C小鼠；应用常规杂交瘤技术将免疫的小鼠脾细胞与Sp2/0小鼠骨髓瘤细胞融合,使用间接ELISA方法筛选分泌抗B7-H4单克隆抗体的杂交瘤细胞株。并对所获得的单克隆抗体进行亚型鉴定,使用Western blotting、免疫沉淀等方法对单克隆抗体特异性进行鉴定。 2.利用制备的小鼠抗B7-H4单克隆抗体,通过免疫组化(IHC)等方法分析B7-H4在胰腺癌组织中的表达特征,并评价其与临床病理特征的关系。结果： 1.获得1株亚型为IgM、轻链为κ型的针对B7-H4的鼠源单克隆抗体：特异性抗B7-H4的单抗3E8能用于免疫沉淀(IP)及IHC等方法检测组织及细胞中的B7-H4表达。 2.免疫组化显示：胰腺癌组织中B7-H4在胞浆和(或)胞膜弥漫表达,表达量显著高于正常胰腺组织(P0.05)。B7-H4在临床肿瘤分级较高患者的胰腺癌组织及有淋巴结转移患者的胰腺癌组织中表达显著增强。结论：成功获得了特异性抗B7-H4的单克隆抗体,B7-H4在胰腺癌组织中表达上调,并与患者肿瘤分级和淋巴结转移密切相关。
[Abstract]:Background and purpose: Pancreatic cancer is a kind of digestive system malignant tumor characterized by occult onset, rapid growth and early metastasis. Pancreatic cancer is one of the highest mortality malignant tumors. There is no effective treatment for pancreatic cancer. Due to the anatomical location of pancreatic tissue, the early symptoms are not obvious and lack of effective means of detection, most of the patients were in the middle and late stage, and the prognosis was poor. The 5-year survival rate is less than 5%. Early detection and early diagnosis is one of the effective measures to improve the prognosis of patients with pancreatic cancer. The highly sensitive tumor molecular markers have important clinical significance for improving the prognosis of pancreatic cancer patients, and can also provide the basis for the new targeted therapy of pancreatic cancer. B7-H4 (also known as B7S1 or B7x) is a newly discovered member of the B7 family, which can inhibit T cell proliferation, cytokine production and cell cycle progression. Thus, inhibiting T cell mediated immune response. B7-H4 plays an important role in tumor immune response. B7-H4 mRNA is expressed in spleen, lung, thymus, placenta, liver, kidney and other normal tissues. However, immunohistochemical staining showed that there was almost no positive expression of B7-H4 protein in normal peripheral tissues, but abundant expression in some tumor tissues, such as breast cancer, ovarian cancer and renal carcinoma. Prostate cancer and non-small cell lung cancer (NSCLC) are closely related to tumor progression. In this study, a hybridoma cell line was prepared by using 3T3-B7-H4 cells stably expressing B7-H4 as immunogen. At the same time, the expression of B7-H4 in pancreatic carcinoma was detected by immunohistochemical staining. To investigate the relationship between the expression of B7-H4 and the clinicopathological features of pancreatic cancer, and to analyze the potential value of B7-H4 in the diagnosis, treatment and prognosis of pancreatic cancer. Methods: 1. Babl/C mice were immunized with 3T3-B7-H4 cells which expressed the extracellular domain of B7-H4 stably. The immunized mouse spleen cells were fused with Sp2/0 mouse myeloma cells by conventional hybridoma technique. The hybridoma cell lines secreting monoclonal antibodies against B7-H4 were screened by indirect ELISA method and the subtypes of the obtained monoclonal antibodies were identified. The specificity of monoclonal antibody was identified by Western blotting and immunoprecipitation. 2. The expression of B7-H4 in pancreatic cancer tissues was analyzed by immunohistochemical method with monoclonal antibody against B7-H4. And to evaluate its relationship with clinicopathological features. Results: 1. A subtype of IgM was obtained. Murine monoclonal antibody against B7-H4 with 魏 type light chain: specific monoclonal antibody 3E8 against B7-H4 can be used for immunoprecipitation (IP). And IHC were used to detect the expression of B7-H 4 in tissues and cells. 2.Immunohistochemistry showed that B7-H4 was diffusely expressed in cytoplasm and / or membrane of pancreatic carcinoma. The expression of P0.05N. B7-H4 in normal pancreatic tissue was significantly higher than that in normal pancreatic tissue. The expression level of B7-H4 was significantly higher in pancreatic cancer tissues with higher clinical tumor grade and in pancreatic cancer tissues with lymph node metastasis. Conclusion: A specific monoclonal antibody against B7-H4 was successfully obtained. The expression of B7-H4 was up-regulated in pancreatic cancer tissues and was closely related to tumor grade and lymph node metastasis.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.9

【共引文献】