本文關鍵詞： B7-H4 單克隆抗體 胰腺癌 免疫組化　出處：《浙江大學》2015年碩士論文　論文類型：學位論文

【摘要】：背景與目的： 胰腺癌是一種以起病隱匿、生長迅速并早期轉移為特征的消化系統惡性腫瘤,是死亡率最高的惡性腫瘤之一,目前尚無有效的治療措施,其發(fā)病率在世界范圍內呈逐年上升趨勢。由于胰腺組織的解剖位置關系,早期癥狀不明顯且缺乏有效的檢測手段,大多數患者就診時已為中晚期,預后差,5年生存率不足5%。早期發(fā)現與早期診斷是改善胰腺癌患者預后的有效措施之一。因此,尋找早期診斷、特異性強、靈敏度高的腫瘤分子標記物對于改善胰腺癌患者的預后具有重要的臨床意義,同時也能為胰腺癌新型靶向治療技術提供依據。 B7-H4(也稱為B7S1或B7x)是新近發(fā)現的B7家族成員,能抑制T細胞增殖、細胞因子產生和細胞周期的進行,從而抑制T細胞介導的免疫應答。B7-H4在腫瘤免疫應答中起重要作用。B7-H4mRNA的在脾、肺、胸腺、胎盤、肝、腎等正常組織中均有表達,然而,免疫組化顯示,B7-H4蛋白在正常外周組織中幾乎沒有陽性表達,而在一些腫瘤組織中有豐富表達,例如乳腺癌、卵巢癌、腎癌、前列腺癌和非小細胞肺癌,并與腫瘤進展密切相關。鑒于目前尚無可用于多種方法檢測B7-H4表達的抗體出售,本研究以穩(wěn)定表達B7-H4的3T3-B7-H4細胞為免疫原,制備了能穩(wěn)定分泌特異性單克隆抗體的雜交瘤細胞株,并對其進行了生物學特性分析。同時,應用免疫組織化學染色檢測B7-H4在胰腺癌組織中的表達,探討B(tài)7-H4的表達與胰腺癌臨床病理特征之間的關系,分析B7-H4在胰腺癌診療及預后判斷等方面的潛在價值。 方法： 1.以穩(wěn)定表達B7-H4胞外段的3T3-B7-H4細胞為免疫原,免疫Babl/C小鼠；應用常規(guī)雜交瘤技術將免疫的小鼠脾細胞與Sp2/0小鼠骨髓瘤細胞融合,使用間接ELISA方法篩選分泌抗B7-H4單克隆抗體的雜交瘤細胞株。并對所獲得的單克隆抗體進行亞型鑒定,使用Western blotting、免疫沉淀等方法對單克隆抗體特異性進行鑒定。 2.利用制備的小鼠抗B7-H4單克隆抗體,通過免疫組化(IHC)等方法分析B7-H4在胰腺癌組織中的表達特征,并評價其與臨床病理特征的關系。 結果： 1.獲得1株亞型為IgM、輕鏈為κ型的針對B7-H4的鼠源單克隆抗體：特異性抗B7-H4的單抗3E8能用于免疫沉淀(IP)及IHC等方法檢測組織及細胞中的B7-H4表達。 2.免疫組化顯示：胰腺癌組織中B7-H4在胞漿和(或)胞膜彌漫表達,表達量顯著高于正常胰腺組織(P0.05)。B7-H4在臨床腫瘤分級較高患者的胰腺癌組織及有淋巴結轉移患者的胰腺癌組織中表達顯著增強。 結論： 成功獲得了特異性抗B7-H4的單克隆抗體,B7-H4在胰腺癌組織中表達上調,并與患者腫瘤分級和淋巴結轉移密切相關。
[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.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R735.9

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