【摘要】： 協(xié)同刺激分子(co-stimulatory molecules)在免疫調(diào)節(jié)中起著重要的作用,B7-H3是新近發(fā)現(xiàn)的協(xié)同刺激分子家族成員。人B7-H3有兩種不同形式的剪切體:B7-H3a和B7-H3b。B7-H3a胞外段由IgV-IgC兩個免疫球蛋白結構域組成,故又稱為2IgB7-H3,而B7-H3b胞外段由IgV1-IgC1-IgV2-IgC2四個免疫球蛋白結構域組成,故又稱為4IgB7-H3。B7-H3mRNA表達水平極其廣泛,在幾乎所有淋巴組織及非淋巴組織均能檢測到該分子mRNA的轉(zhuǎn)錄,其中4IgB7-H3mRNA表達豐度遠高于2IgB7-H3mRNA,因此,既往的研究均認為4IgB7-H3是B7-H3的主要表達形式。但由于沒有特異性識別人4IgB7-H3的單克隆抗體,所以無法明確4IgB7-H3在組織中的蛋白表達譜。本研究制備了一株可以特異性識別人4IgB7-H3的鼠源性抗人B7-H3單克隆抗體,繼而研究和分析了該分子的表達譜。 目的:研制特異性識別鼠抗人4IgB7-H3單克隆抗體并對其進行了初步應用。 方法:以高表達人4IgB7-H3的293T細胞為免疫原,常規(guī)免疫小鼠、細胞融合和篩選。L929/4IgB7-H3基因轉(zhuǎn)染細胞為抗體篩選陽性細胞,L929/2IgB7-H3基因轉(zhuǎn)染細胞為抗體篩選陰性細胞,經(jīng)間接免疫熒光標記和流式細胞術對雜交瘤細胞進行反復篩選和多次亞克隆化,流式細胞術分析抗體對B7家族不同成員基因轉(zhuǎn)染細胞的識別。經(jīng)位點競爭實驗對該抗體抗原表位的識別特異性進行了鑒定。利用兩株抗體采用流式細胞術檢測4IgB7-H3在腫瘤細胞株和免疫細胞上的表達特性以及免疫組織化學方法檢測此兩株抗體在腫瘤細胞株和腫瘤組織上的識別情況。 結果:成功地制備了兩株持續(xù)穩(wěn)定分泌鼠抗人B7-H3單克隆抗體的雜交瘤細胞株4C3和9C3,其中單抗4C3既能識別2IgB7-H3又能識別4IgB7-H3分子,而單抗9C3只能識別4IgB7-H3。蛋白水平上4IgB7-H3相當局限地表達在一些腫瘤細胞株、人外周血單核細胞以及成熟的樹突狀細胞;而B7-H3(2IgB7-H3和/或4IgB7-H3)分子廣泛地表達在多種腫瘤細胞株、單核細胞以及樹突狀細胞。此外,免疫組織化學分析表明4IgB7-H3可在腦膠質(zhì)瘤組織中特異性表達,并隨著膠質(zhì)瘤病理分級的升高而遞增。 結論:獲得了兩株穩(wěn)定分泌鼠抗人B7-H3單克隆抗體的雜交瘤細胞株4C3和9C3。4IgB7-H3單克隆抗體9C3的成功獲得為進一步分析人B7-H3兩種剪切體的表達特性以及生物學功能提供了物質(zhì)基礎。
[Abstract]:Costimulator (co-stimulatory molecules) plays an important role in immunomodulation. B7-H3 is a newly discovered member of costimulator family. Human B7-H3 has two different forms of shearing bodies: B7-H3a and B7-H3b.B7-H3a, which are composed of two immunoglobulin domains (IgV-IgC), so they are also called 2IgB7-H3. The extracellular segments of B7-H3b are composed of four immunoglobulin domains (IgV1-IgC1-IgV2-IgC2), so they are also called 4IgB7-H3.B7-H3mRNA. The transcription of mRNA was detected in almost all lymphoid tissues and non-lymphoid tissues, in which the abundance of 4IgB7-H3mRNA expression was much higher than that of 2IgB7-H3mRNAs. Therefore, previous studies have suggested that 4IgB7-H3 is the main expression form of B7-H3. However, there is no specific monoclonal antibody to recognize human 4IgB7-H3, so it is impossible to identify the protein expression profile of 4IgB7-H3 in tissues. In this study, a murine monoclonal antibody against human 4IgB7-H3 was prepared and its expression profile was analyzed. Objective: to develop a specific mouse anti-human 4IgB7-H3 monoclonal antibody and its preliminary application. Methods: 293T cells with high expression of human 4IgB7-H3 were used as immunogen, and mice were immunized by routine immunization. The cells transfected with. L929 / 4IgB7-H3 gene were used as antibody screening positive cells. L929 / 2 IgB7-H3 gene transfected cells were used as antibody screening negative cells. The hybridoma cells were screened repeatedly by indirect immunofluorescence and flow cytometry. The recognition of different members of B7 gene transfection cells was analyzed by flow cytometry. The recognition specificity of the antigen epitope was identified by site competition test. The expression of 4IgB7-H3 in tumor cell lines and immune cells was detected by flow cytometry and the recognition of these two antibodies in tumor cell lines and tumor tissues was detected by immunohistochemical method. Results: two hybridoma cell lines, 4C3 and 9C3, which secreted murine monoclonal antibody against human B7-H3 successfully, were successfully prepared. McAb 4C3 could recognize both 2IgB7-H3 and 4IgB7-H3 molecules, while McAb 9C3 could recognize only 4IgB7-H3. At the protein level, 4IgB7-H3 is expressed in some tumor cell lines, human peripheral blood monocytes and mature dendritic cells, while B7-H3 (2IgB7-H3 and / or 4IgB7-H3) molecules are widely expressed in many tumor cell lines, monocytes and dendritic cells. In addition, immunohistochemical analysis showed that 4IgB7-H3 could be expressed specifically in gliomas and increased with the increase of glioma pathological grade. Conclusion: two hybridoma cell lines, 4C3 and 9C3.4IgB7-H3 monoclonal antibody 9C3, which secrete murine anti-human B7-H3 monoclonal antibody stably, have been successfully obtained, which provide a material basis for further analysis of the expression characteristics and biological functions of human B7-H3.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

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