本文選題：2IgB7-H3 + 4IgB7-H3��； 參考：《蘇州大學》2010年碩士論文

【摘要】： T細胞的有效活化需要兩條獨立的但是互補的信號途徑。第一信號是抗原遞呈細胞(APC)遞呈的抗原肽-主要組織相容性復合物(MHC),與特殊的T細胞抗原受體復合物TCR/CD3識別,并將信號傳遞給T細胞。第二信號是不可缺少的共刺激信號,它由T細胞和APC表面的共刺激分子相互作用所激發(fā)。共刺激分子并不僅僅單獨激發(fā)T細胞的活化,它們還協(xié)同TCR/CD3復合物提供增強信號。T細胞上的CD28和B7(B7-1和B7-2)相互作用產(chǎn)生的信號,是公認的最經(jīng)典的共刺激信號。共刺激信號有B7和CD28兩大超家族成員,它們在調(diào)節(jié)T細胞的活化和耐受中發(fā)揮著關(guān)鍵作用,有望用于臨床治療。這些通路不僅提供促進和維持T細胞應答的正性第二信號,它們同樣能夠下調(diào)T細胞應答,提供必要的負性第二信號。負性共刺激信號在限制、中斷和減弱T細胞應答中發(fā)揮效應,這對于調(diào)節(jié)T細胞耐受和自身免疫非常重要。 人B7-H3由于mRNA剪接的不同存在著2IgB7-H3和4IgB7-H3兩種異構(gòu)體。鑒于B7-H3兩種異構(gòu)體的功能差異尚不清楚,因此值得進一步深入探討。鑒此,本課題制備了2IgB7-H3和4IgB7-H3兩種異構(gòu)體的轉(zhuǎn)基因細胞株,比較分析了B7-H3兩種異構(gòu)體對T細胞的免疫調(diào)控作用。此外,本研究還研制了1株抗人B7-H3單克隆抗體,為進一步研究B7-H3分子的生物學功能奠定基礎(chǔ)。 一、人B7-H3兩種異構(gòu)體基因轉(zhuǎn)染細胞株的建立及對T細胞刺激作用差異比較 目的:建立2IgB7-H3基因轉(zhuǎn)染細胞株和4IgB7-H3基因轉(zhuǎn)染細胞株,探討B(tài)7-H3兩種異構(gòu)體在介導T細胞免疫應答中是否存在差異。 方法:RT-PCR法從誘導成熟的DC細胞中克隆人B7-H3兩種異構(gòu)體基因2IgB7-H3和4IgB7-H3的編碼區(qū),經(jīng)EcoR I和BamH I雙酶切后插入pIRES2-EGFP真核表達載體構(gòu)建pIRES2-EGFP/2IgB7-H3和pIRES2-EGFP/4IgB7-H3重組子,采用脂質(zhì)體法轉(zhuǎn)染腦膠質(zhì)瘤細胞株SHG44。經(jīng)G418抗性篩選,采用免疫熒光標記和流式細胞術(shù)分析2IgB7-H3和4IgB7-H3在SHG44細胞上的表達。繼而,利用MTT法和酶聯(lián)免疫吸附測定法(ELISA)分析兩種異構(gòu)體基因轉(zhuǎn)染細胞株對T細胞體外增殖和細胞因子分泌的影響。 結(jié)果:成功構(gòu)建了可以穩(wěn)定表達2IgB7-H3和4IgB7-H3的基因轉(zhuǎn)染細胞株。體外生物學功能分析表明,與轉(zhuǎn)染空載體的SHG44/mock細胞相比,SHG44/2IgB7-H3和SHG44/4IgB7-H3均能有效抑制T細胞增殖以及對IFN-γ的分泌,且兩者的協(xié)同抑制作用不存在顯著差異。 結(jié)論:B7-H3兩種異構(gòu)體分子均能負性調(diào)控T細胞介導的免疫應答,但兩者的生物學功能并不存在顯著的差異。 二、鼠抗人B7-H3單克隆抗體的制備及鑒定 目的:研制鼠抗人B7-H3單克隆抗體,并對其生物學特性進行鑒定。 方法:以高表達人2IgB7-H3和4IgB7-H3的293T細胞為免疫原,常規(guī)免疫小鼠、細胞融合和篩選。SHG44/2IgB7-H3和SHG44/4IgB7-H3基因轉(zhuǎn)染細胞為抗體篩選陽性細胞,SHG44/mock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(shù)對雜交瘤細胞進行反復篩選和多次亞克隆化,流式細胞術(shù)分析抗體對B7家族不同成員基因轉(zhuǎn)染細胞的識別。流式細胞術(shù)分析B7-H3在各類細胞株上的表達特性。 結(jié)果:獲得了一株持續(xù)穩(wěn)定分泌鼠抗人B7-H3單克隆抗體的雜交瘤細胞株13F8,該單克隆抗體(克隆號13F8)對B7-H3兩種異構(gòu)體均具有較好的識別特異性。 結(jié)論:以獲得的抗人B7-H3單克隆抗體13F8作為檢測手段,發(fā)現(xiàn)B7-H3的表達譜較為廣泛,該抗體的獲得為進一步研究B7-H3的生物學功能與臨床運用奠定了基礎(chǔ)。
[Abstract]:There are two independent but complementary signaling pathways for T cell activation . The first signal is antigen peptide - major Histocompatibility Complex ( MHC ) presented by antigen presenting cell ( APC ) . It is excited by co - stimulatory molecule interaction of T cells and APC surfaces . The co - stimulatory molecules play a key role in regulating the activation and tolerance of T cells . These pathways not only provide positive second signals for the promotion and maintenance of T cell responses , but also provide the necessary negative second signals . Negative co - stimulatory signals exert an effect in limiting , interrupting , and reducing T cell responses , which are important for regulating T cell tolerance and autoimmune diseases .



There are two isoforms of B7 - H3 and 4IgB7 - H3 in human B7 - H3 . In view of the unclear functional differences between the two isoforms of B7 - H3 , the immunoregulatory effects of the two isoforms of B7 - H3 on T cells are discussed .



Establishment of a human B7 - H3 gene transfected cell line and comparison of T cell stimulating effect



Objective : To establish a 2 IgB7 - H3 gene - transfected cell line and a 4IgB7 - H3 gene - transfected cell line , and investigate whether the two isoforms of B7 - H3 are different in mediating T cell immune response .



Methods : The eukaryotic expression vector pIRES2 - EGFP / 2IgB7 - H3 and pIRES2 - EGFP / 4IgB7 - H3 were constructed by RT - PCR from mature DC cells . The pIRES2 - EGFP / 2IgB7 - H3 and pIRES2 - EGFP / 4IgB7 - H3 were transfected into the eukaryotic expression vector pIRES2 - EGFP / 4IgB7 - H3 .



Results : In vitro biological function analysis showed that SHG44 / 2IgB7 - H3 and SHG44 / 4IgB7 - H3 effectively inhibited T cell proliferation and IFN - 緯 secretion compared with SHG44 / mock cells transfected with empty vector .



Conclusion : B7 - H3 can regulate T cell mediated immune response negatively , but there is no significant difference in the biological function of B7 - H3 .



Preparation and identification of mouse anti - human B7 - H3 monoclonal antibody



Objective : To develop a mouse anti - human B7 - H3 monoclonal antibody and to identify its biological characteristics .



Methods : Human 2IgB7 - H3 and 4IgB7 - H3 were used as immunogen , and the cells were fused and screened . SHG44 / 2IgB7 - H3 and SHG44 / 4IgB7 - H3 gene were used to screen the positive cells . SHG44 / 2 IgB7 - H3 and SHG44 / 4IgB7 - H3 gene were used to screen negative cells .



Results : A hybridoma cell line 13F8 stably secreting anti - human B7 - H3 monoclonal antibody against human B7 - H3 was obtained .



Conclusion : The anti - human B7 - H3 monoclonal antibody 13F8 is used as the detection means , and the expression profile of B7 - H3 is found to be more extensive , which lays a foundation for further research on the biological function and clinical application of B7 - H3 .
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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