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

【摘要】： T細(xì)胞的有效活化需要兩條獨(dú)立的但是互補(bǔ)的信號(hào)途徑。第一信號(hào)是抗原遞呈細(xì)胞(APC)遞呈的抗原肽-主要組織相容性復(fù)合物(MHC),與特殊的T細(xì)胞抗原受體復(fù)合物TCR/CD3識(shí)別,并將信號(hào)傳遞給T細(xì)胞。第二信號(hào)是不可缺少的共刺激信號(hào),它由T細(xì)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(dú)激發(fā)T細(xì)胞的活化,它們還協(xié)同TCR/CD3復(fù)合物提供增強(qiáng)信號(hào)。T細(xì)胞上的CD28和B7(B7-1和B7-2)相互作用產(chǎn)生的信號(hào),是公認(rèn)的最經(jīng)典的共刺激信號(hào)。共刺激信號(hào)有B7和CD28兩大超家族成員,它們?cè)谡{(diào)節(jié)T細(xì)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(jìn)和維持T細(xì)胞應(yīng)答的正性第二信號(hào),它們同樣能夠下調(diào)T細(xì)胞應(yīng)答,提供必要的負(fù)性第二信號(hào)。負(fù)性共刺激信號(hào)在限制、中斷和減弱T細(xì)胞應(yīng)答中發(fā)揮效應(yīng),這對(duì)于調(diào)節(jié)T細(xì)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(jìn)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(xì)胞株,比較分析了B7-H3兩種異構(gòu)體對(duì)T細(xì)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(jìn)一步研究B7-H3分子的生物學(xué)功能奠定基礎(chǔ)。 一、人B7-H3兩種異構(gòu)體基因轉(zhuǎn)染細(xì)胞株的建立及對(duì)T細(xì)胞刺激作用差異比較 目的:建立2IgB7-H3基因轉(zhuǎn)染細(xì)胞株和4IgB7-H3基因轉(zhuǎn)染細(xì)胞株,探討B(tài)7-H3兩種異構(gòu)體在介導(dǎo)T細(xì)胞免疫應(yīng)答中是否存在差異。 方法:RT-PCR法從誘導(dǎo)成熟的DC細(xì)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(dá)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(xì)胞株SHG44。經(jīng)G418抗性篩選,采用免疫熒光標(biāo)記和流式細(xì)胞術(shù)分析2IgB7-H3和4IgB7-H3在SHG44細(xì)胞上的表達(dá)。繼而,利用MTT法和酶聯(lián)免疫吸附測(cè)定法(ELISA)分析兩種異構(gòu)體基因轉(zhuǎn)染細(xì)胞株對(duì)T細(xì)胞體外增殖和細(xì)胞因子分泌的影響。 結(jié)果:成功構(gòu)建了可以穩(wěn)定表達(dá)2IgB7-H3和4IgB7-H3的基因轉(zhuǎn)染細(xì)胞株。體外生物學(xué)功能分析表明,與轉(zhuǎn)染空載體的SHG44/mock細(xì)胞相比,SHG44/2IgB7-H3和SHG44/4IgB7-H3均能有效抑制T細(xì)胞增殖以及對(duì)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(fù)性調(diào)控T細(xì)胞介導(dǎo)的免疫應(yīng)答,但兩者的生物學(xué)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(duì)其生物學(xué)特性進(jìn)行鑒定。 方法:以高表達(dá)人2IgB7-H3和4IgB7-H3的293T細(xì)胞為免疫原,常規(guī)免疫小鼠、細(xì)胞融合和篩選。SHG44/2IgB7-H3和SHG44/4IgB7-H3基因轉(zhuǎn)染細(xì)胞為抗體篩選陽(yáng)性細(xì)胞,SHG44/mock基因轉(zhuǎn)染細(xì)胞為抗體篩選陰性細(xì)胞,經(jīng)間接免疫熒光標(biāo)記和流式細(xì)胞術(shù)對(duì)雜交瘤細(xì)胞進(jìn)行反復(fù)篩選和多次亞克隆化,流式細(xì)胞術(shù)分析抗體對(duì)B7家族不同成員基因轉(zhuǎn)染細(xì)胞的識(shí)別。流式細(xì)胞術(shù)分析B7-H3在各類細(xì)胞株上的表達(dá)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(xì)胞株13F8,該單克隆抗體(克隆號(hào)13F8)對(duì)B7-H3兩種異構(gòu)體均具有較好的識(shí)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(cè)手段,發(fā)現(xiàn)B7-H3的表達(dá)譜較為廣泛,該抗體的獲得為進(jìn)一步研究B7-H3的生物學(xué)功能與臨床運(yùn)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(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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