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  本文選題：LIGHT + HVEM　； 參考：《蘇州大學》2010年碩士論文

【摘要】： 正性與負性共信號分子共同參與,實現(xiàn)了對T細胞有效和適度免疫應答的精確調(diào)控作用。共信號分子根據(jù)結(jié)構(gòu)主要分為免疫球蛋白(CD28/ B7)超家族、腫瘤壞死因子(TNF/TNFR)超家族以及細胞因子超家族。人LIGHT(TNFSF14)屬于TNF家族成員,其與TNFR超家族受體分子HVEM作用可介導促進T細胞活化與增殖的正性共刺激信號。近期研究表明,HVEM也可作為配體與免疫球蛋白超家族成員BTLA或CD160作用介導對T細胞的抑制作用,并且相關研究揭示了HVEM/LIGHT和BTLA(CD160)/HVEM間也存在信號的雙向性。因此,LIGHT/HVEM/BTLA(CD160)構(gòu)成了共信號分子中一個微觀的免疫調(diào)節(jié)網(wǎng)絡,探討和分析LIGHT/HVEM信號的特性及其對T細胞的共刺激作用與機制具有重要的理論意義。 本研究旨在建立L929/LIGHT基因轉(zhuǎn)染細胞株,以此為手段探討人LIGHT分子體外對T細胞的共刺激效應,通過制備鼠抗人LIGHT單克隆抗體和人HVEMIg融合蛋白,分析其對LIGHT/HVEM效應的阻斷,從而為闡明LIGHT/HVEM信號對T細胞的協(xié)同刺激作用與機制,進一步深入探討LIGHT/HVEM/BTLA(CD160)對T細胞復雜的調(diào)控機理提供有價值的實驗數(shù)據(jù)。 一.人LIGHT基因轉(zhuǎn)染細胞株的構(gòu)建及其對T細胞的共刺激作用 RT-PCR從經(jīng)PHA活化的T細胞中克隆人LIGHT編碼區(qū)全長基因,雙酶切后插入真核表達載體pIRES2-EGFP構(gòu)建重組子pIRES2-EGFP-LIGHT,測序正確。脂質(zhì)體法以重組表達載體轉(zhuǎn)染小鼠L929細胞。G418加壓篩選并經(jīng)過數(shù)次亞克隆,建立了L929/LIGHT基因轉(zhuǎn)染細胞株,流式細胞術檢測結(jié)果顯示,L929/LIGHT細胞膜上能穩(wěn)定高表達人LIGHT分子;體外細胞共培養(yǎng)試驗表明,與未轉(zhuǎn)染LIGHT基因的L929/mock細胞相比,L929/LIGHT可顯著地促進抗人CD3單抗(mAb)刺激的T細胞增殖;同時L929/LIGHT亦能明顯促進T細胞對IL-2、IFN-γ和IL-10的分泌。 二.鼠抗人LIGHT單抗和人HVEMIg融合蛋白的研制及其生物學特性 1.以L929/LIGHT細胞為免疫原免疫BALB/c小鼠,采用B淋巴細胞雜交瘤技術,將免疫小鼠的脾臟細胞和小鼠骨髓瘤細胞SP2/0進行融合,HAT選擇性培養(yǎng)基培養(yǎng)雜交瘤細胞。以L929/LIGHT為陽性篩選細胞,L929/mock作陰性對照,FACS分析篩選陽性克隆,經(jīng)過4次亞克隆化培養(yǎng),最終得到一株持續(xù)分泌抗人LIGHT單克隆抗體的雜交瘤細胞株(7A8),經(jīng)快速定性試紙條分析法結(jié)果顯示,單抗7A8重鏈屬于IgG2b,輕鏈為κ。體外長期培養(yǎng)和液氮凍存后,復蘇的雜交瘤細胞生長狀態(tài)良好,穩(wěn)定分泌抗體;2.將人IgG(Fc)基因編碼區(qū)基因與人HVEM胞外段基因融合,按照構(gòu)建L929/LIGHT基因轉(zhuǎn)染細胞的技術和方法將HVEMIg融合基因?qū)胝婧思毎?篩選并亞克隆獲得CHO/HVEMIg轉(zhuǎn)染細胞。利用流式細胞術、SDS-PAGE和Western-blot進行鑒定,結(jié)果顯示,HVEMIg融合蛋白分泌表達于CHO/HVEMIg基因轉(zhuǎn)染細胞的培養(yǎng)上清中。大規(guī)模細胞培養(yǎng)后再濃縮上清,通過protein G親和層析技術獲得純品HVEMIg融合蛋白,流式檢測結(jié)果,純化的蛋白與其配體LIGHT具有良好的結(jié)合能力。T細胞體外增殖實驗表明,抗LIGHT單抗和HVEMIg融合蛋白的加入可部分阻斷由基因轉(zhuǎn)染細胞協(xié)同CD3mAb對T細胞增殖的促進作用,并下調(diào)細胞因子的分泌。由此提示LIGHT/HVEM通過介導正性共刺激作用參與活化T細胞的調(diào)節(jié)。 綜上所述,本實驗成功構(gòu)建了L929/LIGHT基因轉(zhuǎn)染細胞株,該轉(zhuǎn)染細胞表達的膜型人LIGHT對T細胞體外增殖和細胞因子分泌具有顯著的促進作用;成功地研制一株鼠抗人LIGHT單抗7A8和HVEMIg融合蛋白。T細胞體外增殖試驗顯示,單抗7A8和HVEMIg對L929/LIGHT介導的刺激T細胞增殖和細胞因子分泌分別具有部分阻斷作用,單抗7A8為鼠抗人LIGHT的阻斷型抗體。上述結(jié)果為深入探討LIGHT分子的共刺激作用及LIGHT/HVEM/BTLA(CD160)調(diào)節(jié)網(wǎng)絡在T細胞免疫應答中的調(diào)控作用及干預策略提供了有價值的物質(zhì)基礎和實驗參數(shù)。
[Abstract]:Both positive and negative co signalling molecules participate in the precise regulation of the effective and moderate immune responses of T cells. Common signal molecules are divided into immunoglobulin (CD28/ B7) superfamily, tumor necrosis factor (TNF/TNFR) superfamily and cytokine superfamily. Human LIGHT (TNFSF14) belongs to the TNF family members, and TNFR (TNFR) is a member of the TNF family. The role of the superfamily receptor molecule HVEM can mediate the positive co stimulation signal that promotes the activation and proliferation of T cells. Recent studies have shown that HVEM can also be used as a ligand to inhibit the inhibition of T cells by the role of BTLA or CD160 in a member of the immunoglobulin superfamily, and the related studies have revealed the presence of signals between HVEM/ LIGHT and BTLA (CD160) /HVEM. Therefore, LIGHT/HVEM/BTLA (CD160) constitutes a microscopic immunomodulatory network in common signal molecules. It is of great theoretical significance to explore and analyze the characteristics of LIGHT/HVEM signals and their co stimulation and mechanism for T cells.
The purpose of this study was to establish a L929/LIGHT gene transfected cell line to explore the co stimulation effect of human LIGHT molecules on T cells in vitro. By preparing mouse anti human LIGHT monoclonal antibody and human HVEMIg fusion protein, the blocking of LIGHT/HVEM effect was analyzed, thus the synergistic stimulation and mechanism of LIGHT /HVEM signal to T cells were elucidated. Further study of LIGHT/HVEM/BTLA (CD160) provides valuable experimental data for the complex regulation mechanism of T cells.
Construction of human LIGHT gene transfected cell line and its costimulatory effect on T cells
The full length gene of human LIGHT coding region was cloned from the T cells activated by PHA, and the recombinant plasmid was inserted into the eukaryotic expression vector pIRES2-EGFP after double enzyme digestion to construct the recombinant pIRES2-EGFP-LIGHT, and the sequencing was correct. The liposome method was transfected with the recombinant expression vector to transfect the mouse L929 cell.G418 by compression screening and after several subclones, the L929/LIGHT gene transfected cells were established. The results of flow cytometry showed that the L929/LIGHT cell membrane could stabilize the high apparent LIGHT molecule on the membrane, and in vitro cell co culture test showed that L929/LIGHT could significantly promote the proliferation of T cells stimulated by the human CD3 monoclonal antibody (mAb) compared with the L929/mock cells without LIGHT gene, and L929/LIGHT also significantly promoted T cells to IL-2, IFN-. The secretion of gamma and IL-10.
Two. Preparation and biological characteristics of mouse anti human LIGHT monoclonal antibody and human HVEMIg fusion protein
1. BALB/c mice were immunized with L929/LIGHT cells. The B lymphocyte hybridoma technique was used to fuse the spleen cells of the mice and the murine myeloma cells SP2/0. The hybridoma cells were cultured on the HAT selective medium. The positive cells were screened with L929/LIGHT as positive, L929/mock was negative control, FACS analysis screened positive clones and passed 4. The hybridoma cell line (7A8) sustained secretory anti human LIGHT monoclonal antibody was obtained. The rapid qualitative test paper analysis showed that the heavy chain of McAb 7A8 was IgG2b and the light chain was kappa. After long-term culture and liquid nitrogen cryopreservation, the resuscitation clutter cells grew well, stable secreted antibodies; 2. the human Ig was Ig. G (Fc) gene coding region gene and human HVEM extracellular gene fusion gene, according to the construction of L929/LIGHT gene transfection cells technology and methods to transfect HVEMIg fusion gene into eukaryotic cells, screening and subcloning to obtain CHO/HVEMIg transfected cells. Using flow cytometry, SDS-PAGE and Western-blot to identify, the results show that HVEMIg fusion protein secretion. It was expressed in the culture supernatant of CHO/HVEMIg gene transfected cells. After large-scale cell culture, the purified supernatant was re concentrated and the pure HVEMIg fusion protein was obtained by protein G affinity chromatography. The flow test results, the purified protein had a good binding ability with the ligand LIGHT, and the.T cell proliferation experiment showed that the anti LIGHT monoantibody and HVEMIg were melted. The addition of synprotein can partially block the synergistic effect of gene transfected cells on the proliferation of T cells with CD3mAb and down regulate the secretion of cytokines, thus suggesting that LIGHT/HVEM participates in the regulation of activated T cells by mediating positive co stimulation.
To sum up, the L929/LIGHT gene transfected cell line was successfully constructed in this experiment. The membrane type human LIGHT expressed by the transfected cell has a significant promotion effect on the proliferation and cytokine secretion of T cells in vitro. A mouse anti human LIGHT monoclonal antibody 7A8 and HVEMIg fusion protein.T in vitro proliferation test was successfully developed, and the monoclonal antibody 7A8 and HVEMIg pairs L9 were shown. 29/LIGHT mediated stimulation of T cell proliferation and cytokine secretion have partial blocking effect respectively. The monoclonal antibody 7A8 is a blocking antibody against human LIGHT. The above results are valuable for exploring the co stimulation of LIGHT molecules and the regulatory role of LIGHT/HVEM/BTLA (CD160) Regulation Network in T cell immune response and intervention strategies. The material basis and experimental parameters.
【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

【參考文獻】

相關期刊論文 前1條

1 ;LIGHT sensitizes IFNγ-mediated apoptosis of HT-29 human carcinoma cells through both death receptor and mitochondria pathways[J];Cell Research;2004年02期

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本文編號：1938934