【摘要】：BAFF(B cell-activating factor belong to the TNF family)是1999年發(fā)現(xiàn)的新分子,主要表達(dá)于單核細(xì)胞、巨噬細(xì)胞、樹突狀細(xì)胞、活化的T淋巴細(xì)胞和中性粒細(xì)胞等。其受體包括特異性結(jié)合受體BAFF-R、TACI和BCMA,三個受體主要表達(dá)于T和B淋巴細(xì)胞。BAFF與其受體共為TNF/TNFR超家族中的重要成員。業(yè)已發(fā)現(xiàn),BAFF信號對B淋巴細(xì)胞的分化、發(fā)育及抗體的類型轉(zhuǎn)換,及生發(fā)中心的維持具有重要作用;對T淋巴細(xì)胞具有共刺激作用;并與多種自身免疫病如SLE、RA和SS等、腫瘤如慢性B淋巴細(xì)胞性白血病、B細(xì)胞性非何杰金氏病及多發(fā)性骨髓瘤等,以及與心臟等器官移植的同種排斥反應(yīng)相關(guān)。但對BAFF的基礎(chǔ)研究尚需深入。為研究BAFF信號,本研究首先克隆了人全長BAFF分子,建立了BAFF基因轉(zhuǎn)染細(xì)胞,并以此為免疫原,成功研制了一株抗人BAFF單克隆抗體,并深入探討了其生物學(xué)特性。分析了健康人外周血來源和胸水來源T淋巴細(xì)胞表面BAFF的表達(dá)情況和意義。與此同時,本研究克隆了受體全長人TACI基因,建立了可溶性表達(dá)TACI-Fc融合蛋白的基因轉(zhuǎn)染細(xì)胞,并探討了所獲得TACI-Fc融合蛋白的生物學(xué)效應(yīng)。 一、人BAFF基因轉(zhuǎn)染細(xì)胞的構(gòu)建和BAFF分子對B細(xì)胞功能的影響 1.人BAFF基因的克隆和逆轉(zhuǎn)錄病毒表達(dá)載體的構(gòu)建 根據(jù)BAFF基因(OeneBank:NM 006573)序列,利用RT-PCR方法從健康人外周血單個核細(xì)胞克隆出全長BAFF基因,裝入pMD18-T載體,轉(zhuǎn)化感受態(tài)大腸桿菌Top10。重組質(zhì)粒T-BAFF經(jīng)測序正確。利用PCR方法,從重組質(zhì)粒T-BAFF擴增BAFF全長基因并引入酶切位點EcoR I和BamH I,裝入逆轉(zhuǎn)錄病毒表達(dá)載體pSIV1,轉(zhuǎn)化感受態(tài)大腸桿菌Top10。重組表達(dá)載體pSIV1-BAFF經(jīng)測序正確。 2.脂質(zhì)體轉(zhuǎn)染法構(gòu)建BAFF基因轉(zhuǎn)染細(xì)胞 pSIV1-BAFF、輔助病毒載體pHIT456和pHIT60混合后,脂質(zhì)體轉(zhuǎn)染法轉(zhuǎn)染包裝細(xì)胞293T細(xì)胞,獲得具有感染力的重組逆轉(zhuǎn)錄病毒。將含有完整BAFF重組逆轉(zhuǎn)
[Abstract]:BAFF (B cell-activating factor belong to the TNF family) is a new molecule found in 1999, mainly expressed in monocytes, macrophages, dendritic cells, activated T lymphocytes and neutrophils. Its receptors include the specific binding receptors BAFF-RnTACI and BCMA.The three receptors are mainly expressed in T and B lymphocytes. BAFF and its receptors are important members of the TNF/TNFR superfamily. It has been found that BAFF signal plays an important role in the differentiation, development and antibody type conversion of B lymphocytes, as well as the maintenance of germinal center, and has co-stimulatory effect on T lymphocytes, and is associated with many autoimmune diseases such as SLERA and SS. Tumors such as chronic B lymphocytic leukemia B cell non Hodgkin's disease and multiple myeloma are associated with allograft rejection of heart and other organs. However, the basic research of BAFF needs to be further studied. In order to study the BAFF signal, we first cloned the human full-length BAFF molecule, established the BAFF gene transfection cells, and used it as the immunogen, successfully developed a monoclonal antibody against human BAFF, and studied its biological characteristics. The expression and significance of BAFF on T lymphocytes from peripheral blood and pleural effusion were analyzed. At the same time, the full-length human TACI gene of the receptor was cloned, and the gene transfected cells expressing TACI-Fc fusion protein were established, and the biological effects of the TACI-Fc fusion protein were discussed. 1. Construction of human BAFF gene transfected cells and effect of BAFF molecule on B cell function. 1. Cloning of Human BAFF Gene and Construction of Retrovirus expression Vector according to the sequence of BAFF Gene (OeneBank:NM 006573), The full-length BAFF gene was cloned from the peripheral blood mononuclear cells of healthy people by RT-PCR method. The gene was inserted into the pMD18-T vector and transformed into the competent E. coli Top10. The recombinant plasmid T-BAFF was sequenced correctly. Using PCR method, the full-length BAFF gene was amplified from the recombinant plasmid T-BAFF, and the restriction sites EcoR I and BamH I were introduced into the retroviral expression vector pSIV1. The recombinant expression vector pSIV1-BAFF was sequenced correctly. 2. BAFF gene transfected pSIV1-BAFF cells were constructed by liposome transfection method. After pHIT456 and pHIT60 were mixed, the packaging cells 293T cells were transfected with liposome transfection method. An infectious recombinant retrovirus was obtained. Will contain full BAFF recombination reversal
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R392

【共引文獻(xiàn)】

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1 王喜梅;李向培;厲小梅;;B細(xì)胞的異常增殖在干燥綜合征中的研究進(jìn)展[J];安徽醫(yī)藥;2009年02期

2 鄭召嶺;蔡輝;;BAFF在自身免疫性疾病中的作用[J];重慶醫(yī)學(xué);2009年03期

3 孟瑞鋒;盛光耀;王利;;淋巴細(xì)胞凋亡異常在AITP患兒中的作用研究[J];醫(yī)藥論壇雜志;2006年14期

4 吳蔚;陳民利;;BAFF在自身免疫性疾病方面的研究進(jìn)展[J];中國比較醫(yī)學(xué)雜志;2012年01期

相關(guān)博士學(xué)位論文 前3條

1 魏瑞華;腺相關(guān)病毒介導(dǎo)IL-10轉(zhuǎn)基因治療對兔自身免疫淚腺炎干眼模型免疫抑制作用[D];天津醫(yī)科大學(xué);2011年

2 高會廣;人BAFF免疫抑制分子的構(gòu)建及功能鑒定[D];第三軍醫(yī)大學(xué);2006年

3 許珂;周期聯(lián)合免疫抑制劑甲氨蝶呤和環(huán)磷酰胺治療誘導(dǎo)型干燥綜合征模型的實驗研究[D];山西醫(yī)科大學(xué);2008年

相關(guān)碩士學(xué)位論文 前9條

1 楊金良;血清BAFF在ITP和AIHA患者中的表達(dá)及臨床意義[D];河北醫(yī)科大學(xué);2011年

2 曹萌;重組人B淋巴細(xì)胞刺激因子（hsBLyS）對B淋巴細(xì)胞的免疫調(diào)節(jié)作用及其機制研究[D];南京師范大學(xué);2005年

3 黃剛;人sBLyS突變體的構(gòu)建及功能研究[D];第三軍醫(yī)大學(xué);2006年

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5 楊波;62例干燥綜合征住院患者臨床病例分析[D];福建醫(yī)科大學(xué);2009年

6 王喜梅;白介素17在原發(fā)性干燥綜合征患者唇腺組織和外周血的表達(dá)[D];安徽醫(yī)科大學(xué);2009年

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8 馬英淳;干燥綜合征患者血清BAFF與IFN-γ、IL-10、IL-4水平相關(guān)性研究[D];蘇州大學(xué);2010年

9 楊西超;干燥綜合征患者唇腺組織與外周血BAFF/BAFF受體的初步研究[D];第四軍醫(yī)大學(xué);2009年

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本文編號：2181876