FADD缺失突变重组体转染的人β胰岛细胞株的建立及其生物学特性的研究  

Establishment of human islet β-cell line of FADD deleted mutation (FADDdel) and studying on character of its biology

作　　者：周华蓉[1] 李鸣[1] 张悦[1] 周新荣[1] 沈关心[1] 

机构地区：[1]华中科技大学同济医学院免疫学教研室,武汉430030

出　　处：《中华微生物学和免疫学杂志》2001年第6期600-603,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 ( 39870 712 )

摘　　要：目的　建立Fas相关死亡域 (FADD)缺失突变体的表达细胞系 ,阻断Fas死亡信号传导 ,以便今后更深入的探讨通过基因水平修饰靶细胞 ,改变靶细胞的反应状态 ,对移植免疫和自身免疫性疾病的可能的影响。方法　用RT PCR从感染患者外周血淋巴细胞中获得缺失FADD死亡域目的基因片段 (FADDdel) ,利用双酶切将FADDdel定向导入真核表达载体pUC pIC ;用电穿孔法将重组子导入人 β胰岛细胞株 (NIT)中 ,采用FACS法检测sFasL所引起的转染前后NIT凋亡情况。同时在In ternet中通过计算机数字化分子模型技术 ,成功模建FADDdel的 3D结构并与FADD的立体结构进行比较与分析。结果　RT PCR法得到约 5 0 0bp的目的基因片段 ,转化子用BglⅡ ,XbaⅠ酶切后得到 3个特异性片段 ,经酶切和测序鉴定为FADD缺失突变的重组体 (pFADDdel)。将pFADDdel导入NIT后 ,筛选出高表达细胞系F15 ,在IFN γ和TNF α存在时F15仍能有效拮抗sFasL诱导的凋亡 ;并对FADD和FADDdel功能结合域和结合位点分析 ,阐明了FADDdel蛋白质分子在阻断细胞凋亡胞内信号传导通路的分子机制。结论　成功建立了FADDdel稳定表达的人胰岛细胞株 ;pFADDdel能有效抑制Fas和TNFR1介导的胞内凋亡信号通路 ;Objective To construct FADDdel plasmid capable of blocking the Fas triggered intracellular signal transduction and to prepare technology ability for the further inquiring into transplantation by modifying the target cells in gene level. Methods The target gene of FADDdel, designated Fas associated death domain protein(FADD) without N terminal death effective domain(DED), was amplified from human blood lymphocyte by RT PCR and then inserted into the pUC pIC vector. The structure was confirmed by digestion and DNA sequencing. After the transfection of human β cell strain(NIT) with pFADDdel, apoptosis of NIT with or without pFADDdel induced by FasL was detected by FACS. The 3 D structure of FADDdel was successfully constructed, analyzed and compared with stereodrawing of the FADD by means of digital molecule mode technology online. Results A 500bp target gene fragment was obtained by RT PCR. Digestion and DNA sequencing showed that the recombinant plasmid pFADDdel was successfully constructed. After transfection, FADDdel high expression cell strain F15 has been built up. pFADDdel prevented the death of F15 induced by sFasL even cultured with IFN γ and TNF α. Analyzing of function structure domain of FADD and FADDdel showed molecule mechanism of FADDdel protein in blocking signal transduction pathway of apoptosis. Conclusions In the present study, we have established human β islet cells strain which can steadily express the pFADDdel. pFADDdel can block the apoptosis signal of Fas and TNF R1. It is a solid base for the further study of transplantation immunity and organ special autoimmune disease. [

关 键 词：FADD缺失突变 β胰岛细胞株 移植免疫 自身免疫性疾病 

分 类 号：R392[医药卫生—免疫学]