CTLA4.FasL免疫抑制效应及对肝前体细胞增殖分化潜能的影响  

CTLA4.FasL′s immunosuppressive property and its effect on the proliferation and differentiation potentials of liver progenitor cells

作　　者：万真[1] 吕毅[1] 张晓刚[1] 

机构地区：[1]西安交通大学医学院第一附属医院肝胆外科先进外科技术与工程研究所,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2014年第2期147-151,162,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.30600575)~~

摘　　要：目的探讨融合蛋白CTLA4.FasL对肝前体细胞(LEPCs)增殖分化潜能的影响及抑制异种排斥反应的效应。方法克隆CTLA4.FasL基因,构建携带CTLA4.FasL基因与红色荧光蛋白(mCherry)双顺反子结构的重组慢病毒载体Lv-CTLA4.FasL-IRES-mCherry。优化慢病毒感染LEPCs的条件,建立高表达CTLA4.FasL的LEPCs,荧光显微镜观察mCherry的表达,Western blot检测CTLA4.FasL的表达,酶联免疫吸附测定法(ELISA)测定细胞培养上清中CTLA4.FasL的浓度,水溶性四氮唑(WST-1)法检测细胞的增殖活性,Real time PCR(RT-PCR)检测干细胞相关基因CK19和c-Kit mRNA的表达,5-溴脱氧尿嘧啶核苷(Brdu)掺入法测定CTLA4.FasL-LEPCs在异种混合淋巴细胞培养体系中对大鼠淋巴细胞增殖的抑制作用。结果构建重组慢病毒载体Lv-CTLA4.FasL-IRES-mCherry,病毒滴度为2×108 TU/mL。在感染复数(multiplicity of infection,MOI)为10,聚凝胺质量浓度是5μg/mL时,感染效率约90%。Western blot证实了CTLA4.FasL的表达,在细胞培养上清中其质量浓度约为(0.72±0.10)μg/mL。CTLA4.FasL-LEPCs细胞增殖活性未受到影响,CK19和c-Kit基因mRNA表达水平无变化。CTLA4.FasL-LEPCs细胞可显著抑制大鼠淋巴细胞的增殖(P<0.05)。结论构建成功的重组慢病毒载体Lv-CTLA4.FasL-IRES-mCherry可介导CTLA4.FasL基因在LEPCs中高效表达;CTLA4.FasL可有效地抑制异种排斥反应,同时不损害LEPCs增殖活性和分化潜能。Objective To study whether CTLA4. FasL gene-modified liver progenitor cells （LEPCs） can inhibit the proliferation of xenogeneic lymphocytes and evaluate the effect of CTLA4. FasL on the proliferation capacity and differentiation potential of LEPCs. Methods CTLA4. FasL gene was amplified by PCR. Lv-CTLA4. FasL-IRES-mCherry, the recombinant lentiviral vector carrying CTLA4. FasL gene and red fluorescent protein （mCherry） gene was constructed. LEPCs were infected with lentiviral supernatant, mCherry expression was visualized under fluorescent microscope and CTLA4. FasL expression was tested by Western blot and ELISA. Proliferation ability of LEPCs was measured by WST-1 and CK19 and c-Kit mRNA expression by RT-PCR. Brdu incorporation assay was performed to evaluate the suppressive effect of CTLA4. FasL-LEPCs on rat lymphocyte proliferation in mixed lymphocyte reaction （MLR）. Results The recombinant lentiviral vector Lv-CTLA4. FasL- IRES-mCherry was successfully constructed with a titer of 2 ×108 TU/mL. As MOI increased to 10 in the presence of 5 μg/mL polybrene; the transduction efficiency of LEPCs reached about 90%. Western blot confirmed the expression of CTLA4. FasL and its concentration in cell medium was （0. 72±0.10）gg/mL. LEPCs showed unchanged proliferation ability and mRNA expression level of CK19 and c-Kit genes after CTLA4. FasL gene modification. Compared with uninfected control cells, CTLA4. FasL-LEPCs inhibited the proliferation of rat lymphocytes significantly in xenogeneic MLR （P〈0.05）. Gorlclusion We successfully constructed the CTLA4. FasL containing lentiviral vector Lv-CTLA4. FasL-IRES-mCherry, which infected LEPCs and expressed CTLA4. FasL effectively. CTLA4. FasL can inhibit the proliferation of xenogeneic lymphocytes significantly, but does not affect the proliferation capacity and differentiation potential of LEPCs.

关 键 词：CTLA4 FASL 肝前体细胞 异种免疫排斥反应 增殖 分化 

分 类 号：R392.4[医药卫生—免疫学]