pRetro-On/LTβR在Jurkat细胞中表达和诱导细胞凋亡的研究  

Expression of pRetro-On/LTβR vector in Jurkat cells and the role of LTβR in apoptosis of Jurkat cells

作　　者：杨静[1,2] 印澄[1] 王慧娟[1] 徐安琪[1] 季晓辉[1] 

机构地区：[1]南京医科大学微生物与免疫学系,江苏南京210029 [2]南京江宁区妇幼保健所检验科,江苏南京211101

出　　处：《南京医科大学学报（自然科学版）》2011年第11期1578-1582,1592,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：南京医科大学科技发展基金重点项目(2010NJMUZ32)

摘　　要：目的:构建pRetro-On/LTβR载体,获得可表达淋巴毒素β受体((lymphotoxin beta receptor,LTβR)的Jurkat细胞,探讨LTβR在T细胞凋亡中的作用。方法:应用PCR技术扩增LTβR cDNA片段,将PCR产物纯化后定向连接入pRetro-On载体;重组质粒转入大肠杆菌,LB-Amp培养基筛选阳性克隆,经质粒抽提、纯化、NotⅠ和BamHⅠ双酶切、测序,验证pRetro-On/LTβR载体的正确构建;通过脂质体转染T细胞传代系Jurkat细胞;强力霉素(doxycycline,DOX)诱导后,Western blot鉴定转染细胞中LTβR蛋白的表达,流式细胞术检测细胞表面LTβR表达;以配体LIGHT刺激表达LTβR的Jurkat细胞后以annexin-V-PI双染色流式细胞法检测细胞的凋亡。结果:双酶切及测序结果证实LTβR cDNA正确插入pRetro-On质粒;Western blot和流式细胞术结果证实了LTβR在Jurkat细胞内经DOX诱导得到了表达;表达LTβR的Jurkat细胞经LIGHT刺激后凋亡增加。结论:成功构建pRetro-On/LTβR质粒,pRetro-On/LTβR在Jurkat细胞中可以表达;表达LTβR的Jurkat细胞可以通过LIGHT-LTβR途径导致细胞凋亡。Objective： To construct pRetro-On/LTβR vector and obtain LTβR-expressing Jurkat cells,and explore the role of LTβR in T cells apoptosis.Methods： LTβR cDNA was obtained by PCR amplification and subcloned into pRetro-On plasmid.Then E.coli was transformed by the recombinant plasmid and positive clones under LB-Amp medium screen were used for recombinant plasmid extraction and purification.The recombinant plasmid pRetro-On/LTβR was identified by NotⅠ and BamHⅠ double enzyme digestion and DNA sequencing.Jurkat cells,a kind of T cell line,was transfected by the recombinant plasmid with Lipofectamine 2000.LTβR expression in Jurkat cells was induced by doxycycline（DOX） and identified by Western blot analysis and flow cytometry.The apoptosis of Jurkat cells induced by interaction of LTβR with the ligand,LIGHT,was detected by the method of annexin-V-PI double-stain flow cytometry.Results： Double enzyme digestion and DNA sequencing results confirmed that LTβR cDNA was inserted correctly into pRetro-On plasmid.The results of Western blot analysis and flow cytometry confirmed the LTβR expression in Jurkat cells.Apoptosis of transfected Jurkat cells significantly increased after LIGHT stimulation.Conclusion： The pRetro-On/LTβR was successfully constructed,and LTβR could be expressed in Jurkat cells using the recombinant plasmid.The apoptosis of LTβR-expressing Jurkat cells can be induced through LIGHT-LTβR pathway.

关 键 词：淋巴毒素β受体 JURKAT细胞 LIGHT 凋亡 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]