LentiviralHGF的构建及其体外对β_2m^-/Thy-1^+ BDLSCs影响的  

Construction of lentiviral containing rat HGF gene and transfection of β_2m-/Thy-1^+ BDLSCs in vitro

作　　者：胡俊杰[1] 孙超[1] 兰玲[1] 李定国[1] 

机构地区：[1]上海交通大学医学院附属新华医院消化内科,上海200092

出　　处：《胃肠病学和肝病学杂志》2009年第9期795-799,共5页Chinese Journal of Gastroenterology and Hepatology

基　　金：国家自然科学基金资助项目(30500236);上海市科委医学临床研究重点科技攻关项目(064119527);上海市教委优秀青年教师科研专项基金(18040)

摘　　要：目的构建携带大鼠HGF基因的慢病毒载体,并观察慢病毒对体外分选的β2m-/Thy-1+BDLSCs转染效果及影响。方法采用RT-PCR法从大鼠肝脏中提取HGF基因,并将其克隆到穿梭质粒TG006,与包装质粒一起在293T细胞中重组产生LentiviralHGF慢病毒。通过免疫磁珠法体外分选出β2m-/Thy-1+BDLSCs;采用荧光显微镜检测LentiviralHGF对BDLSCs的转染效率;采用ELISA法检测大鼠HGF的蛋白分泌水平;采用MTT法和免疫荧光法分别检测病毒对增殖和分化的影响。结果从纤维化大鼠肝脏中成功克隆出HGF基因,并构建LentiviralHGF慢病毒;慢病毒能高效转染BDLSCs。转染慢病毒的BDLSCs向胞外分泌高浓度的HGF。转染LentiviralHGF后,细胞增殖能力提高,ALB表达阳性。结论LentiviralHGF能高效转染BDLSCs,两者结合为肝纤维化的研究奠定了基础。Objective To construct the LentiviralHGF,and to evaluate the efficiency of the lentiviral in transfecting BDLSCs in vitro.Methods Rat HGF gene was constructed into shuttle plasmid TG006 after being cloned from rat liver,then transferred with lentiviral backbone vector into 293T cells.β2m-/Thy-1＋BDLSCs were isolated by magnetic bead cell sorting（MACS）.LentiviralHGF transfection efficiency was observed by fluorescent microscopy after transduced LentiviralHGF.The levels of HGF secreted from BDLSCs were detected by ELISA.The proliferation and albumin expression of BDLSCs were detected by MTT and immunofluorescence staining.Results Rat HGF gene was cloned from liver and then LentiviralHGF was constructed successfully.A high transfection efficiency was observed when BDLSCs were transfected with lentiviral.HGF expression in cell culture fluid could be detected at a high level,which could stimulate BDLSCs proliferation.Conclusion HGF gene delivered into β2m-/Thy-1＋BDLSCs by lentiviral can be expressed with high efficiency.It might be an effective tool for stem cell transplantation by gene modified donor cells.

关 键 词：β2m-/Thy-1+骨髓源性肝干细胞 大鼠肝细胞生长因子 慢病毒 基因转导 

分 类 号：Q813[生物学—生物工程] R333.4[医药卫生—人体生理学]