摘要
目的以慢病毒载体介导猿猴病毒40(simian virus 40,SV40)大肿瘤抗原(large tumor antigen,TAg)转染原代人肝星状细胞(hepatic stellate cell,HSC)获得永生化HSC株。方法以含SV40 TAg cDNA序列的SV40tsA58质粒为模板,PCR获取全长SV40 TAg序列,经TOPO克隆进入pENTR^(TM)TOPO~?载体,PCR鉴定获得pENTR-SV40 TAg Entrez质粒,再经LR重组反应将SV40 TAg cDNA克隆到目标慢病毒载体pLenti4/V5-DEST,PCR鉴定pLenti4/V5-GW/SV40 TAg质粒,进一步用该质粒转染工程细胞293FT进行假病毒包装,用含包装病毒的细胞培养上清液转染原代人HSC,经博来霉素筛选获得稳定表达SV40 TAg的人HSC株(命名为WM07),并对细胞进行SV40 TAg及α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达的检测和鉴定。结果对pENTR-SV40 TAg Entrez质粒及pLenti4/V5-GW/SV40 TAg慢病毒表达质粒进行PCR测序,结果证实所构建的质粒含ATG转录起始位点及SV40 TAg cDNA序列。对经过转染、筛选获得的WM07行免疫荧光染色,结果显示SV40 TAg在细胞核表达和定位,细胞质内均表达活化的HSC标志物α-SMA。结论 pLenti4/V5-GW/SV40 TAg质粒构建成功。经质粒转染,获得永生化HSC株WM07,可稳定传代并用于肝纤维化研究。
Objective To obtain an immortalized human hepatic stellate cell(HSC)line by lentivirus mediated simian virus 40(SV40)large tumor antigen(TAg)gene transfection into primary isolated HSC.Methods The full-length SV40 TAg c DNA sequence was obtained by PCR amplification from an original plasmid SV40 ts A58 and cloned into pENTRTMTOPO?entrez vector.The sequence was then recloned into a target lentivirus vector by LR recombinant reaction to obtain SV40 TAg expression plasmid p Lenti4/V5-GW/SV40 TAg.This plasmid was packaged by 293 FT engineering cells and the cell supernatant containing SV40 TAg pseudo-virus was collected for transfecting primary isolated HSC.An human HSC line(namely WM07)stably expressing SV40 TAg was then screened out by bleomycin selection.SV40 TAg andα-smooth muscle actin(α-SMA)expression in WM07 was detected by immunofluorescence staining.Results PCR sequencing results showed that the constructed p ENTRSV40 TAg Entrez plasmid and p Lenti4/V5-GW/SV40 TAg plasmid contained ATG transcription start site and SV40 TAg cDNA.By immunofluorescence staining,the expression of SV40 TAg was detected in the nuclei of WM07.The cells were all positive for the staining of activated HSC markerα-SMA.Conclusion p Lenti4/V5-GW/SV40 TAg plasmid was successfully constructed.By the plasmid transfection,an immortalized human HSC line WM07 was established,which can be used in multiple studies on liver fibrosis.
作者
郭津生
王满
GUO Jin-sheng;WANG Man(Department of Gastroenterology,Zhongshan Hospital,Fudan University,Shanghai 200032,China)
出处
《复旦学报（医学版）》
CAS
CSCD
北大核心
2020年第6期893-898,共6页
Fudan University Journal of Medical Sciences
基金
国家自然科学基金(91129705,81070340,30570825)
上海市浦江人才计划(09PJ1402600)。
