转FasL基因卵巢癌HO-8910细胞系的建立

Construction of HO-8910-FasL, a Cell Line with Gene Transfer of Fas L of Ovarian Cancer

目的建立表达转FasL基因卵巢癌细胞系,为以FasL为靶点治疗卵巢癌提供理想细胞模型。方法应用分子克隆技术,将人FasLcDNA片段正向插入逆转录病毒载体pLXSN构建重组质粒pL(hFasL)SN,转染包装细胞PA317收获假病毒上清,磷酸钙法转染卵巢癌HO-8910细胞筛选建系,酶切PCR鉴定、流式细胞仪(FCM)法检测。结果酶切鉴定证实重组质粒pL(hFasL)SN中存在FasL,PCR方法从转染细胞HO-8910-FasL基因组中扩增出0.86kb片段,FCM检测显示FasL表达增加40.32%,HL60细胞与HO-8910-FasL细胞共培养HL60细胞凋亡增加25.65%。结论应用逆转录病毒法建立了稳定高表达FasL并具功能的HO-8910-FasL细胞系。

Objective To construct a HO-8910 cell line with gene transfer of FasL of ovarian cancer and to give a good model for studying ovarian cancer.Methods Human FasL cDNA was inserted into reombinant retrovirus vector plasmidpLXSN by subcolone method.By using calcium phosphate-DNA co-precipitation technique,recombinant plasmid was transfected into retrovirus package cell line PA317 and the culture supernatant containing recombinant retrovirus collected to infect the cell line HO-8910 (HO-8910-FasL).The gene transferred cells were selectively cultured with G418 and identified by polymerase chain reaction PCR, and flow cytometer(FCM) techniques.Results The recombinant PL(hFasL)SN was correctly constructed and confirmed by restriction endonuclease analysis.The presence of FasL cDNA in genome of HO-8910-FasL cell line was detected by PCR and FCM, that showed FasL has raised 40.32% gene expression in HO-8910-FasL.HO-8910-FasL cells could induce apoptosis of HL-60 cells in culture assay and apoptosis of HL-60 cells was significantly increased 25.65%.Conclusion A gene transferred HO-8910-FasL cell line,which can express FasL gene highly and have biological characteristics is successfully established.