以非复制型HIV为载体进行目的基因转移的实验研究

Study on High-efficiency Gene Transfer of Pseudotyped HIV Vector

目的探讨非复制型HIV作为基因转移载体的有效性,为卵巢癌基因治疗转移方法提供新思路。方法磷酸钙沉淀法将非复制型HIV基因转移载体(pHR'CS)系统中的3种成分质粒共转染入病毒包装细胞293T,荧光显微镜、电镜、荧光分光光度计检测病毒产生情况。病毒上清过滤、浓缩后感染卵巢癌细胞SKOV3和正常人成纤维细胞GF,荧光显微镜下观察感染效果。PCR、RT-PCR鉴定目的基因HSV-tk在靶细胞中的整合转录结果。光镜观察及MTT法测定GCV的体外杀伤效应,计算细胞生长抑制率(GIR)和半数抑制浓度(IC50)。结果荧光显微镜下观察到293T细胞中有大量GFP表达,透射电镜下观察到大量浓缩后病毒颗粒,荧光分光光度计检测到510nm处有一高吸收峰,此非复制型HIV感染卵巢癌细胞SKOV3和正常人成纤维细胞GF后,PCR、RT-PCR均显示600bp的HSV-tk阳性条带。光镜观察到细胞在GCV作用后出现明显形态变化;MTT法显示GCV对转导HSV-tk的SKOV3和GF细胞有显著杀伤作用。结论非复制型HIV可将目的基因高效转移至卵巢癌细胞中,为一种有效的基因转移载体。

Objective To investigate the high-efficiency of pseudotyped HIV as gene transfer vector.Metho-ds Three plasmids of pseudotyped HIV gene transfer vector system were transferred into packaging cell line293T by Ca 3 (PO 4 ) 2 precipitation method.GFP (Green Fluorescence Protein)or HSV-tk gene was constructed in the plasmid pHR'CS respectively(pHR'CS.GFP or pHR'CS.HSV-tk).The pse udo-typed HIV particles were observed through electronic microscopy and were measured through spectro-fluorophotometer.High titer pseudotyped HIV was harvested from volume of virus-producing cell super-natant and concentrated.Ovarian epithelial cancer cell line SKOV3and normal human gingival fibro-blast cell GF were infected by pseudotyped HIV.PCR and RT-PCR were resorted to demonstrate the su ccessful transduction and transcription of the HSV-tk gene.After administration of GCV,the ch ange s of those cells and apoptosis were observed through optical microscopy.The cytotoxicity efficacy of HSV-tk/GCV system was evaluated by MTT method.The growth inhibition rate(GIR)of cells and inhi -bition concentration50(IC 50 )were counted.Results The above plasmids were effe ctively tra nsferr ed into293T cell.A lot of pseudotyped HIV particles were observed through ele ctronic mi croscopy.The virus supernatant had a high absorbing value at510nm through spectrofluorophotom eter,which proved the existence of virus.After pseudotyped HIV infection,SKOV3and GF had remarkable infection rate.600bp strand was seen through PCR and RT-PCR.Changes and apoptosis of cells followed by administration of GCV were observed.The MTT method sh owed that the cytotoxicity efficacy of GCV was high to SKOV3and GF cell.Conclusions The pseudotyped HIV is a high-efficiency gene tra n-sfer vector.