SV40T/LoxP等外源性基因修饰与肝细胞永生化的研究

Study on gene modification such as SV40T/LoxP with immortalized liver cell

目的构建永生化肝细胞系,观察其生长状态及检测其相关生物免疫学特性。方法拟利用基因重组克隆方法构建含有SV40T、EGFP基因片段以及LoxP序列,插入Cre基因片段,采用脂质体转染法将新载体pSV-GFP-Cre-LOX转染至来源于成人原代供肝细胞,建立新型肝细胞体系进行细胞培养;利用转化肝细胞能够表达绿色荧光蛋白特性观察该体系中转化肝细胞的生长状况,最后通过系列生化免疫反应检验转化肝细胞的生化免疫学特性;通过Cre重组酶定点切割导入的SV40T基因使转化肝细胞回复到永生化前的状态,并检测其细胞学相关特性。结果转染SV40T基因的肝细胞具备正常肝细胞生物学特性,且增殖较快,体外易培养。结论新建永生化肝细胞系可以为进一步肝细胞移植提供理想细胞材料作出研究基础。

Objective To establish an immortalized liver cell line,and to observe its living status and test related biolgical immunology characteristics.Methods To establish a new carrier pSV-GFP-Cre-LOX containing SV40T （Simian Virus 40）,EGFP（ fluorescence protein gene） and LoxP（self - cut off） sequence by gene recombination cloning.And by using of fiposomes transfer method for new carrier pSV-GFP-Cre-LOX to supplier liver cell of first generation,new liver cell line mmortalized was constructed and was cultured;and observe new liver cell growing condition because liver cell can express green fluoreseeneee, and finally test its related cytology characteristics by serial biochemistry immune method;then make immortalized liver cell restore to the pre-immortalized status through gene SV40T cut off by Cre reeombinase.Results Liver cell containing SV40T has biochemistry characteristics of normal liver cell,multiplicate quickly, and culture easily out of body.Immortalized liver cell can restore to original liver characteristics after cutting off the SV40T.Conclusion To establish a new immortalized liver cell line can supply idealistic cell material for live transplantation for further research.