携带SV40T/HSV-tk基因永生化正常成人肝细胞的构建

Construction of immortalized human liver cells with SV40T/HSV-tk gene

  • 摘要: 目的 构建永生化正常成人肝细胞为生物型人工肝提供安全有效的人肝细胞源。方法 转染包装细胞,获得逆转录病毒;将该病毒感染正常成人肝细胞HL-7702;更昔洛韦毒性实验检测转染后肝细胞内HSV-tk基因;RT-PCR法检测转染后细胞内SV40T的mRNA;Western Blot法和免疫荧光染色检测转染后细胞内SV40T基因的蛋白表达。结果 更昔洛韦检测转染后的肝细胞存活良好,而转染前的肝细胞死亡。RT-PCR法显示转染后的肝细胞内存在SV40T mRNA;Western Blot法和免疫荧光染色法显示转染后肝细胞内存在SV40T蛋白。结论 转染后的正常成人肝细胞中含有SV40T和HSV-tk基因,并表达相关蛋白。

     

    Abstract: Objective To provide safe and effective artificial liver human liver cells by constructing immortalized normal adult liver cells. Methods Artificial liver human liver cells were transfected and packaged to obtain retrovirus which was used to infect the normal adult liver cells HL-7702.Liver cells with the HSV-tk gene were detected by ganciclovir toxicity test.SV40T mRNA-transfected liver cells were detected by RT-PCR.SV40T gene expression in transfected liver cells was detected by Western blot with immunofluorescence staining. Results Ganciclovir toxicity test showed that the transfected liver cells survived well while the untransfected liver cells died.Immortalized SV40T mRNA was detected by RT-PCR in the transfected liver cells,while SV40T protein was detected by Western blot in the transfected liver cells with immunofluorescence staining. Conclusion SV40T and HSV-tk genes can be detected in transfected normal adult liver cells and express their associated proteins.

     

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