全反式维甲酸增强携单纯疱疹病毒胸苷酸激酶基因的超声微泡对SMMC-7721细胞的旁观者效应

目的观察经全反式维甲酸(all-trans retinoic acid,ATRA)诱导后,单纯疱疹病毒胸苷酸激酶/丙氧鸟苷系统(HSV-TK/GCV)对人肝癌细胞SMMC-7721旁观者效应的影响。方法免疫组化法观察经全反式维甲酸诱导前后SMMC-7721细胞连接蛋白Cx32的表达;将含有HSV-TK基因的质粒通过静电吸附在脂质微泡表面上,采用超声辐照转染并用倒置显微荧光镜及RT-PCR观察TK基因的转入及表达;MTT法观察经全反式维甲酸诱导后HSV-TK/GCV对肝癌细胞SMMC-7721的旁观者效应。结果经全反式维甲酸诱导后SMMC-7721细胞膜上Cx32表达明显增加,而胞质内仅少量表达;通过超声辐照后HSV-TK基因可顺利转入SMMC-7721细胞中并稳定表达;与对照组相比,经全反式维甲酸诱导组可明显提高HSV-TK/GCV对肝癌细胞SMMC-7721的旁观者效应(P<0.05)。结论全反式维甲酸可提高SMMC-7721细胞中连接蛋白Cx32的表达且能正确定位于细胞膜上,这可能与其能增强HSV-TK/GCV系统对肝癌细胞SMMC-7721的旁观者效应有关。

All-trans retinoic acid enhances bystander effect of HSV-TK/GCV transfected by ultrasound microbubbles in SMMC-7721 cells

Objective To investigate the bystander effect of herpes simplex virus thymidine kinase(HSV-TK) transfected by ultrasound microbubbles and ganciclovir(GCV) in SMMC-7721 hepatoma cells after the treatment of all-trans retinoic acid(ATRA).Methods MTT assay was used to test the proper concentration of ATRA or GCV in treatment of SMMC-7721 cells.Immunohistochemistry was used to survey the expression of Cx32 protein in SMMC-7721 cells after ATRA treatment.Fluorescence microscopy and RT-PCR were used to detect the expression of TK in SMMC-7721 cells transfected with pIRES2-EGFP-TK with ultrasound microbubbles under ultrasound wave irradiation,and MTT assay was used to survey the changed bystander effect of SMMC-7721 on HSV-TK/GCV after ATRA treatment.Results The proper concentrations of ATRA and GCV were 10-6 mol/L and 40 μg/ml respectively in the treatment of SMMC-7721 cells.After the cells exposure to 10-6 mol/L ATRA,Cx32 protein was in high expression on the cell membrane but in low expression in the cytoplasm.HSV-TK was transfected into SMMC-7721 cells by ultrasound wave irradiation and expressed stably.ATRA treatment enhanced the bystander effect of HSV-TK/GCV on SMMC-7721 cells compared with the cells without ATRA treatment(P<0.05).Conclusion ATRA can improve the expression of Cx32 protein and lead Cx32 protein to cell membrane,which might be involved in ATRA enhancing the bystander effect of HSV-TK/GCV on SMMC-7721 cells.