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  

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

作　　者：廖伟[1] 李生伟[1] 刘长安[1] 龚建平[1] 丁雄[1] 蔡宇[1] 周世骥[1] 

机构地区：[1]重庆医科大学附属第二医院肝胆外科,重庆400010

出　　处：《第三军医大学学报》2011年第15期1582-1585,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30872977);重庆市自然科学基金(CSTC2008BB5229)~~

摘　　要：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。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 MT-F 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 ceils by ultrasound wave irradiation and expressed stably. ATRA treatment enhanced the bystander effect of HSV-TK/GCV on SMMC-7721 cells compared with the ceils 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.

关 键 词：维甲酸 肝癌 超声微泡 连接蛋白32 单纯疱疹病毒胸苷酸激酶/丙氧鸟苷 

分 类 号：R394.2[医药卫生—医学遗传学] R73-362[医药卫生—基础医学]