全反式维A酸对单纯疱疹病毒胸苷激酶基因结合更昔洛韦旁观者效应增强作用及其机制

Bystander effect with its mechanism enchanced by all-trans-retinoic acid in herpes simplex virus thynidine kinase gene/ganciclovir system

目的 通过研究全反式维A酸 (ATRA )对单纯疱疹病毒胸苷激酶基因结合更昔洛韦 (HSV tk/GCV)旁观者效应的增强作用及与Cx4 3表达的关系 ,为提高TK自杀基因的疗效及探索增强作用机制提供实验基础。方法 应用Polybrene转染、荧光定量RT PCR等技术 ,观察ATRA对宫颈癌细胞ME180 ,ME180 /TK转化细胞旁观者效应及诱导Cx4 3mRNA表达的作用。结果 ME180 ,ME180 /TK不同比例的混合细胞随ATRA浓度的增加而存活率降低。ME180 +ME180 /TK混合细胞的存活率随ME180 /TK细胞比例的增加而降低。在 2mg·L- 1更昔洛韦 (GCV)作用下 ,以 10 - 8mol·L- 1ATRA与不加ATRA (0mol·L- 1)的作用相比较 ,在ME180与ME180 /TK不同比例混合的各组中 ,细胞的存活率明显降低 (P <0 .0 5 )。并观察到10 - 8及 10 - 10 mol·L- 1ATRA对GCV旁观者效应的增强作用。RT PCR结果表明 ,经ATRA处理的ME180细胞 ,其Cx4 3mRNA的相对拷贝数比值增高约 1.84倍及 2 .6 5倍。结论 宫颈癌ME180细胞中 ,ATRA在10 - 8～ 10 - 10 mol·L- 1范围内 ,具有明显增强HSV tk/GCV旁观者效应的作用。其主要机制是通过ATRA在转录水平诱导Cx4 3mRNA表达上调 ,导致旁观者效应的增强。

AIM By investigating the bystander effect enchanced by all trans retionic acid (ATRA) in herpes simplex virus thymidine kinase gene/ganciclovir(HSV tK/GCV) system and with relation to Cx43 mRNA expression to provide experimental basis for heightening therapy efficiency of TK suicide gene and exploring mechanism of enchancement role. METHODS Enhancement of the bystander effect in HSV tK/GCV and Cx43 mRNA expression in cervical carcinoma ME180 and ME180/TK transfection cells were examined by using Polybrene transfection, fluor quantitative RT PCR techanques. RESULTS ME180 and ME180/TK mixed cells were suppressed by gradational increase in ATRA concentration in HSV tk/GCV system. The survival rate of ME180+ME180/TK mixed cells decreased with the increase in ME180/TK ratio. In the presence of 2 mg·L -1 GCV, the more lower survival rate was observed at any given mixture of ME180 and ME180/TK cells exposed to 10 -8 mol·L -1 ATRA compared with that in the absence of ATRA (0 mol·L -1 ) (P<0.05). However, enchancement of bystander mediated cell killing occurred only in the concentration range of 10 -8 -10 -10 mol·L -1 of ATRA. The results of RT PCR showed that comparing with non ATRA treated ME180 cells, the ratio of relative copy number of Cx43 mRNA increased by 2.65 and 1.84 fold in ME180 cells exposed to 10 -8 and 10 -10 mol·L -1 ATRA, respectively. CONCLUSION In cervical carcinoma ME180 cell line, ATRA enhances the bystander effect in the HSV tK/GCV system. Its main mechanism of enchancement is up regulation of Cx43 mRNA expression in transcription level.