恢复connexin26表达联合载酵母菌胞嘧啶脱氨酶自杀基因纳泡杀灭膀胱癌细胞  

Efficacy of cationic nanomicrobubbles carrying connexin 26 gene and yeast cytosine deaminase/5-fluorocytosine suicide gene system combined with ultrasonic irradiation in killing bladder cancer cells in vitro

作　　者：陈思乡 姜庆[1] 张俊勇[1] 

机构地区：[1]重庆医科大学附属第二医院泌尿外科,重庆400016

出　　处：《第三军医大学学报》2018年第4期322-327,共6页Journal of Third Military Medical University

基　　金：重庆市卫生计生委重点科研项目(2013-1-020)~~

摘　　要：目的分析载双基因的阳离子纳泡联合超声靶向微泡破坏技术进行基因转染的有效性,探索恢复或上调膀胱癌细胞的缝隙连接蛋白connexin26(Cx26)表达,能否增强自杀基因系统(yeast cytosine deaminase/5-fluorocytosine,YCD/5-FC)的旁观者效应,提高杀灭肿瘤细胞的效率。方法阳离子纳泡结合超声辐照(US)转染人膀胱癌T24细胞,荧光显微镜及流式细胞仪观测转染效率;qRT-PCR和Western blot检测质粒转染后的mRNA或蛋白相对表达量。将实验分为无处理空白对照、载pc DNA3.1-EGFP纳泡组、载Cx26纳泡组、载YCD纳泡组、载YCD+Cx26纳泡组;通过流式细胞术观察恢复Cx26表达后对膀胱癌细胞凋亡的影响。结果 qRT-PCR、Western blot显示纳泡结合超声辐照成功将目的基因转染并有效表达。恢复Cx26表达后,载YCD+Cx26纳泡组的细胞凋亡率为(60.68±2.61)%,明显高于单载YCD纳泡组的(46.42±2.13)%,差异有统计学意义(P<0.01)。结论恢复缝隙连接蛋白Cx26表达,可改善细胞间通讯连接,加强自杀基因系统YCD/5-FC的旁观者效应,促进肿瘤细胞凋亡,提高杀灭膀胱癌细胞的效率。Objective To test the efficacy of cationic nanomicrobubbles carrying connexin 26 gene and yeast cytosine deaminase/5-fluorocytosine （YCD/5-FC） suicide gene system combined with ultrasonic irradiation in enhancing the cell killing effect in bladder cancer cells. Methods Human bladder cancer T24 cells were exposed to cationic nanomicrobubbles carrying both connexin 26 plasmid and the YCD/5-FC suicide gene system followed by ultrasonic irradiation. The gene transduction efficiency was assessed using fluorescence microscopy and flow cytometry, and qRT-PCR and Western blotting were used to verify the expression of the target genes. The effect of nanomicrobubble-mediated transduction of YCD/5-FC and connexin 26 genes on apoptosis of bladder cancer cells was detected by flow cytometry. Results qRT-PCR and Western blotting showed that the target genes were successfully transduced and efficiently expressed in the cells. The cells with nanomicrobubble-mediated transduction of both connexin 26 and YCD/5-FC genes showed an apoptotic rate of （ 60.68 ± 2.61 ） % , which was significantly higher than the rate of （46.42 ± 2.13） % in the cells transduced with YCD gene alone （P 〈 0.01 ）. Conclusion Restoration of connexin 26 expression in bladder cancer cells can improve the intercellular communication, enhance the bystander effect of YCD/5-FC suicide gene system, promote cell apoptosis, and thus markedly improve the efficiency of cell killing.

关 键 词：缝隙连接蛋白connexin26 阳离子纳泡 超声辐照 自杀基因系统yeast CYTOSINE deaminase/5-fluorocytosine 膀胱癌 

分 类 号：R394.33[医药卫生—医学遗传学] R394.3[医药卫生—基础医学]