腺病毒介导的KDR-CDglyTK系统在治疗胃癌中的旁观者效应机制  

Mechanism of bystander effect in therapy of stomach cancer with the KDR-CDglyTK suicide gene system

作　　者：李强[1] 黄宗海[1] 陈飞[1] 俞金龙[1] 

机构地区：[1]南方医科大学珠江医院普外科,广东省广州市510282

出　　处：《世界华人消化杂志》2012年第9期749-753,共5页World Chinese Journal of Digestology

摘　　要：目的:探讨重组腺病毒介导KDR-CDglyTK系统杀伤胃癌SCG7901细胞时细胞缝隙连接与旁观者效应的关系.方法:感染复数(multiplicity of infection,MOI)=100时,将携带融合基因重组腺病毒体外感染SCG7901、HeLa细胞,检测细胞的感染效率以及转基因肿瘤细胞在mRNA水平上融合基因的表达;用荧光漂白恢复技术(fluorescence recovery after photobleaching,FRAP)检测SCG7901、HeLa细胞以及芹黄素作用上述两种细胞的细胞间通讯功能.将已转染细胞和未转染细胞分别按10%∶90%和5%∶95%两种比例混合,用含或不含芹黄素的培养基,培养24 h后,在不同组中分别加入GCV、5-FC两者的混合液,72 h后用MTT法检测细胞存活率.结果:受感染的SCG7901和HeLa细胞中均有绿色荧光蛋白的表达.图像中观察到在芹黄素作用下SCG7901细胞淬灭后荧光强度明显降低,随后随着时间的推移,被淬灭细胞的荧光强度逐渐恢复,在相同时间内其恢复强度较未应用上调剂的SCG7901细胞要强;而在芹黄素作用下的HeLa细胞被淬灭后荧光强度明显降低,随时间的推移荧光强度无明显恢复,在上调剂芹黄素作用下的SCG7901与HeLa细胞间的荧光恢复率有差异.同时,观察到双自杀基因系统在转基因细胞比例分别占5%、10%时,SCG7901细胞在空白组、前药组、芹黄素组、前药+芹黄素组间细胞生存率有差异(F=144.42,P=0.000)、(F=407.83,P=0.000),而HeLa细胞在各组间的细胞生存率无统计学意义(F=0.386,P=0.765)、(F=0.895,P=0.472).结论:FRAP技术证实SCG7901细胞间通讯功能与缝隙连接有一定关系,而HeLa细胞不存在细胞间通讯功能.双自杀基因系统治疗SCG7901细胞时存在旁观者效应而且上调剂可以扩大其旁杀效应;但HeLa细胞不存在旁观者效应.双自杀基因系统治疗胃癌细胞时旁观者效应与细胞间缝隙连接有关.AIM：To study the relation between intercellular gap connection and bystander effect in therapy of stomach cancer with the KDR-CDglyTK suicide system.METHODS：SCG7901 and HeLa cells were infected with adenovirus-based KDR-CDglyTK system（AdKDR-CDglyTK）,and the expression of CDglyTK fusion gene in infected cells was confirmed by RT-PCR.Gap junction intercellular communication was determined by fluorescence recovery after photobleaching（FRAC） in SCG7901 and HeLa cells in the presence or absence of versulin.Infected and non-infected cells were mixed with a proportion of 5% and 95% or 10% and 90%,cultured in the presence or absence of versulin,and then used to detect cell survival by MTT assay.RESULTS：The expression of GFP was observed in both infected SCG7901 cells and HeLa cells.Fluorescence intensity was gradually recovered at different time points after bleaching in SCG7901 cells cultured with versulin.Compared to SCG7901 cells cultured without versulin,the fluorescence recovery of bleached cells cultured with versulin was more obvious at the same time points.In contrast,fluorescence intensity showed no significant changes in HeLa cells cultured with versulin.The mean fluorescence recovery rate had a significant difference between SCG7901 and HeLa cells cultured with versulin.When infected and non-infected cells were mixed at different proportions,cell survival showed a significant difference between groups of SCG7901 cells（F = 144.42,407.83;both P = 0.000）,but had no signif icance difference between groups of HeLa cells（F = 0.386,0.895;P = 0.765,0.472）.CONCLUSION：There is a relation between intercellular communication and gap connection in SCG7901 cells but not in HeLa cells.Versulin can enhance the bystander effect in therapy with the suicide gene system in vitro.The mechanism of bystander effect in therapy with the suicide gene system may involve gap connection.

关 键 词：胃癌 荧光漂白恢复技术 缝隙连接 旁观者效应 自杀基因 

分 类 号：R735.2[医药卫生—肿瘤]