转染缝隙连接蛋白CX43在脑胶质瘤化疗旁观者效应影响的体外实验研究  

Role of Connexin CX43 in Bystander Effect among Glioma Cells Treated with Chemotherapy in Vitro

作　　者：闻海兵[1] 李嘉杰[1] 邹树峰[1] 严剑[1] 魏敏俊[1] 刘先波[1] 颉奎[1] 洪涛[1] 

机构地区：[1]南昌大学第一附属医院神经外科,江西南昌330006

出　　处：《中国神经肿瘤杂志》2011年第2期115-123,共9页Chinese Journal of Neuro-Oncology

摘　　要：背景与目的:肿瘤细胞受损伤时,其旁边未受损肿瘤细胞也出现损伤的现象称为旁观者效应,国内外研究发现多种肿瘤组织在体内外存在该效应,为进一步明确脑胶质瘤在药物化疗时是否存的旁观者效应以及CX43(缝隙连接蛋白43)对该效应的影响,通过构建并转染pCMV-Cx43cDNA质粒入C6细胞,上调缝隙连接蛋白CX43表达,增强缝隙连接功能,探讨转染缝隙连接蛋白CX43对脑胶质瘤化疗体外旁观者效应影响。方法:(1)通过C6细胞培养,并提取总RNA,行RT-PCR、质粒酶切及连接等,构建缝隙连接蛋白CX43真核表达重组质粒pCMV-Cx43cDNA;(2)通过脂质体行重组质粒pCMV-Cx43cDNA转染C6细胞,过表达C6细胞缝隙连接蛋白CX43,RT-PCR及Weston-blot检测CX43 mRNA及蛋白表达水平变化。划痕荷载染料传输实验法检测转染缝隙连接蛋白CX43后缝隙连接功能;(3)通过体外TRASWELL细胞共培养模型,将转染pCMV-Cx43cDNA、空载体细胞分别分成化疗药物替膜唑胺处理组与替膜唑胺未处理组细胞,然后将两组细胞按一定比例分别接种TRASWELL细胞共培养板膜两侧,检测未化疗处理细胞存活率及凋亡率,观察体外转染CX43对体外化疗旁观者影响。结果:(1)成功构建带CX43目的基因的真核表达pCMV-Cx43cDNA重组质粒;(2)成功转染C6细胞并筛选稳定转染过表达CX43细胞株(C6-Cx43)及转染空载体的细胞株(C6-non),C6-Cx43细胞Cx43mRNA及CX43蛋白表达显著增加;(3)在体外TRASWELL细胞共培养模型中,观察到体外未化疗细胞凋亡的旁观者效应,(C6-Cx43)组与对照组差异存在明显统计学意义。结论:(1)C6细胞转染重组质粒pCMV-Cx43cDNA,缝隙连接蛋白CX43表达增高,C6细胞GJIC功能增强;(2)在体外化疗时,C6-Non、C6-Cx43都存在化疗旁观者效应,而C6-Cx43的化疗旁观者效应显著。即转染缝隙连接蛋白CX43能放大体外化疗的旁观者效应。BACKGROUND OBJECTIVE： A bystander effect is usually referred as the damage among cells that have not directly received chemotherapy or irradiation.In this study,we investigated the role of connexin 43（CX43） in the bystander effect among glioma cells treated with chemotherapy in vitro.METHODS： CX43 was introduced into C6 glioma cells with construction and transfection of plasmid pCMV-Cx43cDNA.RT-PCR and Western-blot were employed to detect the expression of CX43.Scratch assay was used to test gap junctional intercellular communication among C6 cells with or without CX43 over-expression.TRASWELL was used to separated cells treated with and without temozolomide.The bystander effect was compared by measuring the apoptosis among untreated C6 cells with and without over-expression of CX43.RESULTS： Over-expression of CX43 was observed after the successful transfection of pCMV-CX43cDNA and the gap junctional intercellular communication was enhanced.More significant bystander effect was observed in the co-culture of C6 cells with over-expression of CX43.CONCLUSON： The gap junctional intercellular communication is enhanced in C6 cells after over-expression of CX43.Over-expression of CX43 can magnify the bystander effect of chemotherapy in C6 cells in vitro.

关 键 词：胶质瘤 化疗 旁观者效应 缝隙连接蛋白CX43 质粒构建 转染 

分 类 号：R739.41[医药卫生—肿瘤]