大鼠增殖抑制基因过表达对C6胶质瘤细胞侵袭的抑制作用及其机制探讨  

Inhibitory effect of rat hyperplasia suppressor gene overexpression on invasion of C6glioma cells and its mechanism

作　　者：王军成[1,2] 邹有瑞[3] 吴桥[1] 霍国进 蒋树财[5] 高鹏[1] 夏明[6] 沈冰[3] 

机构地区：[1]宁夏医科大学临床医学院,宁夏回族自治区银川750004 [2]宁夏医科大学宁夏颅脑疾病重点实验室,宁夏回族自治区银川750004 [3]宁夏医科大学总医院神经外科,宁夏回族自治区银川750004 [4]榆林市第一医院神经外科,陕西榆林719000 [5]滨州市人民医院神经外科,山东滨州256600 [6]徐州市肿瘤医院神经外科,江苏徐州221005

出　　处：《肿瘤》2017年第2期126-134,162,共10页Tumor

摘　　要：目的 :研究大鼠增殖抑制基因(rat hyperplasia suppressor gene,r HSG)过表达对胶质瘤C6细胞侵袭的影响,并探讨其可能的作用机制。方法 :用携带r HSG基因的重组腺病毒Adv-r HSG-GFP感染胶质瘤C6细胞,同时设置Adv-GFP感染的阴性对照组以及仅PBS处理的空白对照组。采用实时荧光定量PCR和蛋白质印迹法检测各组细胞中r HSG的表达变化。然后,采用细胞划痕愈合实验和Transwell迁移及侵袭实验分别检测r HSG过表达对胶质瘤C6细胞迁移和侵袭的影响,细胞免疫荧光法和细胞黏附实验分别分析r HSG过表达后C6细胞骨架形成和黏附能力的变化,实时荧光定量PCR和蛋白质印迹法测定各组细胞中Rho/Rock信号通路关键分子Ras同源基因家族成员A(Ras homolog gene family,member A,RhoA)、Ras相关的C3肉毒底物1(Ras-related C3 botulinum toxin substrate 1,Rac1)、细胞分裂周期蛋白42(cell division cycle 42,Cdc42)、Rho相关卷曲螺旋形成蛋白激酶1(Rho associated coiled-coil forming protein kinase 1,Rock1)和Rock2的表达水平。结果 :重组腺病毒Adv-r HSG-GFP感染48和72 h后,胶质瘤C6细胞中r HSG的表达水平明显升高(P值均<0.01)。与2个对照组相比,Advr HSG-GFP组细胞的划痕愈合率明显降低(P值均<0.01),迁移、侵袭和黏附细胞数均明显减少(P值均<0.05)。r HSG过表达后C6细胞形态发生变化,丝状伪足缩短,部分呈片状;而且细胞中Rho A、Rac1、Cdc42、Rock1和Rock2的m RNA及蛋白表达水平均明显降低(P值均<0.05)。结论 :r HSG过表达可以抑制胶质瘤C6细胞的侵袭,其作用机制可能与阻滞Rho/Rock信号通路有关。Objective： To study the effect of overexpression of rat hyperplasia suppressor gene （rHSG） on invasion of glioma C6 cells, and to investigate its potential mechanism. Methods： The recombinant adenovirus vector Adv-rHSG-GFP carrying rHrHSGSG gene sequence was infected into rat glioma C6 cells. At the same time, C6 cells treated by PBS or infected with Adv-GFP were used as the control groups. The expressions of rHSG mRNA and protein in different groups were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Then, the effects of rHSG overexpression on the migration and invasion of glioma C6 cells were analyzed by scratch wound healing assay, Transwell migration and invasion assay, respectively. The changes of cytoskeleton formation and adhesion ability of C6 cells after rHSG overexpression were analyzed by immunofluorescence and cell adhesion assay, respectively. The expressions of Ras homolog gene family, member A （RhoA）, Ras-related C3 botulinum toxin substrate 1 （Rac1）, cell division cycle 42 （Cdc42）, Rho associated coiled-coil forming protein kinase 1 （Rock1） and Rock2 in Rho/Rock signaling pathway in each group were detected by real-time fluorescent quantitative PCR and Western blotting. Results： The expression of rHSG was significantly up-regulated in C6 cells infected with recombinant adenovirus Adv-rHSG-GFP for 48 and 72 h （both P〈0.01）. Compared with the two control groups, the healing rate of C6 cells was significantly lowered in Adv-rHSG-GFP infected group （both P〈0.01）, while the counts of migratory, invasive and adhesive cells were significantly decreased in Adv-rHSG-GFP infected group （all P〈0.05）. The morphology of C6 cells changed after rHSG overexpresion, showing filopodia shortening or some lamellipodia. The mRNA and protein expressions of RhoA, Rac1, Cdc42, Rock1 and Rock2 were significantly decreased （all P〈0.05） in C6 cells with rHSG overexpression. Conclusion： The overexpression of rHSG can inhibit the in

关 键 词：神经胶质瘤 基因表达调控 肿瘤 细胞运动 RHO相关激酶类 增殖抑制基因 

分 类 号：R730.264[医药卫生—肿瘤]