摘要
目的探讨Ras-Raf-MEK-ERK通路在大鼠增殖抑制基因(r HSG)抑制C6大鼠胶质瘤细胞增殖中的作用。方法用r HSG过表达腺病毒载体(Adv-r HSG-GFP)转染C6细胞后,流式细胞仪分析腺病毒转染效率;Western blot检测r HSG蛋白表达变化;流式细胞仪分析细胞周期;qRT-PCR检测H-ras、N-ras、K-ras、Erk1、Erk2基因mRNA表达变化;Western blot检测H-ras、N-ras、K-ras、p-Erk1/2、Erk1/2、p-Rb、Rb蛋白表达变化。结果腺病毒载体能高效的转染C6细胞并表达GFP蛋白,Adv-r HSG-GFP组r HSG蛋白表达显著高于未转染组(PBS)和AdvGFP组,C6细胞被阻滞于G0/G1期(P<0.01);经IGF-1刺激后,Adv-r HSG-GFP组细胞H-ras、N-ras、K-ras、Erk1、Erk2基因的mRNA和蛋白表达水平与PBS组和Adv-GFP组无明显差异(P>0.05);Adv-r HSG-GFP组细胞过表达r HSG能显著抑制IGF-1诱导的Erk1/2的活化,p-Erk1/2蛋白表达量显著低于其余两组(P<0.01);同时其p-Rb蛋白的表达量显著低于其余两组(P<0.01),而Rb蛋白表达量3组无明显差异(P>0.05)。结论 r HSG可能通过抑制Ras-Raf-MEK-ERK信号通路的激活抗C6恶性胶质瘤细胞增殖。
Objective To explore the effects of Ras-Raf-ERK / MAPK signal pathway in the inhibitive effect of rat hyperplasia suppressor gene（ r HSG） on C6 rat glioma cells. Methods C6 cells were cultured in vitro and infected by r HSG adenovirus over-expression vector（ Adv-r HSG-GFP）,then Western blot was used to determine the expression of r HSG protein; the effect of r HSG on cell cycle was analyzed by flow cytometer. The mRNA expressions of H-ras,N-ras,K-ras,Erk1,Erk2 were identified by qRT-PCR. Western blot was used to determine changes in the expression of H-ras,N-ras,Kras,Erk1 /2,p-Erk1 /2,p-Rb,Rb protein. Results C6 cells could be transfected by adenovirus vector efficiently and express GFP protein. It is observed that in C6 cells of group infected with Adv-r HSG-GFP,the protein expression level of r HSG was more than group of uninfected（ PBS） and group infected with Adv-GFP（ P〈 0. 01）. Tthe results from flow cytometer indicated that the cell cycle was arrested in G0 / G1 phase. After stimulated by IGF-1 in proper condition,there were no difference in the three groups for expressions of mRNA and protein level of H-ras,N-ras,K-ras,Erk1,Erk2（ P〉 0. 05）. The activation of Erk1 /2 induced IGF-1 was inhibited by r HSG protein on cells of Adv-r HSG-GFP group,p-Erk1 /2 protein expression was lower than the other two groups significantly（ P〈 0. 01）; meanwhile,p-Rb protein was the lowest in Adv-r HSG-GFP group（ P〈 0. 01）,there were no differences in three groups for the expression of Rb protein（ P〈 0. 05）.Conclusion Our results suggest that the inhibitive effect of r HSG on malignant glioma could be through inhibiting RasRaf-MEK-ERK signal pathway.
出处
《中风与神经疾病杂志》
CAS
北大核心
2015年第10期884-888,共5页
Journal of Apoplexy and Nervous Diseases
基金
宁夏自然科学基金(No.NZ13129)
