JSRV-Env诱导转染TC-1、TC-1-Hyal2细胞后对AKT和ERK表达的影响  

Changes in the Expression of AKT and ERK after the JSRV-Env-Induced Transfection of TC-1 and TC-1-Hyal2 Cells

作　　者：么宏强[1,2] 孙丽红[1,2] 骆爽[1,2] 纪秀红[1,2] 

机构地区：[1]内蒙古农业大学兽医学院,呼和浩特010018 [2]农业部动物疾病临床诊疗技术重点实验室,呼和浩特010018

出　　处：《病毒学报》2016年第3期283-291,共9页Chinese Journal of Virology

基　　金：国家自然科学基金(项目批准号:31260593);项目名称:JSRV囊膜假病毒诱导肺上皮细胞癌变的信号转导机制研究

摘　　要：为了探索JSRV与其受体相互作用后靶细胞的致瘤机制,本研究应用JSRV Env真核表达质粒分别诱导转染小鼠肺上皮细胞(TC-1)和稳定表达绵羊Hyal-2的小鼠肺上皮细胞(TC-1-Hyal2),而后检测细胞信号转导通路上AKT(丝氨酸/苏氨酸激酶)和ERK(细胞外信号调节激酶)在mRNA及蛋白水平上的表达变化,并分析绵羊Hyal-2在JSRV Env诱导TC-1细胞转化过程中的作用。首先体外培养TC-1和TC-1-Hyal2两种细胞,并分别设立pEGFP-C1-env转染组、pEGFP-C1转染组及未转染质粒组。通过实时荧光定量PCR和Western blot方法检测两关键酶在不同水平上的表达变化。qPCR结果显示:与未转染质粒细胞组比较,转染pEGFP-C1-env的两细胞组中AKT和ERK1/2mRNA表达均显著升高(P<0.05)。Western blot结果显示:与未转染质粒细胞组比较,转染pEGFP-C1-env两细胞组中p-Akt(S473)蛋白表达量显著升高(P<0.05);且p-Akt(T308)和p-Erk1/2蛋白在转染pEGFP-C1-env的TC-1细胞组中表达量也均呈显著升高变化趋势(P<0.05),而这两种蛋白在相同处理的TC-1-Hyal2细胞组中表达量均呈极显著升高(P<0.01)。此外,转染pEGFP-C1空载体的各细胞组与未转染质粒细胞组相比较AKT和ERK mRNA和蛋白含量差异均不显著(P>0.05)。JSRV Env在诱导上述细胞系转化过程中,Ras-Raf-MAPK和PI3K-Akt两条细胞信号转导通路被激活;实验检测得知,转染pEGFP-C1-env的两细胞组中AKT、ERK表达量均升高;相比之下,在TC-1-Hyal2细胞组中其表达量升高更显著。研究结果推测绵羊Hyal-2在JSRV Env诱导TC-1细胞转化过程中起促进作用。This study aims to explore the tumorigenic mechanism of the target cells following JSRV interaction with its receptor.We transfected mouse lung epithelial cells（TC-1）and mouse lung epithelial cells stably expressing sheep Hyal-2（TC-1-Hyal2）with JSRV-Env eukaryotic expression vector,measured the changes in the mRNA and protein expression of AKT（serine/threonine kinase）and ERK（extracellular signal-regulated kinase）in cellular signal transduction pathways,and analyzed the role of sheep Hyal-2in JSRV-Env-induced transformation of TC-1cells.First,TC-1and TC-1-Hyal2 cells were cultured in vitro and were each divided into pEGFP-C1-env transfection group,pEGFP-C1 transfection group,and untransfected group.The expression of key enzymes was determined by PCR and Western blotting.qPCR showed that,for both cell lines,compared with untransfected cells,the expression of AKT and ERK1/2mRNA was significantly increased in the pEGFP-C1-env transfected cells（P〈0.05）.Western blotting showed that,relative to untransfected cells,transfection with pEGFP-C1-env significantly increased p-Akt（S473）protein expression in both cell lines（P〈0.05）.Moreover,p-Akt（T308）and p-Erk1/2protein expression was increased significantly in the pEGFP-C1-env transfected TC-1cells（P〈0.05）,and very significantly in the pEGFP-C1-env transfected TC-1-Hyal2cells（P〈0.01）.Cells of each type transfected with the empty vector pEGFP-C1 and the untransfected cells did not show significant differences in their mRNA and protein levels of AKT and ERK（P 〉0.05）.Thus,the expression of JSRV-Env in the cell lines TC-1and TC-1-Hyal2 activated the cellular signal transduction pathways Ras-Raf-MAPK and PI3K-Akt.The expression of AKT and ERK was significantly increased in pEGFP-C1-env transfected TC-1and TC-1-Hyal2 cells,but a greater increase was seen in the TC-1-Hyal2 cells.We speculate that Hyal2 plays a catalytic role in JSRV-Env-induced transformation of TC-1cells.

关 键 词：JSRV-Env真核表达质粒 TC-1细胞 TC-1-Hyal2细胞 AKT ERK 

分 类 号：S852.65[农业科学—基础兽医学]