【作者】 杨惠； 刘淑英；

【Author】 YANG Hui;LIU Shu-ying;College of Veterinary Medicine, Inner Mongolia Agricultural University/Inner Mongolia Key Laboratory of Basic Veterinary Medicine;

【机构】 内蒙古农业大学兽医学院内蒙古自治区基础兽医学重点实验室；

【摘要】 利用慢病毒载体过表达ex JSRV-env致癌基因,探究ex JSRV-env对绵羊绒毛膜滋养层细胞自噬水平的影响,帮助研究者更好的认识ex JSRV-env致癌基因介导肿瘤发生的分子机制。利用慢病毒p LVX-CMV-ex JSRV-env-EF1a-m Cherry感染绵羊绒毛膜滋养层细胞(Sheeptrophoblastcells,STCs),从感染成功的STCs中抽提总RNA反转录c DNA,并提取细胞总蛋白。通过Eppendorf Realplex荧光定量PCR仪,检测细胞ex JSRV-env基因表达,并检测过表达ex JSRV-env对自噬基因(Microtubule associated protein 1 light chain 3 beta,MAPILCB3)、(Beclin-1,BECN1)、(Sequestosome 1/p62,SQSTM1)表达的影响,利用western blot检测相应自噬蛋白的表达变化,结合Graph Pad Prism 6.0软件分析阳性对照组(空载体质粒组,BLACK)、阴性对照组(STCs,NC)各自噬基因及蛋白表达量间的差异。试验结果表明:荧光场验证5 MOI的p LVX-CMV-ex JSRV-env-EF1a-m Cherry慢病毒载体于72h可以成功转染STCs,RT-q PCR检测结果表明ex JSRV-env基因过表达能导致MAPILCB3、BECN1基因下调;SQSTM1基因表达上调;相应的LC3II、beclin1蛋白减少,P62蛋白增加。从分子学角度考虑,ex JSRV-env基因过表达时,激活Akt/m TOR和MAPK信号通路促使自噬基因MAPILCB3及LC3B蛋白、BECN1及Beclin1蛋白表达受到抑制,与之呈负相关的SQSTM1及P62蛋白表达升高;从细胞学角度考虑,一方面,env基因过表达激活Akt/m TOR和MAPK信号途径促使细胞大量增殖,需要消耗大量能量,但由于细胞自噬水平降低,无法进一步为细胞供能,细胞自身能量平衡被打破;另一方面,env过表达降低细胞自噬水平,细胞通自噬达到抗病毒感染的目的被破坏,细胞稳态被打破却无法立即进入死亡状态,进而促使细胞大量转化。更多还原

【Abstract】 The expression of ex JSRV-env oncogene was overexpressed by lentiviral vector, and the effect of ex JSRV-env on the level of autophagy in sheep chorionic trophoblast cells was explored to help researchers better understand the molecular mechanism of ex JSRV-env oncogene-mediated tumorigenesis. The lentiviral p LVX-CMV-ex JSRV-env-EF1 a-m Cherry was used to infect sheep trophoblast cells(STCs) to extract total RNA reverse transcribed c DNA from infected STCs and extract total cellular proteins. The expression of ex JSRV-env gene was detected by Eppendorf Realplex real-time PCR instrument, and the expression of ex JSRV-env gene on microtubule associated protein 1 light chain 3 beta(MAPILCB3),(Beclin-1, BECN1), The effect of Sequestosome 1/p62, SQSTM1) expression was detected by western blot. The expression of the corresponding autophagy protein was analyzed by Graph Pad Prism 6.0 software. The positive control group(empty vector plasmid group, BLACK) and the negative control group(STCs, NC) were analyzed. Differences in phagocytic and protein expression levels. The results showed that the p LVX-CMV-ex JSRV-env-EF1 a-m Cherry lentiviral vector of 5 MOI successfully infected STCs at 72 h. RT-q PCR analysis showed that overexpression of ex JSRV-env gene could down-regulate MAPILCB3 and BECN1 genes. SQSTM1 gene expression was up-regulated; the corresponding LC3 II, beclin1 protein decreased, and P62 protein increased. From the molecular point of view, when the ex JSRV-env gene is overexpressed, activation of Akt/m TOR and MAPK signaling pathways promotes the expression of autophagy genes MAPILCB3 and BECN1, and the expression of SQSTM1 is negatively correlated with it; from the cytological point of view, On the one hand, the overexpression of env gene activates the Akt/m TOR and MAPK signaling pathways, which promotes the proliferation of cells, which consumes a lot of energy. However, due to the decreased level of autophagy, it is unable to further supply cells, and the cell’s own energy balance is broken., env overexpression reduces the level of autophagy, the cell is autophagic to achieve the purpose of antiviral infection is destroyed, the cell homeostasis is broken but can not immediately enter the state of death, thereby promoting cell transformation.更多还原