【摘要】 目的:研究细胞连接蛋白Lnk对人上皮性卵巢肿瘤细胞中胰岛素PI3K/Akt和MAPK/ERK信号通路的影响及作用机制。方法:稳定转染Lnk WT质粒(空质粒EV作为对照)的TOV112D和OVCA420细胞株,加入人重组胰岛素处理后收获细胞,提取蛋白。Western blot法检测磷酸化和总Akt、ERK和4EBP1信号通路蛋白。并在293T细胞中共转染IR和Lnk WT或EV质粒,收获细胞裂解蛋白。免疫共沉淀(CoIP)检测体外Lnk和胰岛素受体(IR)是否结合。结果:过度表达了Lnk WT的TOV112D和OVCA420细胞中,Akt及其下游信号蛋白4-EBP1的磷酸化蛋白(p-Akt和p-4EBP1)、p-ERK1/2蛋白均较空质粒(EV)对照明显降低;胰岛素刺激下,Lnk WT过表达细胞Akt、4EBP1、ERK1/2磷酸化水平也较EV明显降低;总蛋白则无明显变化。免疫共沉淀实验显示,Lnk与IR在体外可结合。结论:Lnk可通过与IR结合,负性调节对胰岛素反应的PI3K-Akt和MAPK-ERK细胞信号传导;其作为胰岛素信号通路的一个新抑制因子,可能在上皮性卵巢肿瘤及其他胰岛素信号通路异常相关性疾病中起作用。更多还原

【Abstract】 Objective:Effects of Lnk on insulin response PI3 K-Akt and MAPK-ERK1/2 signaling and the possible mechanism were studied in the ovarian cancer cells.Methods:Vectors of Lnk WT and EV were stably transfected in TOV112 D and OVCA420 of epithelial ovarian cancer cells.Incubated with human recombinant insulin and then collected and digested for protein abstraction.Total and phosphorylated proteins of Akt,ERK and 4 EBP1 were detected by Western blot.EV and insulin receptor(IR) were co-transfected into 293 T cell to study whether Lnk could bind to IR,detect by CoIP.Results:In the TOV112 D and OVCA420 cells stably transfected with Lnk WT vectors,phosphorylated proteins of Akt,4 EBP1 and ERK were significantly decreased,compared to the cells stably transfected with EV vectors.Besides,the phosphorylated proteins of Akt,4 EBP1 and ERK in response to insulin show significantly decreased too,in the cells over-expressed with Lnk.Furthermore,Co-IP indicated that Lnk binds to IR.Conclusion:Lnk could inhibit PI3 K-Akt and ERK 1/2 signaling in ovarian cancer cells,and negatively regulate insulin induced PI3 K-Akt and MAPK-ERK signaling via the binding to insulin receptor,which might contribute the genesis of epithelial ovarian cancer and pathology of related diseases of abnormal insulin signaling.更多还原