【摘要】 目的:探讨Wnt5a调控大鼠胰岛β细胞瘤细胞Cyclin D1表达的通路机制。方法:体外培养大鼠胰岛β细胞瘤系细胞INS-1,以人/鼠重组Wnt5a蛋白处理细胞0.5、1、3、6、12 h,进行时间梯度实验;根据处理因素分为溶剂对照组、kn-62处理组、重组Wnt5a蛋白刺激组和kn-62+重组Wnt5a蛋白刺激组。蛋白质印迹法检测磷酸化CaMKⅡ(p-CaMKⅡ)、全细胞β-catenin、Cyclin D1水平变化。结果:经Wnt5a刺激后,INS-1胞内CaMKⅡ磷酸化水平升高,全细胞β-catenin水平下降,Cyclin D1表达减少(P<0.05);kn-62可降低细胞内CaMKⅡ磷酸化水平,拮抗外源性Wnt5a对全细胞β-catenin和Cylcin D1的下调效应(P<0.05)。结论:Wnt5a可通过激活大鼠胰岛β细胞瘤细胞INS-1的CaMKⅡ通路,抑制依赖β-catenin的经典Wnt通路,最终下调细胞Cyclin D1表达。更多还原

【Abstract】 Objective: To investigate the mechanism underlying the pathway via which Wnt5a regulates the expression of Cyclin D1 in rat pancreatic tumor β-cell line. Methods: INS-1 cells,a cell line of rat pancreaticβ-cell tumor,were cultured in vitro and treated with human/mouse recombinant Wnt5a protein for 0.5,1,3,6 and 12 h,respectively,and then subjected to time-gradient experiments.According to treatments,the cells were divided into the solvent control group,kn-62 group,recombinant Wnt5a stimulation group and kn-62 plus recombinant Wnt5a stimulation group. The levels of phosphorylated CaMKⅡ( p-CaMKⅡ),whole cell β-catenin and Cyclin D1 were detected by Western blotting. Results: After Wnt5a stimulation,INS-1 cells showed increased level of intracellular p-CaMKⅡ,and decreased level of whole cell β-catenin and Cyclin D1 expression( P < 0. 05).Treatment with kn-62 decreased the phosphorylation of intracellular CaMKⅡ and antagonized the exogenous Wnt5a-induced down-regulation of whole-cell β-catenin and Cylcin D1( P < 0. 05). Conclusion: Wnt5a may activate the Ca MK II pathway and inhibit the β-catenin dependent canonical Wnt pathway,and finally downregulate the Cyclin D1 expression in INS-1 cells.更多还原