用RNA干扰慢病毒研究Wnt/β-连环蛋白信号通路对小鼠胰岛β细胞增殖的影响  

Construction of β-catenin-targeting RNAi lentivirus and effect of Wnt/β-catenin signaling on proliferation of mice pancreatic beta-cell

作　　者：邹欣[1] 陈刚[1] 沈小燕[1] 方晓文[1] 陈玉芳[1] 胡雅婷[1] 曾月桂[1] 林丽香[1] 

机构地区：[1]福建省立医院内分泌科福建医科大学省立临床医学院内分泌科,福州,350001

出　　处：《中华糖尿病杂志》2010年第2期-,共5页CHINESE JOURNAL OF DIABETES MELLITUS

摘　　要：目的 构建β-连环蛋白特异的RNA干扰(RNA interference,RNAi)慢病毒表达载体,初步探讨Wnt/β-连环蛋白信号通路对小鼠胰岛β细胞增殖的影响.方法 利用体外同源重组技术构建短发夹RNA(shRNA)慢病毒表达载体,与辅助包装质粒一起在293 T淋巴细胞中包装并扩增病毒,逐孔稀释法测定病毒滴度;RNAi慢病毒感染小鼠胰岛β细胞,实时PCR以及Western blot法检测其对β-连环蛋白表达的抑制效应;利用构建的RNAi慢病毒抑制β-连环蛋白表达,四甲基偶氮唑盐法检测小鼠胰岛β细胞增殖活性.组间比较采用方差分析(ANOVA).结果 成功构建了β-连环蛋白特异的RNAi慢病毒表达载体,获得的慢病毒滴度在5.0×10~8TU/ml左右;RNAi慢病毒在3 d后就能有效感染小鼠胰岛β细胞,5 d后使细胞内β-连环蛋白基因mRNA和相应蛋白的表达受到明显抑制;慢病毒感染小鼠胰岛β细胞后,细胞增殖率明显减少.结论 构建的目的RNAi慢病毒表达载体能有效抑制β-连环蛋白基因的表达;Wnt/β-连环蛋白信号通路在小鼠胰岛β细胞增殖中有重要影响.Objective To construct an expressive RNA interference RNAi lentivims specific to β-catenin, and to investigate the regulation of Wnt/β-catenin signaling on the proliferation aspect of mice pancreatic β cell line (Min6 cell). Methods Homologous recombination and cloning techniques were used to create the lentivirual plasmid which contains the RNAi cassette targeting the β-catenin gene. Then, the lentivirual plasmid was transfected with the other two packaging plasmids into 293T cells to product and amplify lentivirus. Dilution assay was used to titer the lentivirual stock. The β-catenin gene silencing effect induced by the RNAi lentivirus in Min6 cell was detected by real time-PCR and Western blot analysis. Finally, we transducted the RNAi lentivims to inhibit the expression of β-catenin protein and to explore the effect of Wnt/β-catenin pathway on proliferation of mice pancreatic β cell, with MTT cell proliferation assay. Results The RNAi lentivirus specific to β-catenin was produced with a titer of about 5.0×10~8 TU/ml. The RNAi lentivirus could be infect Min6 cells efficiently in 3 days, and the inhibition effect was detected in 5 days after infection. The results of MTr assay suggested that inhibiting the β-catenin with the RNAi lentivirus could suppress the proliferation of Min6 cells obviously. Conclusion RNAi lentivirus is an important tool to inhibit the expression of target gene efficiently. The Wnt/β-catenin pathway plays an important role in the regulation of proliferation of mice pancreatic β cells.

关 键 词：RNA干扰 WNT信号 Β-连环蛋白 胰岛Β细胞 增殖 

分 类 号：R587[医药卫生—内分泌]