GSK-3β特异siRNA腺病毒对脐静脉内皮细胞增殖的影响

目的 以糖原合成酶激酶3B(GSK-3β)特异的RNA干扰(RNAi)腺病毒表达载体作为研究工具,初步探讨Wnt/β-连环蛋白(Wnt/β-catenin)信号通路对人脐静脉内皮细胞(HUVEC)增殖的影响.方法 利用体外同源重组技术构建GSK-3β特异的siRNA腺病毒表达载体,在HEK293A细胞中包装并扩增病毒、空斑实验法测定病毒滴度.GSK-3β特异的RNAi腺病毒感染HUVEC,Western印迹和免疫细胞化学法检测其对GSK-3β和β-catenin蛋白表达的影响,以四甲基偶氮唑盐(MTT)法检测细胞增殖.结果 成功构建了GSK-3β特异的siRNA腺病毒表达载体,获得高滴度的腺病毒液.构建的重组腺病毒感染HUVEC显著抑制GSK-3β蛋白的表达,增加β-catenin的蛋白表达.重组腺病毒感染3、5、7 d后,细胞增殖率明显增加(P<0.05或P<0.01).结论 构建的GSK-3β特异的siRNA腺病毒能有效抑制GSK-3β基因的表达,促进β-catenin的蛋白表达,上调Wnt/β-catenin信号通路对人脐静脉内皮细胞增殖有重要的影响.

Effect of GSK-3βtargeting RNAi recombinant adenovirus on proliferation of human umbilical vein endothelial cells

Objective To observe the effect of Wnt/β-catenin pathway on proliferation of human umbilical vein endothelial cell (HUVEC) using the glycogen synthase kinase 3β (GSK-3β)-targeting RNAi recombinant adenovirus vector. Methods Homologous recombination and cloning techniques were used to construct RNAi recombinant adenoviral expressive vectors specific to GSK-3β. Then, the adenovirus plasmids was transfected into HEK 293A cells to produce adenovirus and amplify the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock. The GSK-3β and β-catenin protein expressions were detected by Western blot and immunohistochemistry. The proliferation of HUVEC was detected with MTr assay. Results The RNAi adenovirus vectors specific to GSK-3β were successfully produced with high titer. The expression of GSK-3β protein in HUVEC could be down-regulated efficiently by the RNAi adenovirus, along with increased β-catenin protein expression. The proliferation of HUVEC was significantly increased (P < 0.05 or P < 0.01) after infected with GSK-3β RNAi recombinant adenovirus for 3, 5, 7 days. Conclusion RNAi adenovirus is an important tool that can inhibit the expression of GSK-3β efficiently, along with increased β-catenin protein expression. Up-regulating of the Wnt/β-catenin pathway might play an important role in the proliferation of HUVEC.