GSK-3β特异siRNA腺病毒对脐静脉内皮细胞增殖的影响  

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

作　　者：陈刚[1] 游婷婷[1] 乔玉芳[1] 沈小燕[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学院,350001

出　　处：《中华内分泌代谢杂志》2008年第6期654-657,共4页Chinese Journal of Endocrinology and Metabolism

基　　金：福建省自然科学基金（C0710002）

摘　　要：目的以糖原合成酶激酶3p（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-eatenin）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13-catenin蛋白表达的影响，以四甲基偶氮唑盐（MTr）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-3B蛋白的表达，增加β-catenin的蛋白表达。重组腺病毒感染3、5、7d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。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 MTY 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 adenovlrus, 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. Upregulating of the Wnt/β-catenin pathway might play an important role in the proliferation of HUVEC.

关 键 词：糖原合成酶激酶3 Β-连环蛋白 RNA干扰 人脐静脉内皮细胞 细胞增殖 

分 类 号：R54[医药卫生—心血管疾病]