RNAi对波动性葡萄糖诱导的人脐静脉内皮细胞糖原合成酶激酶-3β、β-联蛋白作用的研究

Effect of RNAi on GSK-3β in human umbilical vein endothelial cells induced by normal-high glucose

目的构建糖原合成酶激酶-3β(glycogen synthase kinase 3β,GSK-3β)特异的RNAi腺病毒表达载体,感染原代人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC),观察其对波动性浓度葡萄糖溶液诱导的HUVEC Wnt信号通路中GSK-3β、β-联蛋白(β-catenin)的影响。方法设计合成针对GSK-3βmRNA不同位点的短发荚RNA(short hairpin RNA,shRNA)编码序列,重组RNAi腺病毒表达载体。实验分为3组,空白对照组、阴性对照组、实验组。空白对照组使用波动性浓度葡萄糖溶液(5.5 mmol/L和30.5 mmol/L两种浓度的葡萄糖溶液每日交替培养,模拟血液中葡萄糖浓度的波动)培养HUVEC;阴性对照组使用波动性浓度葡萄糖溶液培养HUVEC,并使用无携带GSK-3β基因的腺病毒感染培养的HUVEC;实验组使用波动性浓度葡萄糖溶液培养HUVEC,并GSK-3β特异的RNAi腺病毒感染培养的HUVEC。Western印迹法波动性浓度葡萄糖溶液干预前及干预后72小时3组的GSK-3β、β-联蛋白水平。结果波动性浓度葡萄糖溶液干预72小时后,实验组的GSK-3β表达水平(0.1068±0.0034)较空白对照组、阴性对照组低,差异有统计学意义(F=10886.388,P=0.000);实验组的β-联蛋白水平表达水平(0.6653±0.0167)较空白对照组、阴性对照组低,差异有统计学意义(F=315.96,P=0.000)。结论构建的RNAi腺病毒感染HUVEC后可以抑制高糖诱发的GSK-3β蛋白表达、增加细胞内β-联蛋白的蛋白水平。

Objective To observe the effect of GSK-3β-specific RNAi adenovirus expression vector on GSK-3β and β-catenin in the Wnt signaling pathway activated by fluctuation in glucose concentration in HUVEC by constructing glycogen synthase kinase 3β（GSK-3β）-specific RNAi adenovirus expression vectors and infected human umbilical vein endothelial cells （HUVEC） with these vectors. Methods We synthesized short hairpin RNA （shRNAs） targeting different sequences of GSK-3βmRNA and constructed corresponding RNAi adenovirus expression vectors. HUVEC was cultured with daily alternation of 5.5mmol/L and 30.5 mmol/L glucose solutions and fluctuations were simulated in plasma glucose concentration. HUVEC was then infected with GSK-3β-specific RNAi adenovirus expression vectors. Expression levels of GSK-3βandβ-catenin protein were determined using western blot. Results Expression levels of both GSK-3βand β-catenin protein were reduced after infection of GSK-3β-specific RNAi adenovirus expression vectors for 72 hours. Conclusion Infection of HUVEC with our RNAi adenovirus can inhibit high glucose induced-expression of GSK-3βand β-catenin protein.