occludin的RNA干扰慢病毒载体的构建及其在紧密连接中的功能研究

Construction of RNA interfering lentiviral vector of occludin and its role in tight junctions

目的:体外分析occludin在紧密连接中的作用。方法:构建RNA干扰慢病毒载体,转染TM4细胞后,用RT-PCR和Western blot检测其对occuldin的抑制能力,用体外TJ细胞模型分析occuldin在TJ中功能。结果:测序结果表明pLenti 6.3-EGFP-occludin-miR表达载体已经成功构建;RT-PCR结果显示pLenti 6.3-EGFP-occludin-miR-3能够显著抑制occludin的表达(P<0.05);Western印检测分析发现pLenti 6.3-EGFP-occludin-miR-3转染组occuldin的表达值为0.7534±0.089,经ANOVA比较,较空白对照组和pLenti 6.3-EGFP转染组(1.056±0.025)都低,差异显著(P<0.05)。体外细胞TJ模型结果表明pLenti 6.3-EGFP-occludin-miR-3转染组中,TM4表达的occludin的表达受到抑制,紧密连接的紧密程度下降。结论:pLenti 6.3-EGFP-occludin-miR表达载体已经成功构建;occludin是维持紧密连接的功能蛋白之一。

Objective:To investigate the role of occludin in tight junction(TJ)in vitro.Methods:We constructed RNA interfering lentiviral vectors and transfected them into TM4 cells.Then we detected their inhibitory effect on occuldin by RT-PCR and Western blot and analyzed the role of occuldin in TJ using an in vitro TJ cell model.Results:The pLenti 6.3-EGFP-occludin-miR expression vector was successfully constructed.The results of RT-PCR and Western blot showed that pLenti 6.3-EGFP-occludin-miR-3 significantly inhibited the expression of occludin(P<0.05),which was remarkably lower than in the blank control and the pLenti 6.3-EGFP transfection group(0.7534±0.089 vs 1.000 and 1.056±0.025,P<0.05).The expression of occludin was markedly suppressed and the tightness of tight junctions decreased in the TM4 cells transfected with pLenti 6.3-EGFP-occludin-miR-3.Conclusion:The pLenti 6.3-EGFP-occludin-miR expression vector was successfully constructed,and occludin is one of the functional proteins that maintain tight junctions.