sirt1rnai慢病毒载体的构建及鉴定.docVIP

SIRT1对高糖诱导的系膜细胞NF-κB p65乙酰化及MCP-1表达的影响 杜月光1*，柴可夫2，汪丽佩1 浙江中医药大学病理生理学教研室，杭州310053浙江中医药大学中医临床基础研究所，杭州310053省自然基金（Y2110849）Supported by the Natural Science Foundation of Zhejiang Province（Y2110849） [作者简介] 杜月光，博士，教授*通作者Tel: 0571 E-mail: duyueguang@163.com 【摘要】目的SIRT1）对高糖诱导的大鼠肾小球系膜细胞（RMC） p65蛋白乙酰化表达的影响。方法构建干扰SIRT1基因的shRNA慢病毒质粒pTRC-shSIRT1并pTRC-shSIRT1感染4 h后，换用低糖培养液培养）、SIRT1 RNAi+高糖组（添加干扰病毒pTRC-shSIRT1感染4 h后，换用高糖培养液培养），同时设正常对照组和甘露醇高渗对照组。以实时荧光定量PCR检测SIRT1、MCP-1的mRNA，检测SIRT1和NF-κB p65乙酰化蛋白表达，ELISA技术检测MCP-1蛋白含量。结果质粒测序证实干扰SIRT1基因的shRNA慢病毒载体构建成功，且能抑制RMC中SIRT1基因表达。高糖刺激使SIRT1基因表达降低，NF-κB p65蛋白乙酰化增强，MCP-1 mRNA和蛋白水平增高可逆转高糖引起的变化而沉默SIRT1高糖诱导的NF-κB p56乙酰化MCP-1 mRNA和蛋白。结论高糖诱导的RMC MCP-1 mRNA和蛋白的表达，其机制可能与NF-κB P56乙酰化有关。 【关键词】RNA干扰；NF-κB；糖尿病肾病 ffect of SIRT1 on acetylation of NF-κB p65 subunit and the expression of MCP-1 in rat mesangial cells induced by high glucose DU Yue-guang1*, CHAI Ke-fu2;WANG Li-pei1 Department of Pathophysiology，Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, China National TCM Clinical Research Center, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, China 【Abstract】 Objective To explore the effect of silent information regulator 1SIRT1） on acetylation of nuclear factor-κB (NF-κB) p65 subunit and the expression of monocyte chemoattractant protein 1 (MCP-1) in rat mesangial cells (RMC) induced by high glucose. Methods Construct the lentiviral shRNA plasmid pTRC-shSIRT1 to interference SIRT1 gene and identification . The RMCs was classified into high glucose group (high glucose culture medium used), resveratrol + high glucose group (with low glucose culture medium containing 1μmol / L SIRT1 activator resveratrol for 24 h and then switching to high glucose culture medium), SIRT1 RNAi group (4h after viral pTRC-shSIRT1 infection, and then switch to low-glucose culture medium), SIRT1 RNAi + high glucose group (4h after viral pTRC-shSIRT1 infection, and then switch to high glucose culture medium) , While negative control grou