应用RNA干扰技术研究游离脂肪酸对人脐静脉内皮细胞凋亡的影响  

Effects of free fatty acids on apoptosis of human umbilical vein endothelial cell by RNA interference

作　　者：游婷婷[1] 陈刚[1] 林旭[1] 乔玉芳[1] 林丽香[1] 

机构地区：[1]福建省立医院内分泌科,福州,350001

出　　处：《中华糖尿病杂志》2010年第1期-,共5页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：福建省自然科学基金

摘　　要：目的 应用RNA干扰(RNAi)技术探讨游离脂肪酸(FFA)对人脐静脉内皮细胞(HUVEC)凋亡的影响.方法 利用体外同源重组技术构建糖原合成酶激酶-3β(GSK-3β)特异的siRNA腺病毒表达载体,在人胚胎肾293A细胞中包装并扩增病毒,应用空斑实验法测定病毒滴度.选用GsK-3β特异的RNAi腺病毒感染HUVEC,采用Western blot方法检测其对GSK-3β和β-catenin 蛋白水平的影响.FFA贮存液按油酸盐:棕榈酸盐2:1配制,RNAi腺病毒和FFA共同干预HUVEC,采用流式细胞术、Hoechst 33258染色检测细胞凋亡.实验分为0.75 mmol/L FFA组、GSK-3β特异性RNAi腺病毒(Ad-640)+FFA组、未重组RNAi腺病毒(Ad-DEST)+FFA组、正常细胞组.采用方差分析进行数据比较.结果 构建的RNAi腺病毒感染HUVEC可以显著抑制GSK-3β蛋白的表达,上调细胞内β-catenin蛋白水平.0.75 mmol/L FFA具有促进细胞凋亡率的作用,荧光染色可见细胞出现典型的凋亡形态学改变;Ad-640+0.75 mmol/L FFA组与0.75 mmol/L FFA组(F=26.42,P<0.01)及Ad-DEST+0.75 mmol/L FFA组(F=23.34,P<0.01)相比细胞凋亡率显著减少,凋亡形态学改变亦明显减少,而Ad-DEST+FFA组与FFA组细胞凋亡率无显著差异(F=5.56,P>0.05),凋亡形态学改变相似.结论 构建的CSK-3β特异性RNAi腺病毒对FFA作用下的HUVEC具有部分保护作用.Objective To study the role of Wnt/β-catenin signal pathway in high free fatty acid (FFA)-induced apoptosis in human umbilical vein endothelial cell ( HUVEC) by RNA interference ( RNAi). Methods A recombinant adenovirus containing an RNAi cassette targeting the GSK-3p gene was produced and its silencing effect on GSK-3β gene was detected by Western blot analysis in HUVECs. The effect of the RNAi on the protein level of β-catenin was explored by transfecting the RNAi adenovirus to inhibit the expression of GSK-3β proteia The subsequent effect on the Wnt/GSK-3β/β-catenin signal pathway and on apoptosis of HUVECs cultured with FFAs was analyzed by the flow cytometer and Hoechst 33258 in order to explore the possible connection between the signaling pathway and FFA-induced apoptosis. The experimental cells were divided into four groups as followed; 0.75 mmol/L FFA group; RNAi adenovirus ( Ad-640) + FFA group; RNAi adenovirus without recombination ( Ad-DEST) + FFA group; normal cell group. Results The Western blot results showed that the expression of GSK-3β protein in HUVECs was inhibited by the RNAi adenovirus and the protein level of β-catenin was increased by RNAi adenovirus transfection. Apoptosis was observed in HUVECs exposed to 0.75 mmol/L FFAs for 72 h. The apoptotic rate of the Ad-640 + FFA group was significantly reduced with respect to that of the Ad-DEST + FFA and FFA groups whereas there was no significant difference between the Ad-DEST + FFA and FFA groups. Conclusion RNAi adenovirus specific to GSK-3β may partly protect HUVECs from apoptosis induced by FFAs.

关 键 词：RNA干扰 人脐静脉内皮细胞 游离脂肪酸 细胞凋亡 WNT信号通路 

分 类 号：R[医药卫生]