NF—κB信号通路对高糖诱导的人脐静脉内皮细胞凋亡的作用  

Effect of NF-κB pathway on apoptosis of human umbilical vein endothelial cells induced by high glucose

作　　者：陈刚[1] 沈小燕[1] 林旭[2] 游婷婷[1] 乔玉芳[1] 姚瑾[1] 林苗[1] 朱香清[1] 牟伦盼[1] 方晓文[1] 邹欣[1] 林丽香[1] 

机构地区：[1]福建医科大学省立临床学院福建省立医院内分泌科,福州350001 [2]福建医科大学分子医学中心

出　　处：《中华肾脏病杂志》2009年第4期299-304,共6页Chinese Journal of Nephrology

基　　金：卫生部科学研究基金（WKJ2005-2-021）

摘　　要：目的研究高糖尤其是波动性高糖对人脐静脉内皮细胞（HUVEC）凋亡的影响，以及NF-κB信号通路在高糖诱导HUVEC凋亡中的作用。方法构建针对NF-κBp65 mRNA序列1566位点的重组RNAi腺病毒表达载体，并利用RNAi腺病毒抑制HUVEC p65表达。应用流式细胞术及脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）研究NF-κB信号通路在高糖诱导HUVEC凋亡的作用。结果高糖（20．5mmol／L或30．5mmol／L）可以促进HUVEC凋亡。NF-κBp65特异腺病毒感染HUVEC后，可以明显抑制高糖刺激的NF-κBp65核蛋白转录，使核内p65蛋白表达处于基础水平。TUNEL结果示高糖作用后5d，高糖组细胞凋亡率显著高于正常葡萄糖组（25．81％±1．77％比8．20％±0．63％，P〈0．05），Ad—DEST＋高糖组（26．10％±0．98％）与单独高糖组的细胞凋亡率差异无统计学意义（P〉0．05）。Ad-1566＋高糖组的细胞凋亡率（11．49％±0．92％）比Ad—DEST＋高糖组显著下降（P〈0．01）。TUNEL法及流式细胞术检测结果均显示NF-κBp65特异腺病毒可以降低高糖诱导的HUVEC凋亡。结论高糖可以促进HUVEC凋亡。腺病毒感染HUVEC可以明显抑制高糖刺激的NF-κBp65核转录，从而保护高糖作用的HUVEC凋亡。Objective To verify whether the periodic or continuous exposure to high glucose may have different effects on human umbilical vein endothelial cell （HUVEC）apoptosis, and to explore the effect of NF-κB pathway on apoptosis of HUVEC induced by high glucose using the RNAi adenovirus vector. Methods RNAi combinant adenovirus vector which targeted 1566 site of NF-κB p65 mRNA was constructed and the effect of p65 gene knockdown in HUVEC was detected by Western blot analysis. Then, the RNAi adenovirus was transdueted to explore the role of NF-κB pathway on the regulation of apoptosis in HUVEC induced by high glucose. The apoptosis of HUVEC was tested by flow cytometry and TUNEL assay. Results High glucose could induce apoptosis of HUVEC. p65 protein expression of nuclear extracts was significantly increased in high glucose culture as compared to control group, but only slightly increased in NF-κB-specific knockdown group, which maintained at basal state. Compared with normal glucose group, the number of TUNEL-positive cells in high glucose group was significantly increased （25.81%±1.77% vs 8.20%±0.63%, P〈0.05）. The number of TUNEL-positive cells was decreased in 30.5 mmol/L glucose plus Ad-1566 than that in 30.5 mmol/L glucose plus Ad-DEST （11.49%±0.92% vs 26.10%±0.98%, P〈0.01）. Flow cytometry and TUNEL assay showed that the apoptosis of human umbilical vein endothelial cells induced by high glucose was inhibited by the RNAi adenovirus. Conclusion High glucose induces apoptosis of HUVEC. Knockdown of NF-κB p65 may protect HUVEC from apoptosis by preventing high glucose-induced NF-κB nuclear translocation.

关 键 词：RNA干扰 NF-κB 细胞凋亡 高糖 人脐静脉内皮细胞 

分 类 号：R587.2[医药卫生—内分泌] R692[医药卫生—内科学]