蛋白激酶Cβ2通过调控PPARα介导高糖诱导的人内皮细胞损伤  

Protein kinase Cβ2 mediated high glucose-induced human umbilical vein endothelial cells injury via regulation of peroxisome proliferator-activated receptor α

作　　者：林雪波[1] 周波[1] 孙芳[1] 陈芳芳[1] 李启富[1] 邓华聪[1] 

机构地区：[1]重庆医科大学附属第一医院内分泌科,400016

出　　处：《中华内分泌代谢杂志》2010年第1期10-14,共5页Chinese Journal of Endocrinology and Metabolism

基　　金：基金项目：国家自然科学基金资助项目（30570877）

摘　　要：目的研究蛋白激酶C（PKC）β2、PPARα在高糖诱导人脐静脉内皮细胞（HUVECs）表达血管内皮生长因子（VEGF）、血管细胞黏附分子1（VCAM-1）mRNA中的作用及相互关系。方法将培养的HUVECs分为以下8组：正常糖（NG，5mmol／L D-葡萄糖）组、高糖（HG，25mmol／L D-葡萄糖）组、渗透压对照（L，NG＋20mmol／L L-葡萄糖）组、正常糖空载体转染（NN，NG＋Ad5-null）组、高糖PKCβ2转染（HB，HG＋AdS—PKCβ2）组、高糖＋非诺贝特（HF，HG＋40umol／L非诺贝特）组、高糖PKCβ2转染＋非诺贝特（HBF，HB＋40umol／L非诺贝特）组，另以非诺贝特共孵育20min作为HF20组，以上各组细胞均培养6d。以RT-PCR检测VEGF、VCAM-1mRNA的表达水平，用Western印迹法测定PPARα蛋白表达，采用激光共聚焦检测PKCβ2蛋白的表达和转位。结果（1）HG组VEGF、VCAM—1mRNA表达增加，分别为NG组的1．91倍和1．56倍（均P〈0．05）；HB组VEGF、VCAM-1mRNA表达较HG组进一步增加，分别为NG组的2．59倍和2．07倍（均P〈0．05）。HF组VEGF、VCAM-1mRNA表达则明显下调，分别为HG组的68％和74％（均P〈0．05）；与HG组相比，HF20组VEGF、VCAM-1mRNA表达差异无统计学意义。（2）HG组PPARα蛋白表达较NG组减少了20％，HB组PPARα蛋白水平进一步下降，为HG组的78％，与HG组相比，HF组PPARα蛋白水平上调了13％（P〈0．05）。（3）HG诱导PKCβ2核转位激活，定量分析示HG组浆／核荧光强度比值较NG组降低37％（P〈0．05），HB组PKCβ2核转位与HG组相比更加明显。结论高糖通过诱导HUVECsPKCβ2核转位，进而调控PPARα表达，增加VEGF、VCAM-1mRNA的表达。Objective To investigate the roles of protein kinase C （PKC）β2 and PPARα in the mRNA expression of vascular endothelial growth factor（VEGF） and vascular cell adhesion molecule-1 （ VCAM-1 ） in human umbilical vein endothelial cells （HUVECs）exposed to high glucose, and to explore their relationship. Methods The HUVECs were divided into eight groups ： normal glucose （ NG, 5 mmol/L D-glucose） group, high glucose （ HG, 25 mmol/L D-glucose ） group, osmotic control （ L, NG ＋ 20 mmol/L L-glucose ） group, normal glucose transfected with empty vector （ NN, NG ＋ Ad5-null ） group, high glucose transfected with PKCβ2 （ HB, HG ＋ Ad5- PKCβ2 ） group, high glucose plus fenofibrate （ HF, HG ＋ 40 umol/L fenofibrate ） group, and HB plus fenofibrate （HBF, HB＋40 umol/L fenofibrate）group. HUVECs were incubated with fenofibrate for 20 minutes as HF20 group. All cells in various groups were cultured for 6 days. The expressions of VEGF and VCAM-1 mRNA were detected by RT-PCR. PPARα protein expression was tested by Western blot. The expression and translocation of PKCβ2 protein were observed by confocal laser scanning microscope. Results （ 1 ） HG increased VEGF and VCAM-1 mRNA expressions,with 1.91 and 1.56 folds of NG group, respectively （ both P〈0.05）. VEGF and VCAM-1 mRNA expressions in HB group further increased, with 2.59 and 2.07 folds of NG group, respectively （ both P〈0.05 ）. Fenofibrate signifcantly decreased VEGF and VCAM-1 mRNA expressions, with 68% and 74% of HG group, respectively（both P〈0.05）. There were no significant differences in the expressions of VEGF, VCAM-1 mRNA between HF20 and HG groups. （2） The protein expression of PPARα decreased by 20% in HG group compared with NG group,and further decreased in HB group, being 78% of HG group. Compared with HG group, PPARα expression increased by 13% in HF group（ P〈0.05 ）. （3）HG induced PKCβ2 translocation from cytosol to nucleus and quantitative analysis showed the ratio of pla

关 键 词：蛋白激酶Cβ2 过氧化物酶体增殖物激活受体Α 人脐静脉内皮细胞 葡萄糖 视网膜 病变 糖尿病 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]