【作者】 于伟霞；

【导师】 廖端芳； 何淑雅；

【作者基本信息】 南华大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 目的:观察依泽替米贝对平滑肌细胞胆固醇蓄积的影响,并初步探讨PPARγ依赖的信号通路介导依泽替米贝对血管平滑肌细胞胆固醇蓄积的抑制作用。方法:首先以原代培养大鼠主动脉平滑肌细胞(VSMCs)为研究对象,以20mg/L Chol:MβCD作用于细胞48h,造成荷脂细胞模型。然后以不同浓度的依泽替米贝(3μmol/L,10μmol/L,30μmol/L)处理细胞24h,或以30μmol/L依泽替米贝分别处理细胞不同时间(0h,6h,12h,24h,48h),用HPLC检测细胞内总胆固醇(TC)、游离胆固醇(FC)的含量,用油红O染色观察细胞荷脂情况,用Western印迹法检测caveolin-1、nSREBP-1、LXRα蛋白的表达,用免疫荧光法检测caveolin-1及ABCA1的蛋白表达,用RT-PCR检测PPARγ和ABCA1的mRNA变化。最后将带有PPARγsiRNA的质粒转染到VSMCs内,同样以20mg/L Chol:MβCD孵育细胞48h和以30μmol/L依泽替米贝作用24h,再检测细胞内TC、FC的含量,检测caveolin-1、LXRα蛋白的表达及观察ABCA1的mRNA变化。结果:不同浓度依泽替米贝(3μmol/L、10μmol/L、30μmol/L)作用于VSMCs源性荷脂细胞24h,浓度依赖性地降低细胞内TC、FC的含量,30μmol/L作用最强; 30μmol/L依泽替米贝作用于VSMCs源性荷脂细胞不同时间(0h,6h,12h,24h,48h),同样降低细胞内TC、FC和胆固醇酯(CE)的水平,其最佳作用时间在24h时。同时抑制nSREBP-1的表达,明显增加细胞caveolin-1、LXRα蛋白的表达水平及ABCA1 mRNA表达水平,但PPARγ的mRNA水平没有明显变化。用携带有PPARγSiRNA的质粒pSliencer 2.1-U6 hygro转染VSMCs后,细胞内的TC、FC、CE水平较30μmol/L依泽替米贝组有所升高,且caveolin-1、LXRα蛋白表达及ABCA1的mRNA水平明显减少。结论:1.依泽替米贝能降低VSMCs源性荷脂细胞胆固醇蓄积;2.依泽替米贝的作用与其激活PPARγ的活性以增加LXRα的表达进而上调ABCA1的表达有关;3.依泽替米贝的作用还可能与其激活PPARγ及改变SREBP-1的表达进而上调caveolin-1水平有关。更多还原

【Abstract】 AIM: To investigate the influence of ezetimibe on cholesterol accumulation in vascular smooth muscle cells induced by Chol:MβCD and the mechanism of ezetimibe.METHODS: VSMCs taken from rat aorta were respectively treated with different concentration of ezetimib(e0, 3, 10, 30μmol/L)for 24 hours or treated with 30μmol/L of ezetimibe for different time(0, 6, 12, 24, 48 hours). The concentration of total cholesterol and free cholesterol were detected by high performance liquid chromatography. Oil Red O dyeing experiment was used to show the cellular lipid droplets in lipid-loaded cells. The expression of caveolin-1, nSREBP-1, LXRα, ABCA1 and PPARγwere detected by western blotting, RT-PCR and Immunofluorescence methods respectively.Results: Chol:MβCD incubation with VSMCs increased cellular cholesterol accumulation, decreased the expression of caveolin-1, LXRαand ABCA1, and promoted nSREBP-1 expression. Ezetimibe treatment significantly inhibited the expression of nSREBP-1 in concentration-dependent manner. Reversely, ezetimibe enhanced the expressions of caveolin-1, LXRαand ABCA1 in a dose-dependent manner with the peak in 30μmol/ L. When VSMC were treated with 30μmol/ L ezetimibe for different time, the expression peaks of caveolin-1, LXRαand ABCA1 were at 24 hours. But the expression of nSREBP-1 was decreased. Interestingly, the mRNA level of PPARγhad no significant change. When VSMCs were transfected with pSliencer 2.1-U6 hygro-PPARγsiRNA, the level of cellular cholesterol was increased and the expression of caveolin-1, LXRαand ABCA1 were decreased.Conclusion: Ezetimibe inhibits Chol:MβCD-induced cholesterol accumulation of VSMCs via regulating the PPARγ-LXRα-ABCA1 pathway and PPARγ-SREBP 1/Caveolin-1 pathway.更多还原