【摘要】 目的:探讨自噬在高磷诱导的大鼠血管平滑肌细胞(VSMC)钙化过程中的作用。方法:采用磷酸盐(3.2 mmol/L Pi,即高磷状态)建立大鼠VSMC钙化模型。实验分为三组:对照组、钙化组(分为3个亚组:3.2 mmol/L Pi 4d组、3.2 mmol/L Pi 6d组、3.2 mmol/L Pi 8d组);钙化+3-甲基腺嘌呤(3-MA)组(3.2mmol/L Pi 8 d+5 mmol/L 3-MA)。分别通过茜素红S染色法和邻甲酚肽络合酮比色法检测各组细胞钙结节形成及钙含量;蛋白免疫印迹法检测各组细胞转录因子蛋白(Runx2)、α-肌动蛋白(α-SMA)和自噬相关蛋白—膜型微管蛋白1轻链3β(LC3Ⅱ)蛋白表达量。透射电子显微镜观察VSMC内自噬小体形成情况。免疫荧光显微镜下观察VSMC中LC3和Runx2定位表达。结果:与对照组相比,3.2 mmol/L Pi 8 d组钙结节、钙含量、Runx2和LC3Ⅱ蛋白表达量及自噬小体均显著增高,α-SMA蛋白表达量降低,差异具有统计学意义(P<0.05)。与3.2 mmol/L Pi 8 d组相比,钙化+3-MA组细胞钙含量增多,LC3荧光分布量降低,Runx2阳性细胞数增多,差异具有统计学意义(P<0.05)。结论:自噬在磷酸盐诱导的VSMC钙化过程中具有保护作用。更多还原

【Abstract】 Objective:To explore the effect of autophagy on process of high phosphate salt induced vascular smooth muscle cell(VSMC) calcification in experimental rats.Methods: Rats’ model of VSMC calcification was induced by phosphate incubation. VSMC were divided into 3 groups:(1) Control group,(2)Calcification group which included 3 subgroups as 4-day subgroup, the cells were cultured by 3.2 mmol/L phosphate for 4 days, 6-day subgroup and 8-day subgroup,(3) Calcification+ 3-MA(autophagy inhibitor) group, in which the 8-day cells were cultured with 5mmol/L 3-MA. Calcium nodule formation and calcium deposition in VSMC were measured by Alizarin red staining and o-cresolphthaleincomplexone method, protein expressions of Runx2, α-SMA and LC3 Ⅱ were examined by Western blot analysis, autophagosome formation in VSMC was measured by transmission electron microscope and the localization and expression of Runx2 and LC3 Ⅱ in VSMC were observed by immunofluorescent microscope.Results: Compared with Control group, the cells at 8-day subgroup showed more calcium nodules, higher calcium deposition, increased protein expressions of Runx2, LC3 Ⅱ, more autophagosome and decreased α-SMA expression, all P<0.05. Compared with 8-day subgroup, the cells in Calcification+3-MA group presented increased calcium deposition, decreased fluorescence distribution of LC3 Ⅱ and more cells with positive Runx2 protein expression, all P<0.05.Conclusion: Autophagy has the protective effect on process of phosphate induced VSMC calcification in experimental rats.更多还原