高糖刺激血管平滑肌细胞表达cbfα-1和OC的实验研究

Effects of High Glucose on Expression of Core Binding Factor Alpha1 and Osteocalcin in Vascular Smooth Muscle Cells

目的研究高糖在刺激大鼠胸主动脉血管平滑肌细胞(VSMC)转分化过程中对相关骨基质蛋白如核心结合因子α-1(cbfα-1)和骨钙素(OC)表达的影响,并探讨高糖在诱导血管钙化发生过程中可能的作用机制。方法原代培养VSMC,分为正常糖浓度组(5 mmol/L D-葡萄糖)、高糖组(25 mmol/L D-葡萄糖)和甘露醇组(5mmol/L D-葡萄糖+25 mmol/L甘露醇),在不同时间点检测各组VSMC的钙沉积指标;茜素红染色了解各组VSMC钙化程度;实时荧光定量PCR法和免疫印迹法检测各组cbfα-1和OC的mRMA水平和蛋白表达变化;碱性磷酸酶活性检测试剂盒检测碱性磷酸酶(ALP)的活性。免疫组化检测α-平滑肌肌动蛋白(α-SMA)水平。结果与正常糖浓度和甘露醇对照组相比较,高糖组VSMC钙沉积和钙化染色均明显增强,cbfα-1和OC的mRNA和蛋白表达均升高(P<0.05),α-SMA蛋白表达下降(P<0.05),均呈时间依赖性。高糖刺激后VSMC的ALP的活性比对照组增加了近2倍。结论高糖介导的平滑肌细胞钙化可能与高糖促进VSMC分泌骨基质蛋白cbfα-1和OC水平增加有关;高糖在促进VSMC钙化同时,使VSMC的α-SMA表达水平降低,促进其向成骨样细胞转分化。

Objective To investigate the effects of high glucose on expression of core binding factor alpha1(cbfα-1) and osteocalcin(OC) in vascular smooth muscle cells(VSMCs),and discuss the mechanism of small vessels calcification induced by high glucose(GS) in vitro.Methods The primary cultured VSMCs from rats’ aortic segments were divided into three groups,including normal control group(5 mmol/L D-glucose),high glucose group(25 mmol/L D-glucose) and mannitol group(5 mmol/L D-glucose plus 25 mmol/L mannitol).We measured quantitatively the calcium deposition in VSMCs and investigated the calcium extent of VSMCs by alizarin red stain in each group.The mRNA levels of cbfα-1 and OC were measured by real-time PCR,and the protein expression levels of cbfα-1 and OC were examined by Western blot.The activity of alkaline phosphatase was measured by alkaline phosphatase activity testing kit,and the protein level of α-smooth muscle actin(α-SMA) was detected by immunohistochemisty.Results When compared with the normal group and mannitol group,the high glucose group showed that the calcium deposition and calcium extent of VSMCs increased obviously,the mRNA and protein levels of cbfα-1 and OC also increased significantly(P<0.05),while the protein level of α-SMA decreased(P<0.05),which were in a dose-dependent manner. The level of alkaline phosphatase activity of VSMCs was approximately doubled in high glucose group.Conclusion The mechanism of high glucose induced calcification in VSMCs may be due to the increased expression of cbfα-1 and OC.High glucose decrease the expression of α-SMA in VSMCs,which could induce the transdifferentiation from RVSMCs to osteoblast-like cells.