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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 effect of high glucose on the expression of cbfα-1 and osteocalcin (OC) in bone marrow stromal cells (VSMCs) during the process of transdifferentiation of rat aorta vascular smooth muscle cells (VSMCs) To explore the possible mechanism of high glucose in inducing vascular calcification. Methods Primary cultured VSMCs were divided into normal glucose concentration group (5 mmol / L D-glucose), high glucose group (25 mmol / L D-glucose) and mannitol group (5 mmol / L D-glucose + 25 mmol / L Mannitol). Calcium deposition of VSMC in each group was measured at different time points. Alizarin red staining was used to detect the calcification of VSMC in each group. Real-time quantitative PCR and Western blotting were used to detect the levels of mRMA and protein in cbfα-1 and OC The changes of alkaline phosphatase (ALP) activity were detected by alkaline phosphatase activity assay kit. The level of α-smooth muscle actin (α-SMA) was detected by immunohistochemistry. Results Compared with the normal glucose concentration and the mannitol control group, the calcium deposition and calcification staining of VSMC in high glucose group were significantly increased, the mRNA and protein expressions of cbfα-1 and OC increased (P <0.05), the expression of α-SMA protein Decreased (P <0.05), both in a time-dependent manner. The activity of ALP in VSMC increased by nearly 2 folds compared with the control group after high glucose stimulation. Conclusion High glucose-induced calcification of smooth muscle cells may be related to the increase of bone mineral protein (cbfα-1) and OC levels induced by high glucose in high-glucose-induced VSMCs. High glucose may decrease the expression of α-SMA in VSMC while promoting the calcification of VSMC Osteogenic cells transdifferentiation.