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目的:观察高糖对体外培养的大鼠胸主动脉血管平滑肌细胞(vascular smooth muscle cells,VSMCs)功能以及与成骨细胞转分化相关蛋白表达的变化,探讨糖尿病血管病变的可能细胞/分子发病机制。方法:用组织贴块法建立SD大鼠胸主动脉VSMCs的体外培养技术;高糖(葡萄糖浓度:50 mmol/L)作用该细胞,设相同浓度甘露醇为对照组,观察在不同的培养时间下(3、12h和第3、6、9、12d),采用免疫蛋白印迹法和间接免疫荧光法分别检测α平滑肌肌动蛋白(α-SMA),骨桥蛋白(osteopontin,OPN)和成骨细胞特异性核转录因子(core-binging factorα-1,Cbfα-1/RUNX 2)的表达。细胞钙沉积含量测定观察培养细胞钙盐的沉积。结果:组织贴块法培养的大鼠原代胸主动脉VSMCs呈梭型,α-SMA表达强阳性;高糖作用12h,VSMCs的Cbfα-1表达明显上升;高糖作用9d后,VSMCs的α-SMA表达量较高糖作用前降低近90%;免疫荧光染色显示VSMC细胞呈低强度的α-SMA染色阳性;同时,高糖作用下的VSMCs能够检测到OPN蛋白的表达,与对照组相比差异显著;高糖作用下细胞钙沉积含量明显增加。结论:持续的高糖作用能够使动脉VSMCs发生向类似成骨样细胞的转分化,提示高糖诱导的VSMCs转分化可能参与糖尿病血管病变的发生发展。
OBJECTIVE: To observe the function of vascular smooth muscle cells (VSMCs) and the expression of osteoblast transdifferentiation related proteins in cultured rat thoracic aortas induced by high glucose, and to explore the possible cellular / molecular pathogenesis of diabetic vascular disease . Methods: The rat thoracic aorta VSMCs culture in vitro was established by tissue patch method. The cells were treated with high glucose (glucose: 50 mmol / L), and the same concentration of mannitol was used as the control group. The expressions of α-SMA, osteopontin (OPN) and osteogenesis in osteoblasts were detected by Western blotting and indirect immunofluorescence assay at 3,12h and 3,6,9 and 12 days respectively. The expression of core-binging factor alpha-1 (Cbfα-1 / RUNX 2). Cell Calcium Deposition Assay The deposition of calcium in cultured cells was observed. Results: VSMCs of rat primary thoracic aorta cultured in tissue block method were spindle-type, and the expression of α-SMA was strongly positive. The expression of Cbfα-1 in VSMCs was significantly increased after high glucose treatment for 12 h. The expression of OPN protein in VSMCs was detected by immunofluorescent staining. The expression of OPN protein in VSMCs was detected by immunofluorescence staining. Compared with control group Than the significant difference; high glucose under the cell calcium deposition increased significantly. CONCLUSION: Sustained hyperglycemia can induce VSMCs to transdifferentiate to osteoblast-like cells, suggesting that high glucose-induced transdifferentiation of VSMCs may be involved in the development of diabetic vascular disease.