肿瘤坏死因子α介导人脐动脉血管平滑肌细胞成骨样钙化

目的 观察肿瘤坏死因子α（TNF-α）对体外培养人脐动脉血管平滑肌细胞（hUASMC）骨特异性碱性磷酸酶（BAP）、骨桥蛋白（OPN）和骨形成蛋白2（BMP-2）等成骨样分化标志蛋白表达以及细胞外钙盐沉积的影响。 方法 植块贴壁法原代培养人hUASMC。予以TNF-α刺激，甲O-酚酞络合酮方法测定细胞外基质钙盐沉积；Von Kossa法观察钙化染色；实时定量PCR法测定血管平滑肌细胞BAP和OPN mRNA表达水平；免疫印迹法观察血管平滑肌细胞BAP、OPN和BMP-2蛋白表达水平。 结果 一定浓度范围内的TNF-α可促进hUASMC增殖；增加细胞外基质钙盐沉积。TNF-α 50 μg/L刺激可在第3天上调血管平滑肌细胞BAP和OPN mRNA表达水平（BAP mRNA：1.908±0.034比1.000±0.033，OPN mRNA： 3.600±0.073比1.000±0.079，均P < 0.05）。TNF-α 50 μg/L刺激可在第5天上调血管平滑肌细胞BAP、OPN和BMP-2蛋白表达水平（BAP蛋白：3.394±0.083比1.000±0.030，OPN蛋白： 1.967±0.134比1.000±0.070，BMP-2蛋白：2.745±0.289比1.000±0.208， 均P < 0.05）。 结论 一定浓度范围的TNF-α可刺激hUASMC增殖；介导细胞成骨样分化；增加细胞外基质钙盐沉积，从而参与血管钙化的发生发展。

Ossific calcification of human umbilical artery vascular smooth muscle cells induced by TNF-α

Objective To investigate the effect of TNF-α on the expression of bone alkaline phosphatase (BAP), osteopontin (OPN) and bone morphogenetic protein 2 (BMP-2), and the deposit of extracellular calcium in human umbilical artery smooth muscle cells (hUASMC). Method hUASMC were cultured primarily with tissue explants-attached method. The mRNA expression of BAP and OPN was determined by real time-PCR. The protein expression of BAP, OPN and BMP-2 was determined by Western blotting. The calcium deposit was detected by O-cresolphthalein complexone method. Results TNF-α promoted the proliferation of hUASMC and increased the calcium deposit in a time- and dose-dependent manner. The mRNA expression of BAP and OPN in hUASMC was up-regulated by TNF-α（50 μg/L） treatment at day 3(BAP mRNA 1.908±0.034 vs 1.000±0.033, OPN mRNA 3.600±0.073 vs 1.000±0.079, all P<0.05). The protein expression of BAP, OPN and BMP-2 in hUASMC was up-regulated by TNF-α（50 μg/L） treatment at day 5 (BAP protein 3.394±0.083 vs 1.000±0.030, OPN protein 1.967±0.134 vs 1.000±0.070, BMP-2 protein 2.745±0.289 vs 1.000±0.208, all P<0.05）. Conclusion TNF-α of certain concentrations can promote the proliferation of hUASMC, induce the ossific calcification of hUASMC, increase the calcium deposit, and participate in the development of vascular calcium