摘要
目的:探讨高磷及高钙诱导血管平滑肌细胞发生钙化的分子机制。方法:以人血管平滑肌细胞(VSMCs)为模型,采用不同钙磷浓度的培养基与细胞共培养12 d,将细胞分为正常钙、磷组(对照组)、高磷组、高钙组和高磷+高钙组,分别采用免疫印迹(western-blot)技术、逆转录-聚合酶链反应(RT-PCR)技术检测人血管平滑肌细胞核心结合因子α1(Cbfα1)、骨钙素、α-平滑肌肌动蛋白(α-SMA)蛋白质和mRNA的表达;高磷+高钙条件孵育下的细胞加入ZVAD-FMK干预后,再次采用western-blot技术检测Cbfα1、骨钙素蛋白质表达的情况。结果:试验组Cbfα1、骨钙素表达增强(P=0.013;P=0.007),而肌成纤维细胞的特异性标志物α-SMA表达下降(P=0.009),对照组上述3个指标无明显变化(P=0.055、P=0.074、P=0.63);加入ZVAD-FMK与高磷+高钙组细胞共培养后,Cbfα1、骨钙素蛋白质的表达受到明显抑制。结论:高磷或(和)高钙通过使血管平滑肌细胞转化为成骨样细胞而导致钙化的发生;ZVAD-FMK可以在一定程度上抑制这一病理生理进程,提示凋亡和成骨分化均参与了钙化过程。
Objective： To investigate the effect and molecular mechanism of high phosphate and high calcium on calcification of human vascular smooth muscle cells（ VSMCs）. Methods： The human VSMCs were co-cultured with culture medium of different dose of phosphate and calcium in vitro for 12 days and according to dosage of calcium and phosphate the cells were divided into 5 groups as follows：normal calcium and phosphate group（ control group,P 1. 4 mmol / L,Ca 2. 0 mmol / L）,high phosphate group（ P 2. 0 mmol / L,Ca2. 0 mmol / L）,high calcium group（ P 1. 4 mmol / L,Ca 2. 8 mmol /L）,high calcium and high phosphate group（ P 2. 0mmol / L,Ca 2. 8mmol / L）. Western-blot and RTPCR were adopted to detect the protein and mRNA expression of Cbfα1,osteocalcin,α-SMA respectively. In high calcium and high phosphate group,the nonspecific inhibitor ZVAD-FMK of caspase was added in. After this intervention,western-blot was adopted again to detect protein expression of Cbfα1and osteocalcin. Results： Compared with control group,the expression of Cbfα1 and osteocalcin increased in all experimental groups while the expression of α-SMA,which was the specific marker of myofibroblast,decreased（ P 0. 05）. There were no obvious changes in the above-mentioned 3 indicators in the control group（ P 0. 05）. After ZVAD-FMK was added in and co-cultured with cells of high calcium and high phosphate group,the protein expression of Cbfα1and osteocalcin were inhibited obviously. Conclusion： High concentration of phosphate or（ and） calcium can induce calcification of human VSMCs through turning VSMCs into osteoblast like cell（ OLCs）. ZVAD-FMK can inhibit this pathophysiological process to a certain extent,which reveals that both apoptosis and osteogenic differentiation of VSMCs involve in the calcification process.
出处
《贵阳医学院学报》
CAS
2015年第6期603-607,共5页
Journal of Guiyang Medical College
