氧化型低密度脂蛋白对心脏瓣膜成肌纤维细胞的增殖及骨相关蛋白表达的影响

Effect of oxidized low-density lipoprotein on proliferation of aortic valvular myofibroblasts and expression of bone related proteins

目的通过观察猪主动脉瓣成肌纤维细胞在氧化型低密度脂蛋白(ox-LDL)作用下时,细胞的增殖变化以及是否表达骨相关蛋白,探讨心脏瓣膜钙化的病理机制。方法以原代培养的猪主动脉瓣成肌纤维细胞作为研究对象,用与心脏瓣膜钙化有关的病理因素ox-LDL处理细胞,将细胞随机分为4组:对照组(Ctrl)、氧化型低密度脂蛋白组(25μg/ml ox-LDL)、氧化型低密度脂蛋白+阿托伐他汀组(25μg/ml ox-LDL+50μg/ml Ator)、阿托伐他汀组(50μg/ml Ator)。采用流式细胞学技术测定各组细胞在24、48和72 h的增殖变化,利用Western blot和Real-time PCR检测72 h各组细胞的α-平滑肌蛋白(α-SMA)活性以及骨形成蛋白2(BMP2)、碱性磷酸酶(ALP)的基因表达;了解细胞是否异常激活以及是否向成骨细胞样表型转化。结果 ox-LDL分别处理成肌纤维细胞24、48和72 h后,细胞显示出时间依赖性的增殖加速(P<0.05);ox-LDL能使成肌纤维细胞α-SMA的表达明显增加(P<0.05)而成为活性细胞;骨相关蛋白BMP2、ALP的蛋白和m RNA在对照组和Ator组仅有低水平表达,而在ox-LDL组表达显著升高(P<0.01);Ator对ox-LDL的这种病理性影响可以起到一定的抑制作用(P<0.05)。结论 ox-LDL促使心脏瓣膜成肌纤维细胞激活、分化、增殖以及向成骨细胞样表型转化,进而导致心脏瓣膜的钙化。阿托伐他汀可能通过阻遏成肌纤维细胞的增殖起到对心脏瓣膜钙化的防治作用。

[Objective] To explore the potential pathophysiological mechanisms by which oxidized low- density lipoprotein （ox-LDL） could contribute to the progression of aortic valve calcification. [Methods] Valvular myofibroblasts isolated from porcine aortic valve leaflets were treated with ox-LDL （25 I^g/ml） in the presence or absence of Atorvastatin （25 ~g/ml）, and with PBS or Atorvastatin （25 lxg/ml） alone as controls. Cells were then harvested at different time points （24, 48 and 72 h）. Cell proliferation was checked by flow cytome- try. Protein and gene expression levels of a-smooth muscle actin （ct-SMA）, bone morphogenetic protein 2 （BMP2） and alkaline phosphatase （ALP） were analyzed by Western blot and real-time PCR. [Results] Com- pared with the control group, ox-LDL treatment for 24, 48 and 72 h time-dependently activated aortic valvu- lar myofibroblasts by promoting their proliferation （P〈 0.05） and in the meanwhile induced increased expression of ct-SMA （P〈 0.05） and bone related proteins BMP2 and ALP （P〈 0.01）, while the presence of Atorvastatin prevented this process （P〈 0.05）. [Conclusion] ox-LDL can cause aortic valve calcification by promoting the activation, differentiation, proliferation and osteoblast-like phenotype transformation of aortic valvular myofi- broblasts, whereas Atorvastatin can reverse it to a certain extent, implicating its potential for medical therapy for this disease process.