OSX基因沉默对醛固酮诱导的小鼠血管平滑肌细胞钙化的影响

Study on the influence of OSX silenced by siRNA on calcification of vascular smooth muscular cells induced by aldosterone

目的研究成骨相关转录因子(OSX)沉默对醛固酮诱导的血管平滑肌细胞(VSMC)成骨样分化和钙化的影响。方法体外原代培养小鼠的VSMC。针对小鼠OSX基因设计合成小分子干扰RNA(siRNA)序列,使用Lipo 2000为载体,转染体外培养的VSMC;FAM荧光标记siRNA优化转染条件。实时PCR检测OSX mRNA表达。将siRNA序列转染VSMC,细胞分为4组:1正常组;2醛固酮组(1.5 mmol/L);3siRNA转染组:醛固酮+OSX-siRNA;4阴性转染对照组:醛固酮+阴性对照siRNA。实时RT-PCR及免疫印迹法检测OSX、整联结合涎蛋白(Ibsp)基因和蛋白表达;茜素红染色观察细胞钙盐沉积。结果与醛固酮组相比,siRNA转染组OSX mRNA表达在转染后24 h及48 h均明显低于醛固酮组(均P<0.01);OSX蛋白表达在转染后48 h和72 h均低于醛固酮组(均P<0.01),以48 h最明显。siRNA有效沉默OSX基因表达后,OSX转染组Ibsp mRNA和蛋白的表达低于醛固酮组(均P<0.01),而且细胞钙盐沉积低于高磷组。结论 OSX-siRNA可以有效抑制VSMC OSX基因和蛋白的表达,从而抑制醛固酮诱导的VSMC成骨样分化和细胞钙化。OSX可以成为CKD血管钙化治疗的靶点。

Objective To explore the effect of OSX gene silenced by siRNA on osteogenic differentiation and calcification of vascular smooth muscle cells（ VSMC）,induced by aldosterone in vitro. Methods VSMCs were cultured in vitro and passaged 4 to 9 times. Transfection condition had been optimized by FAM fluorescent labeling- siRNA. After transfection with siRNA sequence,VSMCs were divided into 4 groups：① control,② aldosterone（ 1. 5 mmo L /L）,③ siRNA transfection： aldosterone ＋ OSX- siRNA,④ negative transfection control： aldosterone ＋negative control siRNA. OSX and Ibsp mRNA or protein expression had been detected by realtime- PCR and Western blot. Calcium deposition was visualized by Alizarin staining. Results At 24 and 48 h after transfection,the expression of OSX mRNA in siRNA transfection group was significantly lower than that of aldosterone group（ all P〈0. 01）. At 48 and 72 h after. transfection,the expression of OSX protein in siRNA transfection group was significantly lower than that of aldosterone group（ all P〈0. 01）. While OSX gene was silenced by siRNA in siRNA transfection group,the mRNA and protein expression of Ibsp was significantly declined（ all P〈0. 01） and calcium deposited. Conclusion OSX- siRNAKi SS- 1 can suppress the expression of VSMC OSX gene and protein,therefore,osteogenetic differentiation and calcification of vascular smooth muscle cells induced by aldosterone is inhibited.