外源性硫化氢对人脐静脉平滑肌细胞钙化的影响及其可能机制

The Effects of Exogenous Hydrogen Sulfide on Calcification in Human Umbilical Vein Smooth Muscle Cell and the Underlying Mechanism

目的探寻外源性硫化氢对人脐静脉平滑肌细胞(HUSMC)钙化的影响及机制。方法在成功复制HUSMC钙化模型基础上,给予硫氢化钠(Na HS)进行培养,实验分为6组(n=6):对照组、钙化组、单纯Na HS组(8.0×10-6mol/L Na HS)、钙化+Na HS高剂量组(4.0×10-5mol/L Na HS)、钙化+Na HS中剂量组(8.0×10-6mol/L Na HS)、钙化+Na HS低剂量组(1.6×10-6mol/L Na HS)。对各组HUSMC进行Von Kossa染色、显微图像分析,并检测各组碱性磷酸酶活性、钙离子含量,荧光定量PCR法检测细胞中骨桥蛋白mRNA的表达,放射免疫法检测培养液中骨桥蛋白含量。结果硫化氢可明显减少HUSMC钙结节的聚集生长,Von Kossa染色显示细胞聚集处棕褐色钙结节较钙化组明显减轻,差异有统计学意义(P<0.05);钙化+Na HS高剂量组、钙化+Na HS中剂量组、钙化+Na HS低剂量组钙化细胞百分比、细胞钙含量和碱性磷酸酶活性较钙化组明显降低,差异有统计学意义(P<0.05),培养液中骨桥蛋白含量及其细胞内骨桥蛋白mRNA表达均较钙化组减少,差异有统计学意义(P<0.05)。结论硫化氢可以减轻HUSMC钙化的程度,其机制可能是通过下调细胞内骨桥蛋白mRNA的表达,进而导致骨桥蛋白生成减少而实现的。

Aim To explore the effects and underlying mechanism of the exogenous hydrogen sulphide(H2S)donor hydrosulfide( Na HS) on calcification( CAL) in human umbilical vein smooth muscle cell( HUSMC). Method Using Na HS intervention on the basis of successfully copied HUSMC calcification model,and experiment were randomly divided into 6 groups( n = 6) : the control group,calcification group,pure Na HS group( Na HS),CAL-Na HS-H group(4. 0× 10- 5mol / L Na HS),CAL-Na HS-M group(8. 0 × 10- 6mol / L Na HS),CAL-Na HS-L group(1. 6 × 10- 6mol / L Na HS).Calcium deposition in HUSMC were detected by Von Kossa staining,and calculated Von Kossa staining by micro-image analysis system; the total calcium contents were calculated by ultraviolet spectrophotometry. The alkaline phospharase(ALP) activity were calculated by automated enzyme-linked immunosorbent; The osteopontin mRNA expression were detected by real-time fluorescence quantitative PCR. The osteopontin content were calculated by radioimmunoassay.Results H2 S can significantly reduce the calcification of the HUSMC. Compared with CAL group,Von Kossa staining showed brown nodules were significantly reduced in CAL-Na HS-H group,CAL-Na HS-M group and CAL-Na HS-L group( P< 0. 05). Compared with CAL group,the percentage of positive cells in calcification,the calcium content,ALP levels and OPN content and mRNA expression was significantly reduced in CAL-Na HS-H group,CAL-Na HS-M group and CALNa HS-L group respectively( P < 0. 05). Conclusion H2 S could inhibit the HUSMC calcification and the mechanism may be downregurating expression of OPN mRNA in the intracellular,thereby causing OPN generated to reduce.