碱性环境对慢性肾衰竭大鼠血管钙化的影响

Effect of alkalinization on vascular calcification in rat with chronic renal failure

目的:探讨碱性环境对慢性肾衰竭大鼠血管钙化的影响及可能机制。方法:将21只雄性SD大鼠随机分为4组:假手术组(甲基纤维素灌胃)、慢性肾衰竭组(甲基纤维素灌胃)、钙化组(甲基纤维素加骨化三醇灌胃)、碱干预组(甲基纤维素及骨化三醇灌胃及5%碳酸氢钠腹腔注射)。采用von Kossa染色及邻甲酚酞络合酮比色法检测大鼠胸主动脉钙化情况;免疫组织化学方法检测胸主动脉Runx2表达。体外采用组织块贴壁法培养原代大鼠胸主动脉平滑肌细胞,利用10 mmol/Lβ-甘油磷酸钠制备血管平滑肌细胞钙化模型。使用HCl和NaHCO3调节培养基pH值。细胞随机分为3组:正常对照组(pH 7.4)、钙化组(10mmol/Lβ-甘油磷酸钠+pH7.4)、碱干预组(10mmol/Lβ-甘油磷酸钠+pH 7.7),共培养12d。采用茜素红染色及邻甲酚酞络合酮比色法检测细胞钙化情况,RT-PCR和Western Blot法检测Runt相关转录因子-2(runt-related transcription factor 2,Runx2)的表达。结果:体内实验中,成功制备了慢性肾衰竭血管钙化的大鼠模型,与钙化组相比,给予碳酸氢钠干预后,大鼠血管钙盐沉积均明显增加(P<0.05);免疫组织化学结果显示碱性环境可明显升高Runx2表达(P<0.05)。体外实验中,与正常对照组相比,钙化组钙盐沉积明显增加(P<0.05);与钙化组相比,碱干预组钙盐沉积明显增加(P<0.05)。RT-PCR和Western Blot显示,与正常对照组相比,钙化组Runx2表达明显增加(P<0.05);与钙化组相比,碱干预组Runx2表达明显增加(P<0.05)。结论:碱性环境可能通过促进类骨表型转化进而促进慢性肾衰竭大鼠血管钙化。

Objective：To explore the effect of alkalinization on vascular calcification in rat with chronic renal failure.Method：In vivo,Male SD rats（n=21）were randomly divided into four groups：sham operated group（n=5）,5/6Nx（n=5）,5/6Nx＋calcitriol（n=5）,5/6Nx＋calcitriol＋alkalosis（n=6）.Vascular calcification was measured by von Kossa staining and quantification of calcium.Runx2 in aorta was determined by immunohistochemistry.Vascular smooth muscle cells（VSMCs）were obtained from rat aortic and identified by immunocytochemistry,then randomly divided into control group,calcification group and alkalinization group（pH=7.4or 7.7adjusted with HCL and NaHCO_3,VSMCs were treated with 10mmol/Lβ-glycerophosphate）.Calcium deposition was measured by Alizarin red staining and quantification of calcium.RT-PCR and Western blot were used to observe VSMCs Runx2 expression.Result：Compared with 5/6Nx＋calcitriol group,calcification was all significantly increased（P〈0.05）in 5/6Nx＋calcitriol＋alkalosis group.Immunohistochemistry showed that the expression of Runx2 was upregulated by alkalinization（P〈0.05）.In vitro,compared with control group,calcium deposition in VSMCs were significantly increased in calcification group after incubation for 12days（P〈0.05）,the expression of Runx2 was increased as well（P〈0.05）,and calcium deposition and the expression of Runx2 in alkalinization group were more than that in calcification group（P〈0.05）.Conclusion：Upregulation of osteogenic differentiation may contribute to promoting VSMCs calcification in alkalinization.