目的：探讨不同pH值环境下骨形态发生蛋白2（BMP-2）信号通路对高磷诱导大鼠血管平滑肌细胞（VSMCs）钙化的影响。方法选取5～8周龄健康雄性SD大鼠，体外分离培养大鼠胸主动脉VSMCs，取第4代VSMCs进行实验，设正常对照组、pH7.4+高磷组、pH7.1+高磷组和pH7.7+高磷组。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，酶联免疫吸附法检测细胞碱性磷酸酶（AKP）活性，用反转录聚合酶链反应（RT-PCR）法检测BMP-2、Smad1及Runt相关转录因子2（Runx2）mRNA的表达。结果与正常对照组相比，pH7.4+高磷组大鼠VSMCs的钙含量增加、茜素红染色钙化结节增多，BMP-2、Smad1、Runx2 mRNA表达及AKP活性均增高（均P<0.05）；与pH7.4+高磷组相比，pH7.1+高磷组大鼠VSMCs的钙含量减低、茜素红染色钙化结节减少，BMP-2、Smad1、Runx2 mRNA表达及AKP活性均降低（均P<0.05），pH7.7+高磷组大鼠VSMCs的钙含量增加、茜素红染色钙化结节增多，BMP-2、Smad1、Runx2 mRNA表达及AKP活性均增高（均P<0.05）。结论胞外酸性环境（pH7.1）抑制高磷诱导的大鼠VSMCs钙化，胞外碱性环境（pH7.7）促进高磷诱导的大鼠VSMCs钙化，其机制可能是通过BMP-2信号通路介导VSMCs表型转化。%Objective To explore the effect of different pH values on calcification of rat vascular smooth muscle cells (VSMCs) through bone morphogenetic protein (BMP)-2 signaling pathway. Methods Healthy male SD rats aged 5-8 weeks were selected in the study. VSMCs from rat thoracic aorta were cultured in vitro, and then identified by immunocytochemistry. The VSMCs were randomly divided into 4 groups by random sampling method:normal group (pH 7.4), pH7.4+high phosphorus group, pH 7.1+high phosphorus group, and pH 7.7+high phosphorus group. Calcium deposition and alkaline phosphatase (AKP) activity were measured by alizarin red staining and enzyme linked immunosorbent assay. The expressions of BMP-2, Smad1 and Runx2 mRNA were detected by RT-PCR. Results Compared with the control group, the calcification staining was increased in pH 7.4+high phosphorus group, calcium content was increased and expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were also increased (P<0.05). While compared with the pH 7.4+high phosphorus group, calcification staining, calcium content, expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were decreased in pH 7.1+high phosphorus group (P<0.05). The calcification staining, calcium content, expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were increased in pH 7.7+high phosphorus group (P<0.05). Conclusion The extracellular acidic environment (pH 7.1) can inhibit high-phosphotus-induced VSMCs calcification, whereas extracellular alkaline environment (pH 7.7) induce high-phosphotus-induced VSMCs calcification. The mechanism is presumably that VSMCs calcification is induced by influencing BMP-2 pathway, which may be mediated by VSMCs phenotype transdifferentiation of BMP-2 signaling pathway.
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