Klotho在高磷诱导的血管平滑肌细胞钙化中的表达研究

Klotho expression in hyperphosphate-induced calcified vascular smooth muscle cells

目的:探讨Klotho mRNA及蛋白在高磷诱导的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)钙化中的表达变化。方法:体外培养大鼠原代VSMCs,利用细胞计数试剂-8(cell counting kit-8,CCK-8)试剂盒检测不同浓度磷刺激条件下VSMCs的增殖情况。高磷培养基(3 mmol/L)刺激细胞3 d或6 d后,采用茜素红染色评估VSMCs钙化情况,用碱性磷酸酶(alkaline phosphatases,ALP)试剂盒和钙含量试剂盒分别检测细胞内ALP活性和钙含量,实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)和蛋白印迹法检测钙化相关蛋白骨形态发生蛋白2(bone morphogenetic protein 2,BMP-2)、Runt相关转录因子2(Runt related transcription factor 2,Runx2)和Klotho mRNA及蛋白的表达变化。结果:培养基磷浓度升高抑制了VSMCs增殖;而高磷刺激VSMCs 6 d后,茜素红染色可见钙化结节明显形成,细胞内钙含量显著增加[(1.52±0.14)mmol/g比(0.03±0.01)mmol/g,P=0.043],成骨相关蛋白BMP-2、Runx2的mRNA和蛋白表达均增加,而Klotho的mRNA和蛋白表达均下降。结论:高磷可刺激VSMCs钙化形成和表型转换,Klotho可能参与了高磷诱导的VSMCs钙化机制。

Objective： To investigate the changes of Klotho expression in rat vascular smooth muscle cells（VSMCs）calcification induced by high phosphate. Methods： Rat VSMCs was cultured in vitro, the proliferation of VSMCs induced by different concentrations of phosphate was detected by cell counting kit-8（CCK-8）. VSMCs were treated with high phosphate（3 mmol/L） for 3 days or 6 days, and the calcification of VSMCs was evaluated by alizarin red staining. The activity of alkaline phosphatase（ALP） and calcium content in VSMCs were detected by ALP assay kit and calcium colorimetric assay kit, respectively. The expression of m RNA and protein of bone morphogenetic protein 2（BMP-2）, Runt related transcription factor 2（Runx2） and Klotho were detected by quantitative real-time polymerase chain reaction（qrt-PCR） and Western blotting（WB）. Results： Higher-phosphate media inhibited the proliferation of VSMCs. After being incubated in high-phosphate media for 6 days, calcified nodules were observed with alizarin red staining and calcium content was significantly increased; qrt-PCR demonstrated that levels of BMP-2 and Runx2 m RNA and protein were increased and the expressions of Klotho m RNA and protein levels were decreased. Conclusions： Klotho might be involved in the hyperphosphate-induced calcification and phenotype changes in VSMCs.