Klotho RNA干扰对人主动脉平滑肌细胞钙化和表型转化的影响

目的探讨抗衰老因子(Klotho)RNA干扰对人主动脉血管平滑肌细胞钙化和表型转化的影响。方法采用慢病毒抑制人主动脉平滑肌细胞的Klotho表达,通过荧光显微镜、荧光定量PCR及Western印迹法验证转染效率。采用微量法检测细胞内钙离子含量及碱性磷酸酶活性,酶联免疫吸附试验检测骨桥蛋白水平,采用荧光定量PCR检测α-平滑肌肌动蛋白(SMA)、平滑肌(SM)22α、骨桥蛋白等相对表达量。结果慢病毒转染人主动脉平滑肌细胞后Klotho表达明显下调。转染Klotho能显著增加人主动脉平滑肌细胞内钙水平,增加碱性磷酸酶活性,提高α-SMA、SM22α及表型转化标记物骨桥蛋白的mRNA表达水平,同时细胞裂解液中骨桥蛋白水平也显著增加。结论Klotho表达减少可促进人主动脉平滑肌细胞钙化,并促进细胞从收缩型向分泌型转化。

Effects of Klotho RNA interference on calcification and phenotypic transformation in human aortic smooth muscle cells

Objective To investigate the effects of Klotho RNA interference on calcification and phenotypic transformation in human aortic vascular smooth muscle cells(HA-VSMC).Methods Lentivirus was used to inhibit the expression of Klotho in HA-VSMC,and the transfection efficiency was verified by fluorescence microscopy,fluorescence quantitative PCR and Western blot.The intracellular calcium content and alkaline phosphatase activity were detected by the micromethod.The level of osteopontinin cell lysate was detected by ELISA.The relative expressions ofα-SMA,SM22αand osteopontin were detected by fluorescence quantitative PCR.Results The expression of Klotho in HA-VSMC was significantly downregulated after lentivirus transfection.Compared with the control group,Klotho transfection significantly increased the intracellular calcium level,alkaline phosphatase activity and mRNA expressions ofα-SMA,SM22αand osteopontin,which were phenotypic transformation marker of HA-VSMC.In addition,the level of osteopontin in cell lysate was also significantly increased.Conclusions Klotho deficiency could promote the calcification of HA-VSMC and promote phenotypic transformation from contractile type to secretory type.