不同浓度β-甘油磷酸盐对人脐静脉平滑肌细胞钙化的影响

Effects of Different Concentration β-glycerophosphate on Calcification of Human Umbilical Vein Smooth Muscle Cells

目的探寻β-甘油磷酸盐诱导原代培养人脐静脉平滑肌细胞(HUSMC)钙化的适宜浓度。方法采用原代培养人脐静脉平滑肌细胞,根据培养液中β-甘油磷酸盐浓度不同依次分为8组:正常对照组、10mM组、12mM组、14 mM组、16 mM组。以更换培养液当天记为第1天,培养10d,von kossa染色观察细胞钙沉积情况、显微图像分析法测量钙化阳性细胞百分比、比色法检测细胞内钙含量和碱性磷酸酶活性。结果对照组SMC银染无钙结节,显微图像分析阳性细胞为(0.1060±0.05)%;12mM组SMC银染可见明显钙结节存在,钙化阳性细胞数为(4.7009±1.35)%,钙含量和碱性磷酸酶活性较对照组明显升高(P<0.05)。10mM组、16mM组、20mM组、30mM组与对照组相比钙含量和碱性磷酸酶活性差异无统计学意义(P>0.05),但10mM组、16mM组钙化阳性细胞百分比升高明显(P<0.05),20mM组、30mM组与对照组相比钙化阳性细胞百分比差异无统计学意义(P>0.05);40mM组钙含量和碱性磷酸酶活性明显降低(P<0.001),灰度扫描钙化阳性细胞随之降低。结论12mMβ-甘油磷酸盐可刺激人脐静脉平滑肌细胞出现钙盐沉积,且人脐静脉SMC钙化对β-甘油磷酸盐无浓度依赖性。

Objective To explore the the appropriate concentration of β-glycerol phosphate induced primary cultured human umbilical vein smooth muscle cells（HUSMC） calcification.Methods culturing Human umbilical vein smooth muscle cells in the presence of β-glycerophosphate（β-GP） were divided into eight different concentrations： normal control group（culture medium without β-GP）,10mM group（culture medium containing 10mM β-GP）,12mM group（culture medium containing 12mM β-GP）,14 mM group（culture medium containing 14mM β-GP）,16mM group（culture medium containing 16mM β-GP）,20 mM group（culture medium containing 20mM β-GP）,30 mM group（culture medium containing 30mM β-GP）,40 mM group（culture medium containing 40mM β-GP）.The day in which the culture medium was replace was recorded as d1,and cultured for 10 days.Calcification was confirmed by von kossa staining,applied colorimetry detection of intracellular calcium content and alkaline phosphatase activity changes.Results In control group,there was non-calcium nodule with SMC silver staining;12mM group of SMC silver staining could be seen the existence of calcium nodules clearly,calcium content and alkaline phosphatase activity in 12mM group was significantly higher than that in control group（P0.05）.10mM group,16mM group,20mM group,30mM group and control group,calcium content and alkaline phosphatase activity showed no significant difference（P0.05）;40mM group calcium content and alkaline phosphatase activity showed significantly lower（P0.01）.Conclusion Concentrations of 12mM β-glycerol phosphate may directly stimulate human umbilical vein smooth muscle cells to be calcified,and human umbilical vein SMC calcification showed no dose-dependent on β-glycerol phosphate.