辛伐他汀对大鼠成骨细胞经典Wnt信号通路相关因子表达的影响

目的观察不同浓度、不同时间辛伐他汀对体外培养的大鼠成骨细胞经典Wnt通路Wnt3a、LRP5和β-catenin的m RNA表达的影响,探讨辛伐他汀抗骨质疏松的可能机制。方法用酶消化法培养大鼠颅骨成骨细胞,采用倒置相差显微镜及碱性磷酸酶染色和茜素红染色观察和鉴定成骨细胞。用浓度分别为10^-8mol/L、10-7mol/L、10^-6mol/L和10^-5mol/L的辛伐他汀干预大鼠成骨细胞24 h、48 h和72 h,并设无辛伐他汀为对照组,采用RT-PCR检测经典Wnt信号通路Wnt3a、LRP5和β-catenin的m RNA表达情况。结果镜下观察及染色结果均显示具有成骨细胞典型特征。RT-PCR结果显示：10^-5mol/L辛伐他汀组干预24 h后较对照组Wnt3a（2.09±0.12）vs（1.82±0.09）]、LRP5（1.42±0.07）vs（1.29±0.07）]和β-catenin（1.10±0.06）vs（0.95±0.06）]的m RNA表达显著增加;干预48 h和72 h后各组Wnt3a（F=37.78和96.52,P=0.000）、LRP5（F=10.32,P=0.001和F=22.02,P=0.000）的m RNA表达随着辛伐他汀浓度的增大而显著增加,β-catenin的m RNA表达较对照组显著增加（F=6.67和21.90,P〈0.05）;10^-8-10^-5mol/L辛伐他汀各组中分别干预24 h、48 h和72 h,随着干预时间延长Wnt3a和LRP5的m RNA表达均增加;干预48 h与24 h比较,β-catenin的m RNA表达差异无统计学意义（1.08±0.09）vs（1.02±0.06）,P〉0.05],而干预72 h较24 h显著增加（1.23±0.10）vs（1.02±0.06）,P〈0.05],且辛伐他汀浓度为10^-6mol/L和10^-5mol/L干预72 h较48 h显著增加（1.32±0.07）vs（1.11±0.05）和（1.47±0.08）vs（1.19±0.04）,P〈0.05]。结论辛伐他汀可促进体外培养大鼠成骨细胞经典Wnt信号通路Wnt3a、LRP5和β-catenin的m RNA表达。

Effect of simvastatin on the related factors in expression of canonical Wnt signaling pathway in osteoblasts of rats

Objective To investigate the effect of different concentrations, multi-treatment course of simvastatin on the m RNA expression of Wnt3 a, LRP5 and β-catenin in osteoblasts of rats and the potential mechanism.Methods Osteoblasts were obtained from calvaria. Morphology of osteoblasts was observed under phase contrast microscope after ALP staining and Alizarin red staining. Rat osteoblasts were treated by different concentrations of simvastatin（10^-8mol/L, 10-7mol/L, 10^-6mol/L and 10^-5mol/L）, and Wnt3 a, LRP5 and β-catenin m RNA expression were detected using RT-PCR after 24 h, 48 h and 72 h. Results The cultured cells had the typical features of osteoblasts. Compared with that in control group, the result of RT-PCR showed that the expression of Wnt3 a （2.09±0.12） vs（1.82±0.09）], LRP5（1.42±0.07） vs（1.29±0.07）] and β-catenin （1.10±0.06） vs（0.95±0.06）] m RNA in 10^-5mol/L simvastatin group treated after 24 h, and those of Wnt3a（F=37.78 and 96.52, P=0.000） and LRP5（F=10.32, P=0.001 and F=22.02, P=0.000）increased significantly with the increase of concentration of simvastatin in each group treated after 48 h and 72 h（P〈0.05）. After 24 h, 48 h and 72 h intervention, the expression of Wnt3 a and LRP5 m RNA increased significantly in each group as the intervention time got longer. In group 10^-6mol/L and 10^-5mol/L, compared with that after simvastatin intervention 48 h, β-catenin m RNA expression increased significantly after 72 h （1.32±0.07） vs（1.11±0.05） and（1.47 ± 0.08） vs（1.19 ± 0.04）, P〈0.05]. Conclusion Simvastatin could promote Wnt3 a, LRP5 and β-catenin m RNA gene＇s expression of canonical Wnt signaling pathway in osteoblast of rats.