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. 2016 Aug 8;30(8):1038-1043.
doi: 10.7507/1002-1892.20160208.

[REGUL ATORY EFFECT OF SIMVASTATIN ON MIDDLE/L ATE STAGES OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS VIA p38MAPK PATHWAY]

[Article in Chinese]
Kezhong Zhang  1 , Guangyuan Liu  1 , Faming Tian  1 , Liu Zhang  1
Affiliations

[REGUL ATORY EFFECT OF SIMVASTATIN ON MIDDLE/L ATE STAGES OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS VIA p38MAPK PATHWAY]

[Article in Chinese]
Kezhong Zhang et al. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. .

Abstract
in English, Chinese

Objective: To investigate the regulatory effect of simvastatin on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at middle/late stages by p38MAPK pathway under condition of osteoinductive environment.

Methods: The bone marrow of bilateral femur and tibia were harvested from 20 4-week-old female Sprague Dawley rats. BMSCs were isolated and cultured with whole bone marrow culture method; the second generation of cells were randomly divided into 5 groups: control group (complete medium, CM), simvastatin group (simvastatin medium, SIM), osteogenic induction group (osteogenic induction medium, OM), simvastatin and osteogenic induction group (simvastatin+osteogenic induction medium, OM+SIM), and blocker group (SB203580+simvastatin+osteogenic induction medium, OM+SIM+SB). MTT assay was used to detect the cell activity in CM group and SIM group at 2, 3, 4, 5, and 6 days, ELISA method to measure the content of alkaline phosphatase (ALP) in OM group and OM+SIM group at 7 and 14 days. The mRNA and protein expressions of osteocalcin (OCN) were detected by real-time quatitative PCR and Western blot after 1, 12, and 24 hours of osteogenic induction at 21 and 28 days. The protein expressions of phospho-p38 (p-p38) and p38 in OM group, OM+SIM group, and OM+SIM+SB group were detected by Western blot at the best induction time of simvastatin.

Results: MTT assay showed that no significant difference was found in absorbance (A) value between CM group and SIM group at each time point (P>0.05), indicating no effect of 1×10-7 mol/L simvastatin on cell viability. ELISA results showed that ALP content significantly increased in OM+SIM group when compared with OM group at 7 and 14 days; the ALP content was significantly higher at 7 days than 14 days in OM group and OM+SIM group (P<0.05). OCN mRNA and protein expressions at 12 hours were significantly higher than those at other time points in each group (P<0.05), and the expressions of OM+SIM group was significantly higher than those of OM group (P<0.05). The best induction time of simvastatin was 12 hours. At 12 hours after blocking intervention, the p-p38/p38 in OM+SIM+SB group was significantly lower than that in OM group and OM+SIM group (P<0.05), and the p-p38/p38 in OM+SIM group was significantly higher than that in OM group (P<0.05).

Conclusions: Simvastatin can increase the mRNA and protein expression levels of OCN and the protein of p-p38 in osteogenic differentiation of BMSCs at middle/ late stages, and its best induction time is 12 hours.

目的: 探讨在成骨诱导环境下，辛伐他汀对BMSCs成骨分化中晚期的作用和对p38MAPK信号通路的影响。.

方法: 取20只雌性SD大鼠双侧股骨和胫骨骨髓，全骨髓培养法分离培养BMSCs并传代，取第2代细胞进行实验。实验分为5组，对照组（CM组）：完全培养基培养；辛伐他汀组（SIM组）：含终浓度为1×10-7 mol/L辛伐他汀的完全培养基培养；成骨诱导培养基组（OM组）：含10 mmol/L β甘油磷酸钠和50 μg/mL抗坏血酸的成骨诱导培养基培养；辛伐他汀和成骨诱导培养基组（OM+SIM组）：含终浓度为1×10-7 mol/L辛伐他汀成骨诱导培养基培养；阻断剂组（OM+SIM+SB组）：先用p38MAPK信号通路阻断剂SB203580干预30 min后，再采用含终浓度为1×10-7 mol/L辛伐他汀成骨诱导培养基培养。MTT法检测CM组和SIM组细胞活性；ELISA法检测OM组及OM+SIM组培养7、14 d细胞ALP含量。应用实时定量PCR和Western blot方法检测OM组和OM+SIM组第21、28天开始成骨诱导培养1、12、24 h后，骨钙素(osteocalcin，OCN)蛋白和mRNA表达水平，从而确定辛伐他汀作用最佳时间点，随后检测该时间点OM组、OM+SIM组和OM+SIM+SB组p38、磷酸化p38（phospho-p38，p-p38）蛋白表达，计算p-p38/p38。.

结果: MTT检测示各时间点，SIM组与CM组吸光度（A）值比较差异无统计学意义（P＞0.05），提示辛伐他汀对BMSCs活性和增殖能力无明显影响。与OM组相比，OM+SIM组7、14 d ALP含量均明显增高，且两组组内7 d ALP含量均高于14 d，差异均有统计学意义（P＜0.05）。实时定量PCR及Western blot检测示，第21、28天开始成骨诱导培养后，各组OCN mRNA及蛋白在12 h时表达量高于其他时间点（P＜0.05），且OM+SIM组明显高于OM组（P＜0.05）；确定辛伐他汀诱导成骨干预的最佳起效时间为12 h。阻断剂干预后，12 h时OM+SIM+SB组p-p38/p38明显低于其他两组（P＜0.05），OM+SIM组高于OM组（P＜0.05）。.

结论: 辛伐他汀的最佳起效时间为给药后12 h，其可以提高BMSCs成骨分化中晚期OCN蛋白、mRNA和p-p38蛋白表达量。.

Keywords: Bone marrow mesenchymal stem cells; Osteocalcin; Osteogenic differentiation; Phospho-p38; Simvastatin.

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