论文部分内容阅读
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p 38 MAPK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/Lβ甘油磷酸钠和50μg/m L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p 38 MAPK信号通路阻断剂SB 203580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m RNA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p 38、磷酸化p 38(phospho-p 38,p-p 38)蛋白表达,计算p-p 38/p 38。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 m RNA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-p 38/p 38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、m RNA和p-p 38蛋白表达量。
Objective To investigate the effect of simvastatin on mid-late osteogenic differentiation of BMSCs and the effect on p38 MAPK signaling in osteogenic environment. Methods Twenty female Sprague-Dawley rats were divided into two groups. BMSCs were isolated and passaged by whole bone marrow culture. The second passage cells were used for experiments. The experimental group was divided into 5 groups, the control group (CM group): complete culture medium; the simvastatin group (SIM group): the complete medium containing simvastatin at the final concentration of 1 × 10-7 mol / L; Induction medium group (OM group): osteogenic induction medium containing 10 mmol / L sodium β-glycerophosphate and 50 μg / mL ascorbic acid; simvastatin and osteogenic induction medium group (OM + SIM group) (1 × 10-7 mol / L simvastatin osteogenic induction medium); Blocker group (OM + SIM + SB group): first with p38 MAPK signal pathway blocker SB203580 intervention for 30 min, And then with the final concentration of 1 × 10-7 mol / L simvastatin osteogenic induction medium culture. Cell viability was measured by MTT assay in CM and SIM groups. ALP levels in OM and OM + SIM groups at 7 and 14 days were detected by ELISA. The mRNA and protein expression of osteocalcin (OCN) and m RNA in OM group and OM + SIM group were detected by real-time PCR and Western blot at 1, 12 and 24 h after osteoblast induction at 21 and 28 days, respectively The best time point of simvastatin was determined. The expression of p 38 and phospho-p 38 (pp 38) protein in OM group, OM + SIM group and OM + SIM + SB group at this time point was detected, and pp 38 / p 38. Results MTT assay showed no significant difference in absorbance (A) between SIM and CM groups at all time points (P> 0.05), suggesting that simvastatin had no effect on BMSCs activity and proliferation. Compared with the OM group, ALP levels in the OM + SIM group increased significantly at 7 and 14 d, and the levels of ALP in the two groups were all higher than those at 14 d (P <0.05). Real-time quantitative PCR and Western blot showed that OCN m RNA and protein expression in each group were higher than those at other time points (P <0.05) after osteoblasty induction on the 21st and 28th day, and OM + SIM group Significantly higher than that of OM group (P <0.05). The optimal onset time of simvastatin induced osteogenesis was 12 h. The p-p38 / p38 of OM + SIM + SB group was significantly lower than that of the other two groups (P <0.05) after 12 h intervention, and OM + SIM group was higher than OM group (P <0.05). Conclusion The best onset time of simvastatin is 12 h after administration, which can enhance the expression of OCN protein, m RNA and p-p38 protein in osteogenic differentiation of BMSCs.