金匮肾气丸调控FNDC5、BMP2对BMSCs成骨分化的作用

摘要

目的 研究金匮肾气丸通过调控FNDC5、BMP2基因对BMSCs成骨分化的影响.方法 采用全骨髓贴壁法培养BMSCs,分别用大鼠空白血清和高、低剂量金匮肾气丸干预BMSCs成骨分化过程.采用CCK8检测金匮肾气丸对BMSCs的增殖、毒性作用;AKP活性检测细胞上清成骨分化活性;ALP染色检测细胞成骨分化能力;反转录-实时荧光定量技术(RT-qPCR)检测FNDC5、BMP2 mRNA表达;Western-blotting检测FNDC5、BMP2蛋白表达.结果 CCK8结果示,从第1天到第7天,各组细胞增殖整体呈上升趋势,7天后各组细胞增殖呈下降趋势,且各组增殖(OD值)无统计学差异.含药血清干预BMSCs 3天时,SG组AKP活性明显高于CON(P ＜0.01),SD组与CON组差异虽无统计学意义,但高于CON组;7天时,SG和SD两组AKP活性均高于CON组(P＜0.05);ALP染色结果示:SG组和SD组ALP染色阳性率均高于CON组,而SG组ALP染色率高于SD组.干预3天时,SD组FNDC5 mRNA表达较CON、SG组显著上调(P＜0.05);7天时,SD、SG组FNDC5mRNA表达均较CON组显著上调(P＜0.05),SD组高于SG组(P＜0.05).3、7天时SG、SD组BMP2 mRNA表达均较CON组显著上调(P＜0.01).干预第3、7天时,SG、SD组FNDC5、BMP2蛋白水平均明显高于CON组(P＜0.05).结论 金匮肾气丸含药血清可能通过上调FNDC5、BMP2基因水平来促进BMSCs成骨增殖、分化.%Objective To investigate the effect of Jinkui Shenqi Pills on osteogenic differentiation of BMSCs by regulating FNDC5 and BMP2 genes.Methods BMSCs were cultured by whole bone marrow adherence method.The osteoblast differentiation process of BMSCs was treated with blank serum and high and low dose of Jinkui Shenqi Pills,respectively.The proliferation and toxicity of Jinkui Shenqi Pills on BMSCs were detected by CCK8.The osteogenic differentiation activity of cell supernatant was detected by AKP activity.The osteogenic differentiation ability of cells was detected by ALP staining.RT-qPCR was used to detect the mRNA expression of FNDC5 and BMP2.Western blotting was used to detect the protein expression of FNDC5 and BMP2.Results CCK8 result showed that the cell proliferation in each group increased from day 1 to day 7,and the proliferation of each group showed a decreasing trend after 7 days,and there was no significant difference in proliferation (OD).The activity of AKP in SG group was significantly higher than that in the CON groups (P ＜ 0.01).The difference between SD group and CON group were not statistically significant,but SD group was higher than CON group.At the 7th day,the activity of AKP of SG and SD groups were higher than CON group (P ＜ 0.05).The positive rate of ALP staining in SG group and SD group were higher than that in CON group,while the ALP staining rate in SG group was higher than that in SD group.The expression of FNDC5 mRNA in SD group were significantly higher than that in CON group and SG group (P ＜ 0.05).While there was no significant difference between SD group and CON group (P ＜ 0.05),SD group was higher than SG group (P ＜ 0.05).The expression of BMP2 mRNA in the SG and SD groups were significantly higher than that in CON group at the 3th and 7th day (P ＜0.O1).The levels of FNDC5 and BMP2 in SG and SD groups were significantly higher than those in CON group (P ＜ 0.05).Conclusion The serum of Jinkui Shenqi Pills may promote the osteogenesis and differentiation of BMSCs by up-regulating FNDC5 and BMP2 gene levels.