巴戟天多糖通过p38 MAPK信号通路促间充质干细胞成骨分化的体外研究

Analysis of the Osteogenetic Effects Exerted on Mesenchymal Stem Cells by Morindae Officinalis Polysaccharide via p38 MAPK Signaling Pathway in Vitro

目的:探讨巴戟天多糖对间充质干细胞(BMSCs)促成骨作用的影响及该作用与p38MAPK信号通路的相关性。方法:体外骨髓贴壁法培养BMSCs,取第3代细胞,流式细胞仪鉴定细胞表面抗原。实验分为3组:1)对照组(经典成骨诱导),用地塞米松,β-甘油磷酸钠,VitC和无药血清的DMEM/F12培养基体外培养第3代的BMSCs;2)巴戟天多糖(MOP)组,用含巴戟天多糖药物血清的DMEM/F12培养基体外培养第3代的BMSCs;3)阻滞剂组(MOP+SB203580),用p38 MAPK的特异阻滞剂SB203580预干预细胞1h后,加入含巴戟天多糖药物血清的DMEM/F12培养基体外培养第3代的BMSCs。体外培养3d,蛋白印迹法检测p38及其磷酸化产物的蛋白表达及用SB203580阻断p38MAPK信号后巴戟天多糖对RUNX2,OPN的影响;体外培养14d,ALP活性试剂盒检测BMSCs的成骨分化情况;体外培养21d,茜素红染色检测BMSCs钙结节的情况。结果:100g/L巴戟天多糖药物血清培养基为促(BMSCs)成骨的最佳浓度,差异有统计学意义(P<0.01);MOP组钙结节数量明显高于对照组和阻滞剂组,差异有统计学意义(P<0.01);与对照组比较,巴戟天组能提高细胞RUNX2,OPN的蛋白表达,差异有统计学意义(P<0.01),提高了p38蛋白磷酸化表达,差异有统计学意义(P<0.05);p38特异性阻滞剂SB203580阻断p38 MAPK信号后巴戟天多糖促成骨作用明显下降,差异有统计学意义(P<0.01)。结论:巴戟天多糖能促进BMSCs成骨分化,其作用可能与激活p38蛋白的表达有关。

Objective：To investigate the effect of Morindae Officinalis polysaccharide（MOP）in promoting osteogenesis of rat bone marrow mesenchymal stem cells（MSCs）and the correlation with p38 MAPK signaling pathway.Methods：Rat BMSCs were cultured by adherence in vitro.The third generation of cells was collected.Cell surface antigen was identified by flow cytometry.The study includes three groups.1）The control group（classically osteogenic induction method）：the third generation of cells were cultured by DMEM/F12 medium with dexamethasone,β-glycerophosphate,VitC and drug free serum in vitro.2）The MOP group：the third generation of cells were cultured by DMEM/F12 medium with MOP in vitro.3）The blocker group（MOP＋SB203580）：the third generation of cells was pre-intervention by p38 MAPK specific blocker SB203580 for one hour then were cultured by DMEM/F12 medium with MOP in vitro.Western-blot was used to observe the protein and phosphorylation expression of p38 and the influence of MOP on RUNX2,OPN after p38 MAPK blocked by SB203580 after treatment with MOP for 3 days.After culture with different concentration of MOP for 14 days in vitro.Osteogenic differentiation of MSCs were detected by alkaline phosphatase（ALP）activity.Alizarin red staining was used to observe mineralized nodules of MSCs after treatment with MOP for 21 days.Results：MOP at a dose of 100g/L,have the best ability to promote osteogenic differentiation on BMSCs with a significant statistically difference（P〈0.01）.Mineralized nodules of MOP group were obviously higher than the control group with a significant statistically difference（P〈0.01）and RUNX2,OPN of MOP group were obviously higher than the control group with a significant Statistically difference（P〈0.01）and Phospho-p38 of MOP group were higher than the control group with a Statistically difference（P〈0.05）.The indexs of the RUNX2,OPN in blocker group（MOP＋SB203580）were less than those of MOP group（P〈0.01）。Conclusion：MOP promotes differentiation of the mesenchymal stem cells into osteoblasts and its effect is related to the activation of p38 expression in the MAPK signaling pathway.