目的 研究炎性微环境下转化生长因子-β1(transforming growth factor-β1,TGF-β1)对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的影响及作用机制.方法 应用肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)体外模拟炎性微环境,检测不同浓度TGF-β1对BMSC增殖活性的影响.实验分为对照组[BMSC+成骨诱导液(os-teogenic medium,OM)]、实验组(BMSC+TGF-β1+OM)和抑制组[BMSC+SB431542(TGF-β1 拮抗剂)+OM].应用 ALP 染色、茜素红染色、实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)及 Western blot 法对BMSCs成骨分化能力和TGF-β信号通路因子Smad2、Smad3进行检测并比较3组间的差异.结果 低浓度TGF-β1作用下BMSCs增殖活性较高,高浓度(50ng/ml)抑制BMSCs增殖活性;成骨诱导7天和10天时,实验组与其他两组比较,ALP染色较深、面积最大;茜素红染色结果显示,实验组体外矿化结节形成均高于其他两组;RT-qPCR检测结果显示,实验组成骨分化基因OPN、RUNX2与ALP表达均增高,TGF-β信号通路中Smad3在实验组表达最高,抑制组表达最低,差异有统计学意义(P＜0.01).Western blot法检测结果显示,实验组内OPN、RUNX2与ALP蛋白表达水平增高,差异有统计学意义(P＜0.05);TGF-β信号通路中Smad3蛋白表达在3组中差异不明显,而p-Smad3在实验组中表达明显增高、抑制组中明显降低,差异有统计学意义(P＜0.01).结论 炎性微环境中TGF-β1通过激活TGF-β/Smad3信号通路促进了 BMSCs成骨分化.
TGF-β1 Promotes the Osteogenic Differentiation of BMSCs through TGF-β1/Smad3 Pathway in Inflammatory Microenviroment
Objective To investigate the effect of transforming growth factor-β1(TGF-β1)on osteogenic differentiation of bone marrow mesenchymal stem cell(BMSC)in inflammatory microenvironment and its mechanism.Methods Tumor necrosis factor-α(TNF-α)was used to stimulate the inflammatory microenvironment in vitro to detect the influence of different concentrations of TGF-β1 on the proliferative activity of BMSCs.The experiment was divided into three groups:control group with BMSCs+osteogenic medium(OM),experimental group with BMSCs+TGF-β1+OM,and inhibitor group with BMSCs+SB431542(TGF-β1 antagonist)+OM.The osteogenic differentiation ability of BMSCs and the key factors Smad2 and Smad3 in the TGF-β signal pathway were tested by alkaline phosphatase(ALP)staining and alizarin red staining,real-time quantitative polymerase chain reaction(RT-qPCR)and West-ern blot,and the differences between the three groups were compared.Results Low concentration of TGF-β1 promoted proliferative ac-tivity of BMSCs,while high concentration of TGF-β1(50ng/ml)inhibited proliferative activity of BMSCs.The ALP staining test showed that a stronger staining and larger area in experiment group were seen at 7days and 10days of osteogenic induction(P＜0.01)compared with the other two groups.The results of alizarin red staining also confirmed the formation of mineralized nodules in experimental group was higher than that in the other two groups.The results of RT-qPCR analysis showed that the expression levels of osteogenic differentiation genes OPN,RUNX2 and ALP were increased in the experimental group.The expression of Smad3 in TGF-β signaling pathway was the highest in the experimental group and the lowest in the inhibition group,and the difference was statistically significant(P＜0.01).West-ern blot results showed that the expression levels of OPN,RUNX2 and ALP protein in the experimental group were increased,and the differences were statistically significant(P＜0.05);The protein expression of Smad3 in TGF-β signaling pathway was no significantly difference among the three groups,while the expression of p-Smad3 was significantly increased in the experimental group and significant-ly decreased in the inhibition group,and the differences were statistically significant(P＜0.01).Conclusion TGF-β1 could promote osteogenic differentiation of BMSCs by activating TGF-β/Smad3signal pathway in inflammatory microenvironment.
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