CD4+Foxp3+调节性T细胞对骨髓间充质干细胞成骨分化能力的影响

Effect of CD4+Foxp3+ regulatory T cell on the osteogenic differentiation of bone marrow mesenchymal stem cells

目的研究CD4+Foxp3+调节性T细胞对骨髓间充质干细胞(BMMSC)成骨分化能力的影响。方法实验分为三组,对照组为CD4+初始T细胞和BMMSC;实验组为调节性T细胞与BMMSC;抑制组为调节性T细胞与BMMSC并加入GW4869(外泌体抑制剂)。三组均进行矿化诱导。在0、7 d时碱性磷酸酶(ALP)染色和活性检测ALP表达及活性,在0、21 d时茜素红染色和钙含量分析检测矿化结节形成及钙含量,在7 d时PCR检测骨向分化基因ALP、OCN和Runx2 mRNA水平。结果实验组ALP染色面积及ALP活性高于对照组(P<0.01),茜素红染色矿化结节形成及钙含量高于对照组(P<0.01);ALP、OCN和Runx2 mRNA水平高于对照组(P<0.01)。抑制组ALP染色面积及ALP活性低于实验组(P<0.001),茜素红染色矿化结节形成及钙含量低于实验组(P<0.01),ALP、OCN和Runx2 mRNA水平低于实验组(P<0.01)。结论调节性T细胞能够促进BMMSC成骨分化,其外泌体可能参与了这一过程。

Objective To investigate the effect of CD4+Foxp3+regulatory T cell on the osteogenic differentiation of bone marrow mesenchymal stem cell(BMMSC).Methods The experiment was divided into three groups,the control group consisted of CD4+T cells and BMMSC;the experimental group consisted of regulatory T cells and BMMSC;the inhibitory group consisted of regulatory T cells and BMMSC and added GW4869(exosome inhibitor).Mineralization induction was performed in all three groups.ALP expression and activity were detected by alkaline phosphatase(ALP)staining and activity at day 0 and 7,mineralization nodules formation and calcium content were detected by alizarin red staining and calcium content analysis at day 0 and 21,levels of bone differentiation genes ALP,OCN,and Runx2 mRNA were detected by PCR at day 7.Results the ALP staining area and ALP activity in the experimental group were higher than those in the control group(P<0.01).The formation and calcium content of mineralized nodules stained with alizarin red were higher than those in the control group(P<0.01),and the expression levels of ALP,OCN,and Runx2 mRNA were higher than those in the control group(P<0.01).The ALP staining area and ALP activity in the inhibitory group were lower than those in the experimental group(P<0.01),the formation and calcium content of mineralized nodules stained with alizarin red were lower than those in the experimental group(P<0.01),and the expression levels of ALP,OCN,and Runx2 mRNA were lower than those in the experimental group(P<0.01).Conclusion Tregs could promote the osteogenic differentiation of BMMSC,and Tregs exosomes may participate in this process.