Toll样受体激活对间充质干细胞免疫调节功能的影响

Effect of Toll-like Receptor Activation on Immunomodulatory Function of Mesenchymal Stem Cells

目的探讨Toll样受体(Toll-like receptor,TLR)激活对骨髓和脐带两种不同来源间充质干细胞(mesenchymal stem cells,MSC)免疫调节功能的影响,同时比较骨髓MSC与脐带MSC中TLR的表达情况。方法从关节置换术患者废弃的关节中取得骨髓,分离培养骨髓MSC。从健康产妇废弃的脐带中分离培养脐带MSC。采用TLR4激动剂脂多糖(lipopolysaccharide,LPS)、TLR3激动剂聚肌胞苷酸[polyinosinic-polycytidylic acid,Poly(I:C)]分别预处理骨髓MSC或脐带MSC,然后与健康志愿者外周血单个核细胞(peripheral blood mononuclear cells,PBMC)共培养,共培养结束后流式细胞术检测PBMC增殖情况。以实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测骨髓MSC和脐带MSC中TLR4、TLR3的基因表达水平。流式细胞术检测骨髓MSC和脐带MSC中TLR4、TLR3的蛋白表达水平。结果骨髓MSC与PBMC共培养实验结果显示,LPS预处理可以明显降低骨髓MSC对PBMC增殖的抑制效应,Poly(I:C)预处理对骨髓MSC免疫调节功能无显著影响。脐带MSC与PBMC共培养实验结果显示,LPS或Poly(I:C)预处理对脐带MSC抑制PBMC增殖的影响均不显著。与骨髓MSC比较,脐带MSC中TLR4、TLR3的基因和蛋白表达水平均明显更低。结论TLR4激活可以干扰骨髓MSC的免疫调节功能,但是TLR4或TLR3激活对脐带MSC免疫调节功能的影响并不显著,可能与脐带MSC中TLR4、TLR3的基础表达量低有关。

Objective To investigate the effect of Toll-like receptor(TLR)activation on the immunomodulatory function of mesenchymal stem cells(MSC)from bone marrow and umbilical cord,and to compare the expression of TLR in bone marrow MSC and umbilical cord MSC.Methods Bone marrow was obtained from the abandoned joints of patients undergoing joint replacement,and bone marrow MSC were isolated and cultured.Umbilical cord MSC were isolated and cultured from the abandoned umbilical cord of healthy pregnant women.Bone marrow MSC or umbilical cord MSC were pretreated with TLR4 ligand lipopolysaccharide(LPS)and TLR3 ligand polyinosinic-polycytidylic acid[poly(I:C)]respectively,and then co-cultured with peripheral blood mononuclear cells(PBMC)of healthy volunteers.After co-culture,the proliferation of PBMC was detected by flow cytometry.The gene expression levels of TLR4 and TLR3 in bone marrow MSC and umbilical cord MSC were detected by real-time quantitative polymerase chain reaction(RT-qPCR).The protein expression levels of TLR4 and TLR3 were detected by flow cytometry.Results The co-culture experiment of bone marrow MSC and PBMC showed that LPS pretreatment could significantly reduce the inhibitory effect of bone marrow MSC on the proliferation of PBMC,and poly(I:C)pretreatment had no significant effect on the immunomodulatory function of bone marrow MSC.The co-culture experiment of umbilical cord MSC and PBMC showed that LPS or poly(I:C)pretreatment had no significant effect on the inhibition of PBMC proliferation by umbilical cord MSC.Compared with bone marrow MSC,the gene and protein expression levels of TLR4 and TLR3 in umbilical cord MSC were significantly lower.Conclusion TLR4 activation can interfere with the immunomodulatory function of bone marrow MSC.However,TLR4 or TLR3 activation has no significant effect on the immunomodulatory function of umbilical cord MSC,which may be related to the low expression of TLR4 and TLR3 in umbilical cord MSC.