外泌体通过NF-κB信号通路调节骨关节炎的炎症反应

Exosomes modulate immune responses in osteoarthritis through NF-κB signaling pathway

目的:探讨脐带间充质干细胞来源的外泌体对骨关节炎软骨细胞的免疫调节作用。方法:提取并鉴定脐带间充质干细胞与外泌体;脂多糖(LPS)构建骨关节炎细胞模型;实验分为软骨细胞组(Control组)、软骨细胞+40 ng/ml LPS组(LPS组)、软骨细胞+40 ng/ml LPS+10μg/ml外泌体组(LPS+Exo组),流式细胞术、ELISA分别检测三组细胞凋亡、炎症因子表达;qRT-PCR、Western blot检测三组软骨生成相关基因和蛋白表达;细胞免疫荧光检测NF-κB信号通路的激活,Western blot检测相关蛋白表达。结果:成功提取并鉴定脐带间充质干细胞及其分泌的外泌体;与LPS组相比,LPS+Exo组软骨细胞凋亡率降低,IL-6、TNF-α、NO表达量明显减少(P<0.05),软骨细胞CoL2A mRNA表达水平显著升高(P<0.05),CoL2A、AGC-1蛋白表达水平显著提高(P<0.05),MMP-13蛋白表达水平显著降低(P<0.05);细胞核中p65蛋白明显增多,p-p65与p65蛋白比值降低。结论:脐带间充质干细胞的外泌体对骨关节炎软骨细胞具有免疫调节作用,并可抑制NF-κB信号通路激活。

Objective:To investigate immunomodulatory effect of umbilical cord mesenchymal stem cell-derived exosomes on osteoarthritis chondrocytes.Methods:Umbilical cord mesenchymal stem cells and exosomes were extracted and identified;lipopolysaccharide(LPS)was used to construct osteoarthritis cell model.Experiment was divided into chondrocyte group(Control group),chondrocyte+40 ng/ml LPS group(LPS group)and chondrocyte+40 ng/ml LPS+10μg/ml exosome group(LPS+Exo group).Cell apoptosis and inflammatory cytokines expressions were detected by flow cytometry and ELISA in three groups,respectively.qRT-PCR and Western blot were used to detect expressions of chondrogenesis related genes and proteins in three groups.Activation of NF-κB signaling pathway was detected by immunofluorescence,and expressions of related proteins were detected by Western blot.Results:Umbilical cord mesenchymal stem cells and exosomes were successfully extracted and identified.Compared with LPS group,apoptosis rate of chondrocytes in LPS+Exo group was decreased,expression levels of IL-6,TNF-αand NO were significantly decreased(P<0.05),expression level of CoL2A mRNA in chondrocytes was significantly up-regulated(P<0.05),protein expressions of CoL2A and AGC-1were significantly increased(P<0.05).Expression level of MMP-13 protein was significantly decreased(P<0.05);p65 protein was significantly increased in nucleus,and ratio of p-p65 to p65 protein was decreased.Conclusion:Exosomes of umbilical cord mesenchymal stem cells have immunomodulatory effects on osteoarthritis chondrocytes,and can inhibit the activation of NF-κB signaling pathway.