PPARβ/δ激动剂联合人自体关节液来源的间充质干细胞治疗骨关节炎的实验研究

Peroxisome proliferator-activated receptor β/δ agonists combined with autologous mesenchymal stem cells derived from human synovial fluid in the treatment of osteoarthritis

目的探讨过氧化物酶体增殖物激活受体(PPAR)β/δ激动剂(GW0742)对人关节液来源的间充质干细胞(hSF-MSC)成软骨分化及炎性环境下炎症因子释放的影响。方法抽取膝关节骨关节炎患者患侧关节腔内的关节液并分离培养及鉴定。设置空白对照组(不完全成软骨诱导培养基)、转化生长因子(TGF)-β1组(含浓度为10ng/mL TGF-β1的成软骨诱导培养基)、TGF-β1+GW0742组(含10ng/mL TGF-β1、1μmol/L GW0742的成软骨诱导培养基)及GW0742组(含浓度为1μmol/L GW0742的成软骨诱导培养基),采用Pellet法诱导hSF-MSC成软骨分化。通过比较各组成软骨分化相关标志物SOX-9基因表达和蛋白合成以及阿利新蓝、甲苯胺蓝染色等来评估GW0742对hSF-MSC增殖及成软骨分化能力的影响。另设置空白对照组(20%关节液+80%完全培养基)、hSF-MSC组(20%关节液+80%完全培养基+1×105个/mL hSF-MSC)、hSF-MSC+GW0742组(20%关节液+80%完全培养基+1μmol/L GW0742+1×105个/mL hSF-MSC),通过比较第1、3天各组培养液中炎症因子肿瘤坏死因子(TNF)-α表达来评估GW0742+hSF-MSC组免疫抑制能力。结果应用直接贴壁法成功从炎性关节液中分离培养hSF-MSC。TGF-β1、TGF-β1+GW0742及GW0742对hSF-MSC增殖能力无明显影响;聚合酶链反应(PCR)和Western Blot检测表明,GW0742组hSF-MSC成软骨分化作用最为明显;甲苯胺蓝和阿利新蓝染色显示,GW0742组hSF-MSC能分泌较多的蛋白聚糖,且较TGF-β1+GW0742组细胞分泌的蛋白聚糖更为致密;hSF-MSC+GW0742组炎症因子表达显著低于hSF-MSC组。结论 PPARβ/δ激动剂GW0742不仅能提升hSF-MSC成软骨分化能力,还能增强hSF-MSC抗炎能力。

Objective To investigate the effect of peroxisome proliferator-activated receptor(PPAR)β/δagonists,GW0742,on the chondrogenic differentiation of human synovial fluid-derived mesenchymal stem cells(hSF-MSCs)and on the release of inflammatory cytokines in inflammatory milieu.Methods Synovial fluid was obtained from the human osteoarthritic knee joints.hSF-MSCs were isolated,cultured and identified.Cultured according to the Pellet method simulating3D condition for chondrogenic differentiation,hSF-MSCs were divided into4groups:blank control group,transforming growth factor(TGF)-β1group(chondrogenic induction medium containing10ng/mL TGF-β1),GW0742group(chondrogenic induction medium containing1μmol/L GW0742),and TGF-β1+GW0742group(chondrogenic induction medium containing10ng/mL TGF-β1+1μmol/L GW0742).The effect of GW0742on the proliferation and chondrogenic differentiation of the hSF-MSCs were evaluated by comparing the changes in the cell proliferation,the cartilage-special marker SOX9gene expression and protein synthesis and Alcian Blue/toluidine blue staining.In another experiment containing the blank control group(20%synovial fluid+80%complete medium),hSF-MSCs group(20%synovial fluid+80%complete medium+1×10^5cells/mL hSF-MSC),hSF-MSCs+GW0742group(20%synovial fluid+80%complete medium+1×10^5cells/mL hSF-MSC+1μmol/L GW0742),the level of TNF-αin the culture medium on day1and day3were measured to assess the immunosuppressive effect of GW0742.Results hSF-MSCs were successfully isolated from the osteoarthritic knee joints and then identified.No significant differences in terms of hSF-MSCs proliferation were found in the TGF-β1group,TGF-β1+GW0742group or GW0742group.According to the expression result of SOX9in polymerase chain reaction(PCR)and Western Blot,GW0742group had the most significant effect on chondrogenic differentiation.However,cells in the GW0742group could secrete more aggrecan than the TGF-β1+GW0742group on the basis of the staining findings.Furthermore,the level of inflammatory cytokines in the hSF-MSCs+GW0742group was significantly lower than those in the hSF-MSCs group.Conclusion PPARβ/δagonist GW0742may not only improve the chondrogenic differentiation of hSF-MSCs,but also enhance the anti-inflammatory ability.