骨髓间充质干细胞通过抑制破骨细胞相关因子改善膝关节骨性关节炎

Bone marrow mesenchymal stem cells improve osteoarthritis of the knee by inhibiting osteoclast-related factors

目的探讨骨髓间充质干细胞是否通过抑制破骨相关因子改善膝关节骨性关节炎。方法将40只健康SPF级SD大鼠随机分为空白组、模型组、甲氨蝶呤组和骨髓间充质干细胞组,每组8只,剩余8只用于骨髓间充质干细胞的分离。除空白组外,其余组采用木瓜蛋白酶关节腔注射建立膝关节骨性关节炎大鼠模型。造模成功后第2天起空白组和模型组大鼠膝关节腔注射生理盐水0.3 ml;甲氨蝶呤组大鼠腹腔注射甲氨蝶呤(0.5 mg/kg);骨髓间充质干细胞组大鼠膝关节腔内一次性注射骨髓间充质干细胞0.3 ml,同时每周1次膝关节腔内注射生理盐水0.3 ml,每周1次,共4次。4次干预结束后各组大鼠进行关节指数(AI)评分;苏木素-伊红(HE)染色观察软骨组织病理学变化;酶联免疫吸附法(ELISA)检测血清RANKL、OPG、CXCL10含量;实时荧光定量(qRT-PCR)检测软骨组织RANKL、OPG、TRAF6、CXCL10、CXCR3 mRNA表达;蛋白质印迹法(Western blot)检测软骨组织RANKL、OPG、TRAF6、CXCL10、CXCR3蛋白表达。结果与空白组比较,模型组大鼠AI评分,血清RANKL、OPG、CXCL10含量,软骨组织RANKL、OPG、TRAF6、CXCL10、CXCR3 mRNA和蛋白表达明显升高(P<0.05)。与模型组比较,甲氨蝶呤组大鼠AI评分、血清OPG、CXCL10含量及软骨组织TRAF6、CXCL10、CXCR3蛋白表达明显降低(P<0.05);骨髓间充质干细胞组大鼠AI评分、血清RANKL、OPG、CXCL10含量、软骨组织RANKL、OPG、TRAF6、CXCL10、CXCR3 mRNA和蛋白表达明显降低(P<0.05)。与甲氨蝶呤组比较,骨髓间充质干细胞组大鼠AI评分、血清RANKL、OPG、CXCL10含量、软骨组织RANKL、OPG、TRAF6、CXCL10、CXCR3 mRNA和蛋白表达均差异无统计学意义(P>0.05)。结论骨髓间充质干细胞明显改善膝关节骨性关节炎病理损伤,作用效果与甲氨蝶呤作用效果相当,可能通过调节RANKL/OPG/TRAF6信号通路发挥对膝关节骨性关节炎大鼠的治疗作用。

Objective To investigate whether bone marrow mesenchymal stem cells(BMSCs)improve the knee osteoarthritis by inhibiting osteoclast-related factors.Methods Forty healthy SPF-grade SD rats were randomly divided into control group,model group,methotrexate group and bone marrow MSC group,with 8 rats in each group,and the remaining 8 rats were used for the isolation of BMSCs.Except for control group,the rats in the other groups were injected with papain to establish the rat model of knee osteoarthritis via joint cavity.From the second day after successful modeling,rats in control group and model group were injected with saline 0.3 ml into the knee joint cavity;rats in methotrexate group were injected intraperitoneally with methotrexate(0.5 mg/kg);and rats in bone marrow MSC group were injected with BMSCs 0.3 ml into the knee joint cavity,and simultaneously injected with saline 0.3 ml into the knee joint cavity once a week for 4 weeks.The joint index(AI)score of rats was performed after the intervention,HE staining was performed to observe the histopathological changes of cartilage,ELISA was performed to detect serum RANKL,OPG and CXCL10,qRT-PCR was performed to detect the expression of RANKL,OPG,TRAF6,CXCL10 and CXCR3 mRNA in cartilage tissue,and Western blot was performed to detect the expression of RANKL,OPG,TRAF6,CXCL10 and CXCR3 in cartilage tissue.Results Compared with control group,AI score,serum RANKL,OPG,CXCL10 contents,and mRNA and protein expression of RANKL,OPG,TRAF6,CXCL10,CXCR3 in cartilage tissue were significantly increased in model group(P<0.05).Compared with model group,AI score,serum OPG,CXCL10 contents and TRAF6,CXCL10 and CXCR3 protein expression in cartilage tissue were significantly reduced in methotrexate group(P<0.05).Compared with model group,AI score,serum RANKL,OPG,CXCL10 contents,and RANKL,OPG,TRAF6,CXCL10,CXCR3 mRNA and protein expression in cartilage tissue were significantly reduced in bone marrow MSC group(P<0.05).Compared with methotrexate group,there were no statistically significant changes in AI score,serum RANKL,OPG,CXCL10 contents,RANKL,OPG,TRAF6,CXCL10,CXCR3 mRNA and protein expression in cartilage tissue in bone marrow MSC group(P>0.05).Conclusion BMSCs can significantly improve the pathological damage of knee osteoarthritis,and the effect is equivalent to that of methotrexate.BMSCs may exert therapeutic effects on knee osteoarthritis by regulating the RANKL/OPG/TRAF6 signaling pathway.