腺相关病毒介导核心结合蛋白因子2对小鼠退行性膝关节炎的保护作用

Protective effect of related transcription factor-2 mediated by adeno-associated virus on degenerative knee arthritis in mice

目的观察腺相关病毒介导核心结合蛋白因子2(Runx2)对小鼠退行性膝关节炎的修复作用。方法60只大鼠(购自上海斯莱克实验动物有限公司)随机分为假手术组、模型组和Runx2组,模型组和Runx2组小鼠通过小鼠右后肢膝关节前交叉韧带切断手术,建立退行性膝骨关节炎小鼠模型。假手术组小鼠仅做手术,不切断交叉韧带。小鼠建模成功4周后,假手术组和模型组小鼠膝关节腔注射携带空载体的阴性腺相关病毒。Runx2组小鼠膝关节腔注射携带Runx2基因的腺相关病毒。注射21 d后,分析3组小鼠攀爬时间和长距离运动能力;分析关节软骨和滑膜评分的变化;采用酶联免疫吸附试验(ELISA)试剂盒检测3组小鼠肿瘤坏死因子-α(TNF-α)和白细胞介素(IL)-1β水平;采用荧光定量聚合酶链反应(PCR)分析3组小鼠软骨组织中OSX和骨保护素(OPG)mRNA表达水平。计量数据比较采用单因素方差分析,组建两两比较采用LSD法。结果与模型组小鼠攀爬时间和长运动距离[(539.11±79.21)s和(68.20±13.99)m]比较,Runx2组[(1401.72±102.83)s和(201.58±17.92)m]明显增加,差异有统计学意义(t=9.102,P<0.05;t=6.119,P<0.05)。与模型组小鼠膝关节软骨评分和膝关节滑膜评分[(7.51±1.82)分和(7.25±1.93)分]比较,Runx2组[(5.98±1.86)分和(6.01±1.62)分]明显下降,差异有统计学意义(t=6.195,P<0.05;t=5.018,P<0.05)。与模型组小鼠外周血TNF-α和IL-1β水平[(251.50±43.19)ng/L和(271.57±32.19)ng/L]比较,Runx2组[(78.54±8.19)ng/L和(98.40±10.32)ng/L]明显下降,差异有统计学意义(t=7.001,P<0.05;t=7.193,P<0.05)。与假手术组和模型组骨桥蛋白(OPN)mRNA水平(1.00±0.19和0.94±0.17)比较,Runx2组(2.55±0.23)显著增加,差异有统计学意义(t=2.209,P<0.05;t=2.413,P<0.05)。与假手术组和模型组OSX mRNA水平(0.98±0.14和1.09±0.21)比较,Runx2组(3.01±0.20)显著增加,差异有统计学意义(t=2.501,P<0.05;t=2.216,P<0.05)。结论腺相关病毒介导Runx2可降低退行性膝关节炎小鼠的炎性因子水平,促进成骨细胞成熟,降低膝关节软骨和滑膜的病理变化,提高小鼠运动能力。

Objective To investigate the repair effect of related transcription factor-2(Runx2)mediated by adeno-associated virus on degenerative knee arthritis in mice.Methods Sixty mice were randomly divided into three groups:sham operation group,model group and Runx2 group.The animals in model group and Runx2 group were operated on by cutting the anterior cruciate ligament of the right hind leg of the mouse to establish the model of degenerative knee osteoarthritis.In the sham operated group,only the operation was performed,and the cruciate ligament was not cut.Four weeks after the successful modeling of mice,the mice in the sham operated group and the model group were injected with negative adeno-associated virus carrying empty vector into the knee joint cavity,and those in the Runx2 group were injected with adeno-associated virus carrying the Runx2 gene.At 21st day after injection,the climbing time and long-distance movement ability of mice in the three groups were analyzed.The changes in articular cartilage and synovium scores were analyzed.The levels of tumor necrosis factor-α(TNF-α)and interleukin(IL)-1βin the three groups were detected by enzyme linked immunosorbent assay(ELISA)kit.The levels of OSX and bone protection element(OPG)mRNA in the cartilage tissues of the three groups were analyzed by fluorescence quantitative polymerase chain reaction(PCR).Results As compared with the model group[(539.11±79.21)s and(68.20±13.99)m],the climbing time and the long exercise distance in Runx2 group[(1401.72±102.83)s and(201.58±17.92)m]significantly increased(t=9.102,P<0.05;t=6.119,P<0.05).As compared with the model group(7.51±1.82 and 7.25±1.93),the score of knee joint cartilage and synovium in Runx2 group(5.98±1.86 and 6.01±1.62)significantly decreased(t=6.195,P<0.05;t=5.018,P<0.05).As compared with the model group[(251.50±43.19)and(271.57±32.19)ng/L],the levels of TNF-αand IL-1βin the Runx2 group[(78.54±8.19)and(98.40±10.32)ng/L]significantly decreased(t=7.001,P<0.05;t=7.193,P<0.05).As compared with sham operated group and model group(1.00±0.19 and 0.94±0.17),osteopontin(OPN)mRNA level in Runx2 group(2.55±0.23)significantly increased(t=2.209,P<0.05;t=2.413,P<0.05).As compared with the sham operated group and the model group(0.98±0.14 and 1.09±0.21),the OXS mRNA level in the Runx2 group(3.01±0.20)significantly increased(t=2.501,P<0.05;t=2.216,P<0.05).Conclusion Adeno-associated virus-mediated Runx2 can significantly reduce the level of inflammatory factors,promote the maturation of osteoblasts,reduce the pathological changes of cartilage and synovium of knee joint,and improve the motor ability of mice.