慢病毒介导的Sox-9基因转染对大鼠关节软骨细胞炎性反应的影响

Influence of inflammatory response in chondrocytes of rats by lentivirus mediated Sox-9 gene transduction

目的评价Sox-9基因过表达对IL-β诱导的大鼠关节软骨细胞(RCs)炎性反应的影响,为临床提供参考依据。方法通过慢病毒(Lv-Sox-9)感染的方法在RCs细胞中过表达Sox-9,RT-PCR,Western blot检测病毒感染后大鼠软骨细胞中Sox-9、CollagenⅡ基因mRNA及蛋白含量;将细胞分为3组,细胞对照组,IL-1β诱导组,对照病毒感染且IL-1β诱导组和Sox-9过表达且IL-1β诱导组,将软骨细胞或者病毒感染后的软骨细胞接种于纳米孔氧化铝膜上,正常条件培养24h后,诱导组细胞培养液中添加5μg/L的IL-1β,继续培养3d,CCK-8法检测细胞增殖活性变化、扫描电镜观察细胞形态及代谢变化。结果通过慢病毒感染的方法可以在大鼠关节软骨细胞中有效上调Sox-9基因的表达,病毒感染后72h,Lv-Sox-9感染组细胞内Sox-9mRNA和蛋白含量均显著高于细胞对照组和对照病毒组;检测结果还显示,Rcs细胞内CollagenⅡ基因在转录和蛋白表达水平均与Sox-9正相关。结论 Sox-9可以作为抗炎治疗的潜在目标基因。

OBJECTIVE To evaluate the effects of lentivirus mediated Sox-9gene transduction on the inflammatory response in rat chondrocytes（RCs）induced by IL-1β,so as to provide evidence for clinic.METHODS Firstly,RCs were infected with Lv-Sox-9.Real Time PCR and Western blotting were performed to quantitatively detect the relative mRNA level and protein level of Sox-9and CollagenⅡ of RCs.The cells were divided into 3groups,cells control,cells induced by IL-1β,cells infected with Lv-control and induced by IL-1β,cells infected with Lv-Sox-9and induced by IL-1β.Then we seeded the RCs or the infected RCs to the nano-pore alumina film,and cultured for24 hours in normal condition.Then the IL-1β（5μg/L）was added into culture solution of induced cells group cultured for 3days on normal condition.Finally,CCK-8was used to detect the changes of cell proliferation activity and scanning electron microscope was used to observe the changes fo cell morphology and metabolism.RESULTS The results showed that lentivirus infection could effectively up-regulate Sox-9expression in RCs.After 72 hof infection,the Sox-9mRNA and protein level in Lv-Sox-9infection group were higher than these in cells control group and control virus group.The detection results showed that the transcription and protein level of CollagenⅡgene in RCs both positively related to Sox-9.CONCLUSIONSox-9might be considered as a potential target for anti-inflammatory treatment.