Kartogenin诱导大鼠骨髓间充质干细胞成软骨分化并促进软骨缺损的修复  

Kartogenin enhances rat cartilage defect repair by promoting chondrogenic differentiation of bone marrow mesenchymal stem cells in rats

作　　者：张博凯 李浩可 夏稳 张超 杨蕊瑗 郑杰徽 王敏 Zhang Bokai;Li Haoke;Xia Wen;Zhang Chao;Yang Ruiyuan;Zheng Jiehui;Wang Min(Department of Orthopaedics,the Second Affiliated Hospital,Army Medical University,Chongqing 400037,China;Department of Jiangbei,the First Affiliated Hospital,Army Medical University,Chongqing 400037,China;The 78156 Troops,400000,China)

机构地区：[1]陆军军医大学第二附属医院骨科,重庆400037 [2]陆军军医大学第一附属医院江北院区(陆军第958医院)派驻通信士官学院门诊部,重庆400037 [3]78156部队,重庆400000

出　　处：《创伤外科杂志》2025年第2期141-148,158,共9页Journal of Traumatic Surgery

基　　金：重庆市研究生创新课题(CYB23289)。

摘　　要：目的研究Kartogenin(KGN)对大鼠软骨缺损修复效果,以及其诱导骨髓间充质干细胞(BMSCs)成软骨分化过程中的具体作用及发挥作用的可能机制。方法12只8周龄SD雄性大鼠,于滑车沟处构建直径为1mm,深度为2mm膝关节软骨缺损模型,随机分为KGN组和对照组,各6只。KGN组缺损处填入含有KGN的水凝胶,对照组则使用单纯水凝胶。分别于4、8周处死大鼠(每组3只),分离股骨髁,采用国际软骨修复协会(ICRC)评分标准评估大体治疗效果。使用HE染色、番红固绿染色检测软骨缺损组织学修复情况。在体外实验中,体外培养BMSCs,KGN组加入终浓度为100 nM的KGN,对照组予以DMSO溶剂对照。通过PCR及Western blot检测ColⅡ、ACAN、核心结合因子β(CBF-β)表达量。同时进行真核细胞转录组分析。结果体内实验,伤后4周KGN组软骨缺损修复ICRS评分(5.5±0.5)分、Wakitan评分(5.5±0.5)分明显优于对照组[(3.0±1.0)分、(11.5±0.5)分],差异有统计学意义(P<0.05)。伤后8周KGN组软骨缺损修复ICRS评分(10.8±0.8)、Wakitan评分(2.5±0.5)分明显优于对照组[(7.2±0.8)分、(6.8±0.3)分],差异有统计学意义(P<0.05)。体外实验,KGN处理BMSCs后,软骨外基质蛋白ColⅡ、ACAN相关mRNA表达和蛋白水平显著增高,提示KGN能促进BMSCs成软骨分化。为探究KGN的作用机制,蛋白免疫印迹及rt-PCR检测发现核心结合因子β(CBF-β)蛋白及mRNA水平在加入KGN后增高,并且免疫荧光提示CBF-β进入细胞核的量显著增加(P<0.05)。真核细胞转录组分析提示,BMSCs经过KGN处理后,成软骨分化关键通路ERK通路被激活。结论KGN能促进大鼠关节软骨缺损修复,其原因可能为通过调控CBF-β表达及入核,促进间充质干细胞成软骨分化。Objective To investigate the specific role and potential mechanisms of Kartogenin(KGN)in the chondrogenic differentiation of rat bone mesenchymal stem cells(BMSCs).Methods A total of 12 eight-week-old male SD rats were used to create a knee joint cartilage defect model(diameter of 1 mm and depth of 2 mm)at the trochlear groove.Rats were randomly divided into KGN group and control group,with 6 rats in each group.The defect site in the KGN group was filled with 20 mg hydrogel containing KGN(1 mg KGN∶31 PBS∶200 mg hydrogel),while the control group was treated with hydrogel only.At 4 and 8 weeks,rats were sacrificed(3 rats at each time point).The femoral condyles were separated,and the gross therapeutic effects were evaluated.The International Cartilage Regeneration&Joint Preservation Society(ICRS)score and Wakitan score as well as HE staining and safranin O-fast green staining were used to assess the repair of the cartilage defect.For the in vitro experiments,BMSCs were cultured:the KGN group with a final concentration of 100 nM KGN and the control group with dimethyl sulfoxide.Expression levels of CollagenⅡ(ColⅡ),Aggrecan(ACAN),and core-binding factorβ(CBF-β)were detected by PCR and western blot.Concurrently,eukaryotic cell transcriptome analysis was performed.Results For the in vivo experiments,the KGN group showed much better cartilage defect repair at both 4 weeks(ICRS score:5.5±0.5 vs.3.0±1.0;Wakitan score:5.5±0.5 vs.11.5±0.5)and 8 weeks after treatment(ICRS score 10.8±0.8 vs.7.2±0.8;Wakitan score:2.5±0.5 vs.6.8±0.3),all P<0.05 compared with control group.For the in vitro experiments,after treatment of BMSCs with KGN,the expression of cartilage extracellular matrix proteins,i.e.,ColⅡand ACAN,significantly increased at both mRNA and protein levels.This suggests that KGN could promote chondrogenic differentiation of BMSCs.To explore the mechanism of action of KGN,protein immunoblotting and RT-PCR detected increased levels of CBF-βprotein and mRNA after the adding of KGN,and immunofluorescence

关 键 词：软骨缺损 Kartogenin 骨髓间充质干细胞 核心结合因子β 大鼠 

分 类 号：R68[医药卫生—骨科学]