AID对激素性股骨头坏死骨髓间充质干细胞的影响  

Effect of AID on bone marrow mesenchymal stem cells in glucocorticoid-induced osteonecrosis of the femoral head

作　　者：孙志博 马中希 叶志伟 刘珍星 杨钟华 张山锋 SUN Zhi-bo;MA Zhong-xi;YE Zhi-wei;LIU Zhen-xing;YANG Zhong-hua;ZHANG Shan-feng(Department of Orthopedics,Peopled Hospital of Wuhan University,Wuhan 430060,China;Department of Orthopedics,Puai Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430034,China)

机构地区：[1]武汉大学人民医院骨科,湖北武汉430060 [2]武汉市第四医院、华中科技大学同济医学院附属普爱医院骨科,湖北武汉430034

出　　处：《中国矫形外科杂志》2021年第23期2168-2172,共5页Orthopedic Journal of China

基　　金：武汉市卫计委资助项目(编号:WX16C28);国家自然科学基金资助项目(编号:81501894);中央高校基本科研业务费专项资金(编号:2042020kf0054)。

摘　　要：[目的]探讨活化诱导胞喃陡核苷酸脱氨酶(activation-induced cytidine deaminase,AID)对激素性股骨头坏死(glucocorticoid-induced osteonecrosis of the femoral head,GC-ONFH)骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-SCs)的影响。[方法]采集16例GC-ONFH和16例股骨颈骨折患者的股骨骨髓,分离培养BMSCs至第3代。细胞分为4组:A组为GC-ONFH源BMSCs,B组为GC-ONFH源BMSCs并经AID重组慢病毒转染,C组为GC-ONFH源BMSCs经慢病毒空载体转染,D组为股骨颈骨折BMSCs。检测比较4组的细胞增殖、线粒体膜电位、细胞内活性氧水平、ABCB1启动子区甲基化,以及AID和ABCB1的mRNA和蛋白表达水平。[结果]8组MTT检测OD值显著高于A和C组(P<0.05),但仍低于D组(P<0.05)。B组线粒体膜电位较A、C组明显提高(P<0.05),但仍低于D组(P<0.05)。B组的ROS水平较A、C组显著降低(P<0.05),但仍显著高于D组(P<0.05)。B组ABCB1启动子区甲基化率较A、C组明显降低(P<0.05),接近于D组水平(P>0.05)。B组的AID和P-gp蛋白表达显著高于A组及C组(P<0.05),稍低于D组(P>0.05)。B组AIDmRNA和AB-CB1 mRNA表达量显著高于A组和C组(P<0.05),接近D组水平(P>0.05)。[结论]与股骨颈骨折相比,激素性股骨头坏死AID表达明显降低,过表达AID能显著地降低ABCB1过甲基化修饰并改善细胞功能。[Objective]To explore the effects of activation-induced cytidine deaminase(AID)on bone marrow mesenchymal stem cells(BMSCs)in glucocorticoid-induced osteonecrosis of the femoral head(GC-ONFH).[Methods]The femoral bone marrows were harvested from 16 patients with GC-ONFH and 16 patients with femoral neck fracture to isolate the BMSCs and culture to the third generation.The cells were divided into four groups:group A was of BMSCs derived from GC-ONFH,group B was of BMSCs from GC-ONFH and transfected with AID recombinant lentivirus,group C was BMSCs derived from GC-ONFH transfected with lentiviral empty vector,and group D was BMSCs form femoral neck fracture.The cell proliferation,mitochondrial membrane potential(MMP),intracellular reactive oxygen species(ROS),and methylation of ABCB1 promoter region,expression of AID and ABCB1 in mRNA and protein levels were assayed and compared among the 4 groups.[Results]The OD value of MTT assay in group B was significantly higher than those in groups A and C,but still lower than that in group D(P<0.05).The MMP of group B was significantly higher than those of groups A and C(P<0.05),nevertheless it was still lower than that of group D(P<0.05).The ROS level of group B was significantly lower than those of groups A and C(P<0.05),whereas was still significantly higher than that of group D(P<0.05).The methylation rate of ABCB1 promoter region in group B was significantly lower than those in groups A and C(P<0.05),which was close to the level of group D(P>0.05).The expression of AID and P-gp protein in group B was significantly higher than those in group A and C(P<0.05),and slightly lower than those in group D(P>0.05).The ex-pression of AID mRNA and ABCB1 mRNA in group B was significantly higher than those in group A and C(P<0.05),and was close to the level of group D(P>0.05).[Conclusion]Compared with femoral neck fractures,the expression of AID in GC-ONFH significantly reduces.Overexpression of AID can significantly reduce ABCB1 hypermethylation and improve cell function.

关 键 词：激素性股骨头坏死 骨髓间充质干细胞 活化诱导胞嘧啶核苷酸脱氨酶(AID) 甲基化 

分 类 号：R318[医药卫生—生物医学工程]