红细胞生成素基因修饰内皮祖细胞移植对大鼠糖皮质激素性股骨头坏死的影响  

Effect of erythropoietin gene-modified endothelial progenitor cell transplantation on steroid-induced femoral head necrosis in rats

作　　者：杨青 吴磊 YANG Qing;WU Lei(Department of Orthopaedics,Wuhan Fifth Hospital,Wuhan 430050,China)

机构地区：[1]武汉市第五医院骨科,湖北武汉430050

出　　处：《中国药理学与毒理学杂志》2020年第9期677-686,共10页Chinese Journal of Pharmacology and Toxicology

摘　　要：目的观察移植红细胞生成素(EPO)修饰的内皮祖细胞(EPC)对大鼠糖皮质激素性股骨头坏死的影响和机制。方法体外复苏并培养EPC,制备EPC悬液并用羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)进行标记。真核表达质粒pcDNA3-EPO转染EPC,转染后2和4周Western印迹法检测EPC中EPO蛋白表达。SD大鼠im给予醋酸泼尼松龙注射液49 mg·kg^-1,连续7 d,观察病理变化确定股骨头坏死模型成功。模型大鼠分为模型组(尾静脉注射L-DMEM 0.5 mL)、EPC组(尾静脉注射EPC 6×10^8L^-1 0.5 mL)和EPO基因修饰EPC组(EPO-EPC,尾静脉注射EPO-EPC悬液6×10^8L^-1 0.5 mL),每天1次,连续7 d。每组分别取5只大鼠的股骨头,RT-PCR和Western印迹法检测股骨头组织中活化胱天蛋白酶3、P53正向细胞凋亡调控因子(Puma)、BCL-XL和EPO基因及蛋白表达水平;TUNEL法观察骨坏死区骨细胞凋亡。4周后进行行为实验检测大鼠的平均速度、总的运动长度、自主活动次数和抓力;HE染色观察股骨头病理变化并进行形态计量学分析;荧光显微镜观察股骨头CFSE标记细胞分布。结果Western印迹结果显示,EPC在转染人EPO基因2和4周后EPO高表达(P<0.01)。RT-PCR和Western印迹结果显示,与模型组相比,EPC组大鼠股骨头坏死区组织活化胱天蛋白酶3和Puma基因及蛋白表达明显降低(P<0.05);BCL-XL和EPO基因和蛋白表达明显升高(P<0.05,P<0.01);与EPC组相比,EPO-EPC组大鼠股骨头坏死区组织活化胱天蛋白酶3和Puma基因及蛋白表达明显降低(P<0.05),BCL-XL和EPO基因及蛋白表达明显升高(P<0.05)。TUNEL检测结果显示,与模型组相比,EPC组骨细胞凋亡率降低(P<0.01);与EPC组相比,EPO-EPC组骨细胞凋亡率降低(P<0.01)。行为学检测表明,与模型组相比,EPC组相关指标升高(P<0.05);与EPC组相比,EPO-EPC组相关指标升高(P<0.05)。HE染色显示,模型组骨小梁形态不规则,明显变窄,变稀疏,骨小梁分割程度亦明显下降;EPC组骨小梁变窄减轻,OBJECTIVE To investigate the effect and mechanism of erythropoietin(EPO)modified endothelial progenitor cell(EPC)(EPO-EPC)transplantation on steroid-induced femoral head necrosis in rats.METHODS The femoral heads of 5 rats were taken from each group,and the gene and protein expressions of activated caspase 3,P53 positive apoptosis regulator(Puma),BCL-XL and EPO in femoral head tissue were detected by RT-PCR and Western blotting.The TUNEL method was employed to observe bone cell apoptosis in areas of osteonecrosis.EPCs were resuscitated and cultured in vitro,and EPC suspensions were prepared and labeled with CFSE.The eukaryotic expression plasmid pcDNA3-EPO was transfected into EPCs,and Western blotting was used to detect EPO protein expression in EPCs at 2 and 4 weeks after transfection.SD rats were injected with prednisolone acetate injection(49 mg·kg^-1,im)for 1 week.Pathological observation of femoral heads confirmed that the model was successful.The model rats were randomly divided into 3 groups,model group(0.5 mL L-DMEM via tail vein injection),EPC group(0.5 mL EPCs 6×10^8 L^-1 via tail vein injection)and EPO-EPC group(0.5 mL EPO-EPCs 6×10^8 L^-1 via tail vein injection).Three rats were randomly sacrificed on the 7 thday after transplantation,and the expressions of cleaved-caspase 3,Puma,BCL-XL and EPO in femoral head tissue were detected by RT-PCR and Western blotting.The TUNEL method was used to evaluate bone cell apoptosis in areas of necrosis.After 4 weeks,the average speed of the rats,the total length of movement,the number of times of autonomous activities and grip strength were detected.HE staining was used to observe the pathological changes of the femoral head and morphometric analysis.The distribution of CFSE labeled cells in the femoral head was observed under a fluorescent microscope.RESULTS Western blotting results showed that EPCs were highly expressed at 2 and 4 weeks after transfection with human EPO gene(P<0.01).The results of RT-PCR and Western blotting showed that compared with the mode

关 键 词：红细胞生成素 基因修饰 干细胞 干细胞移植 股骨头坏死 

分 类 号：R965[医药卫生—药理学] R683[医药卫生—药学]