骨形态生成蛋白2联合转化生长因子β3诱导髓核间充质干细胞向类髓核细胞分化的实验研究  

Effect of combined use of bone morphogenetic protein 2 and transforming growth factor-β3 on nucleus pulposus stem cells differentiation into nucleus pulposus cells

作　　者：程实[1] 应金威 文天用[1] 裴世深 苏凌浩 王德利 阮狄克[1] CHENG Shi;YING Jinwei;WEN Tianyong;PEI Shishen;SU Linghao;WANG Deli;RUAN Dike(Department of Orthopedic Surgery,Navy General Hospital,Beijing 100048;Department of Orthopedie Surgery,Peking University Shenzhen Hospital,Shenzhen 518035,Guangdong,China)

机构地区：[1]海军总医院骨科,北京100048 [2]北京大学深圳医院骨关节科,广东深圳518035

出　　处：《中华骨与关节外科杂志》2018年第9期704-708,共5页Chinese Journal of Bone and Joint Surgery

基　　金：国家自然科学基金资助项目(81470102;81171740)

摘　　要：背景:髓核间充质干细胞(nucleus pulposus mesenchymal stem cells, NPSCs)在椎间盘损伤退变修复中具有重要应用价值,诱导髓核间充质干细胞向类髓核细胞分化是关键步骤。目的目的:探讨联合使用骨形态生成蛋白2(bone mor-phogenetic protein 2, BMP-2)和转化生长因子β3(transforming growth factorβ3, TGF-β3)诱导髓核间充质干细胞向类髓核细胞分化的可行性及效果。方法方法:取3月龄大鼠尾椎髓核组织,分离培养髓核间充质干细胞,并进行形态学观察、三系分化鉴定和流式细胞术检测干细胞表面抗原。将培养获得的NPSCs分为4组,对照组、20 ng/ml TGF-β3组、100 ng/ml BMP-2组和20 ng/ml TGF-β3+100 ng/ml BMP-2组,分别加入不同组合的细胞因子对NPSCs进行诱导培养。采用CCK-8增殖实验检测各组细胞因子毒性,在诱导第7天、第14天提取mRNA进行实时定量RT-PCR检测各组髓核特异基因Aggrecan、SOX-9、Collagen-2、Krt19的相对表达量;在诱导第14天进行阿利新蓝染色检测细胞外基质的表达量,对比各组染色强度来比较各组NPSCs分化程度。结果结果:成功从髓核组织分离NPSCs并进行鉴定。原代细胞培养11 d后传至P3代,贴壁细胞呈纺锤长梭形,旋窝样生长。CCK-8实验结果显示,加入细胞因子组细胞增殖水平在培养3 d后较对照组升高,但3组之间无统计学差异,排除由细胞增殖导致的胞外基质增多。RT-PCR结果显示,在诱导第7天时加入细胞因子组髓核特异基因的表达较对照组均升高(P<0.05),在第14天时,20 ng/ml TGF-β3+100 ng/ml BMP-2组髓核特异基因的表达较20 ng/ml TGF-β3组和100 ng/ml BMP-2组升高(P<0.05)。阿利新蓝染色结果显示,20 ng/ml TGF-β3+100 ng/ml BMP-2组细胞外基质分泌明显高于其他组。结论结论:联合使用TGF-β3和BMP-2能够有效诱导NPSCs向类髓核细胞分化,可为下一步应用NPSCs治疗椎间盘退变打下基础。Background:Nucleus pulposus mesenchymal stem cells (NPSCs)are of great potential in intervertebral disc regeneration when regression and injury occurring,and the key step is the induction of NPSCs into nucleus pulposus cells.Objective:To investigate the feasibility and effect of combined using of transforming growth factor β3(TGF-β3)and bone morphogenetic protein 2(BMP-2)on NPSCs differentiation into nucleus pulposus (NP)cells.Methods:Rats of 3months old were sacririced for NP tissue in caudal vertebra.The NPSCs were isolated and cultured,and were identified by cell morphological observation,three lineage differentiation and flow cytometry.The collected NPSCs were divided into four groups as control group,20 ng/ml TGF-β3group,100ng/mI BMP-2group and 20ng/ml TGF-β3+I00ng/ml BMP-2group,and different combinations of cytokines indicated above were added into respective culture mediums.CCK-8assay was used to detect the toxicity of cyto- kines in each group.Total mRNA was extracted after 7and 14days of induction,then real-time-PCR was used to detect the relative nucleus specific genes such as Aggrecan,SOX-9,Collagen-2,and Krt19in each group.After 14days of induction,al- clan blue staining was used to detect the expression of extracellular matrix,and the degree of NPSCs differentiation in each group were compared by the staining intensities.Results:NPSCs were successfully isolated and identified from nucleus pulposus tissue.The primary cell culture was passed to P3for 11days and the adherent cells were showed spindle-like shape.CCK- 8results showed that the proliferation ability of the other three groups was higher than the control group after 3days of cuhure, but there were no statistical difference among the three groups,excluding the disturbance of the increased extracellular matrix owing to cell proliferation.RT-PCR results showed that the expression of the NP-speciric genes in the other three groups were higher than that in the control group after 7days induction (P<0.05).After 14days induction,the expression o

关 键 词：髓核间充质干细胞 分化 骨形态生成蛋白2 转化生长因子Β3 椎间盘退变 

分 类 号：R681.53[医药卫生—骨科学]