骨髓基质干细胞诱导成雪旺细胞的可行性及促轴突生长的体外功能实验  

Bone mesenchymal stem cells differentiate into a Schwann-cell phenotype and promote neurite out- growth in vitro

作　　者：张莉[1] 王玉[1] 彭江[1] 陈继凤[1] 赵喆[1] 赵斌[1] 薛静[1] 眭翔[1] 杨昱[1] 许文静[1] 卢世璧[1] 

机构地区：[1]解放军总医院骨科研究所,北京100853

出　　处：《中华创伤骨科杂志》2010年第10期945-949,共5页Chinese Journal of Orthopaedic Trauma

基　　金：国家高技术研究发展（863）计划（2009AA03Z312）;军队“十一五”计划（062057）;国家支撑计划（2009BA187B02）

摘　　要：目的 探讨骨髓基质干细胞（BMSCs）向雪旺细胞（SCs）分化的可行性,以及分化成类SCs的表型、分子及功能特征.方法 原代培养F344乳鼠BMSCs,流式细胞仪检测细胞表面特异标记CD29、CD44、CD45的表达;诱导干细胞向成骨细胞和脂肪细胞分化,评价干细胞的生物学特性;采用碱性成纤维细胞生长因子和forskolin等诱导BMSCs向SCs分化,光镜观察诱导后细胞形态的变化;免疫荧光染色鉴定SCs特异性标记物S100和p75的表达;RT-PCR分析诱导前、后SCs相关基因的表达;培养大鼠背根神经节神经元,分别与诱导前后的BMSCs共培养,评价其促轴突生长的功能特性.结果 分离培养的鼠BMSCs CD29、CD44表达呈阳性,CD45表达呈阴性：干细胞诱导的成骨细胞茜素红染色阳性,脂肪细胞油红O染色阳性;BMSCs经过胶质细胞生长因子的作用,光镜下发现诱导的细胞形态与SCs相似;免疫荧光染色S100和p75阳性;RT-PCR结果S100、CD104均表达增强;与背根神经节神经元共培养,诱导后的BMSCs促进轴突生长的距离为（285.3±36.7）μm,与未诱导组BMSCs的[（113.5±11.5）μm]相比,差异有统计学意义（t=8.966,P=0.001）.结论 BMSCs可诱导分化成SCs,其表型、分子及功能特征与SCs相似,诱导分化的BMSCs是一种理想的神经组织工程的种子细胞.Objective To investigate the phenotypic, molecular and functional characteristics of bone mesenchymal stem cells （BMSCs） differentiated along a Schwann cell（SCs） lineage. Methods Cultured BMSCs were isolated from F344 rats, and the undifferentiated BMSCs were confirmed by detection of MSC-specifie cell-surface markers. The BMSCs were differentiated along a glial cell lineage using an established cocktail of growth factors. Following differentiation, we used immunofluorescence staining; RT-PCR and in vitro co-culture model system with rat-derived primary sensory neurons to evaluate the characteristics of differentiated BMSCs. Results BMSCs were successfully isolated from the rat bone marrow. The isolated BMSCs expressed CD29 and CD44 but not CD45. Osteogenic differentiation was detected by Alizarin red staining and adipogenic differentiation was confirmed by Oil-red O staining. BMSCs treated with a mixture of glial growth factors adopted a spindle-like morphology similar to Schwann cells. Immunocytochemical staining and RT-PCR analysis revealed that the treated cells expressed the glial markers S100 and p75 indicative of differentiation. On co-culture with dorsal root ganglia neurons, the differentiated BMSCs enhanced the outgrowth of neurites in dorsal root ganglia neurons. Conclusion Since BMSCs can be differentiated into cells that are Schwann-like in terms of morphology, phenotype, and function, they can be used as a suitable SCs substitute for nerve repair.

关 键 词：骨髓细胞 雪旺细胞 神经节 脊 神经元 

分 类 号：R686[医药卫生—骨科学]