人脐血间充质干细胞体外扩增和向神经元样细胞定向诱导分化的研究  

Study of in vitro expansion and differentiation into neuron-like cells of human umbilical cord blood mesenchymal stem cells

作　　者：侯玲玲[1] 曹华[1] 魏国荣 白慈贤[1] 张涌[2] 吴祖泽[3] 裴雪涛[1] 

机构地区：[1]军事医学科学院输血研究所干细胞室,100850 [2]西北农林科技大学胚胎工程实验室 [3]军事医学科学院放射医学研究所

出　　处：《中华血液学杂志》2002年第8期415-419,共5页Chinese Journal of Hematology

基　　金：国家 8 63计划资助项目 (2 0 0 1AA2 1615 1) ;国家重点基础研究发展规划项目 (973 ) (G19990 5 3 90 3 ) ;北京市 2 48创新工程资助项目 (95 5 0 2 13 80 0 )

摘　　要：目的　探讨人脐血间充质干细胞 (mesenchymalstemcells,MSC)的体外分离、纯化、扩增和向神经元样细胞的定向诱导分化条件。方法　无菌条件下采集正常人脐血 ,肝素抗凝 ,用相对密度为1.0 77的淋巴细胞分离液分离脐血单个核细胞 ,进而获得MSC。用MesencultTM 培养基进行纯化和扩增培养。用流式细胞仪检测MSC的表面标志。取扩增 2 ,5 ,8代的MSC分别向神经元样细胞诱导 ,免疫组化和组化法检测神经元样细胞特异性标志和结构。结果　脐血MSC每扩增一代 ,细胞数量增加约 2～ 3倍。 6 .6× 10 5个人脐血原代MSC在体外扩增 10代后获得 9.9× 10 8个细胞 ,扩增约 1.5× 10 3倍。流式细胞仪检测结果显示 ,脐血MSC不表达CD34 、CD1 1a和CD1 1b,强表达CD2 9,弱表达CD71 ,与骨髓MSC表面抗原特性一致。诱导后的细胞平均有 70 %左右呈现典型的神经元样表型 ,免疫组化法检测发现不同代数的MSC诱导后均表达神经丝蛋白 (neurofilament,NF)和神经元特异性烯醇化酶 (neuron specificenolase ,NSE) ;组织化学法检测可看到神经元特有结构尼氏体。结论　脐血MSC在体外有较强的扩增能力 ,能够向非间充质类细胞分化。Objective To explore the isolation, purification and expansion of human umbilical cord blood mesenchymal stem cells(MSCs) into neuron like cells in vitro. Methods Human cord blood samples were obtained sterilely with 20 U/ml preservative free heparin. MSCs were isolated by lymphocyte separation medium (density 1 077 g/ml), and purified and expanded with Mesencult TM medium. The surface antigen expression of MSCs was detected by flow cytometry. The passage 2, 5 and 8 of the expanded MSCs were induced to differentiate to neuron like cells . Specific markers and structures were detected by immunohistochemistry and histochemistry methods. Results The number of MSCs increased two to three fold with each expanded passage. 6.6×10 5 primary MSCs were expanded ten passages to reach a numbe of 9.9×10 8, and was increased about 1.5×10 3 fold. Flow cytometry showed that MSCs did not express antigens CD 34 , CD 11a and CD 11b , but expressed strongly CD 29 and weakly CD 71 , which was identical to human bone marrow derived MSCs. 70% cells exhibited typical neuron like phenotype after induction. Immunohistochemistry staining showed that all of the induced different passage MSCs expressed neurofilament (NF) and neuron specific enolase (NSE). Special Nissl body was found by histochemistry. Conclusion MSCs in human umbilical cord blood can expand in vitro and differentiate into non mesenchymal cells.

关 键 词：流式细胞仪 细胞替代治疗 基因治疗 脐血 间充质干细胞 体外扩增 神经元样细胞 定向诱导分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]