人骨髓间充质干细胞的免疫调节作用及向神经元样细胞诱导分化  

Immunomodulatory effect and neuronal-like cells differentiation of bone marrow mesenchymal stem cells

作　　者：陆华[1] 苗宗宁[2] 吴卫江[1] 蒋云召[1] 葛风[1] 房文峰[1] 徐杰[1] 朱爱华[1] 陈革[1] 周建宏[1] 陆爻忠[1] 唐志放[1] 王泳[1] 

机构地区：[1]无锡市第三人民医院神经外科,江苏省无锡市214041 [2]无锡市第三人民医院细胞室,江苏省无锡市214041

出　　处：《中国组织工程研究与临床康复》2009年第45期8955-8960,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:骨髓间充质干细胞作为具有多向分化潜能的干细胞,属于未分化的前体干细胞,其表型分化尚不成熟,因此在同种异体移植后无排斥反应或反应较弱。但是在体外条件下通过化学和生物诱导物使骨髓间充质干细胞转化为神经元,影响骨髓间充质干细胞向神经细胞、胶质细胞系统定向分化的因素比较复杂。目的:观察人骨髓间充质干细胞的免疫调节作用,及向神经元样细胞诱导分化的能力。设计、时间及地点:对比观察,于2008-01/2009-03在无锡市第三人民医院细胞室完成。材料:骨髓来源于人髋关节手术时的松质骨碎片或髂骨游离移植。方法:首先分离和培养骨髓间充质干细胞,建立双向混合淋巴细胞反应体系,在双向混合淋巴细胞反应中,将来自2个志愿者的外周血单个核细胞(各1×105/孔)等比例加入96孔板中,根据不同效靶比E/Tratios(骨髓间充质干细胞/外周血单个核细胞)加入丝裂霉素处理过的骨髓间充质干细胞。在骨髓间充质干细胞诱导分化为神经元样细胞实验中分别选择两种诱导培养基,方法1诱导:DMEM+体积分数为10%胎牛血清+1μmol/L全反式维甲酸+20μg/L碱性成纤维细胞生长因子+20μg/L表皮生长因子;方法2诱导:DMEM+2%二甲基亚砜+100μmol/L丁羟茴醚。主要观察指标:3H掺入法测定淋巴细胞增殖率,观察骨髓间充质干细胞对淋巴细胞增殖的影响;通过条件培养基定向诱导,免疫荧光和免疫组织化学染色观察其分化为神经细胞的能力。结果:①骨髓间充质干细胞与混合淋巴细胞反应体系共同培养抑制了淋巴细胞增殖,其增殖抑制率与加入骨髓间充质干细胞的数量呈正比。②方法1诱导2h后,光镜下可见骨髓间充质干细胞的细胞质向核收缩,呈典型核周体形态。3～5h后多数细胞能形成神经元样细胞形态,但细胞数目无明显增加。3d后大多数细胞转变为双极或多极神经元细胞样�BACKGROUND： As a kind of undifferentiated precursor cells, the phenotypic differentiation of bone marrow mesenchymal stem cells （BMSCs） remains immaturity, thus it presents weak rejection following transplantation. However, the in vitro directional differentiation of BMSCs into neuronal cells is easy affected by various factors. OBJECTIVE： To observe the immunomodulatory effect and the potential of BMSCs differentiate into neuronal-like cells. DESIGN, TIME AND SETTING： A contrast observation was conducted at the Department of Cytology, Third People＇s Hospital ol Wuxi from January 2008 to March 2009. MATERIALS： Bone marrow was harvested from chips of cancellous or ilium bone during hip joint surgery. METHODS： Firstly, the BMSCs were separated and cultured to establish mixed lymphocyte reaction （MLR） system. Secondly, 2 samples of peripheral blood mononuclear cells （1×10^5/well） were added into 96-well plate, and then BMSCs treated by mitomycin were added according to different ratios （BMSCs/peripheral blood monouclear cells）. At the end, the cells were cultured as follows： Method 1： DMEM＋1 0% fetal calf serum＋l pmol/L RA ＋20 pg/L basic flbroblast growth factor＋20 μg/L epidermal growth factor, Method 2： DMEM＋2% dimethyl sulfoxide ＋100 μmol/L butylated hydroxyanisole. MAIN OUTCOME MEASURES： The growth rate of lymphocyte was measured by ^3H-Thymidine, and the effect of BMSCs on lymphocyte proliferation was observed. Additionally, the differentiation potential of BMSCs into neuronal cells was determined by immunofluorescence and immunohistochemistdcal staining. RESULTS： ①The BMSCs inhibited lymphocyte proliferation in MLR system and the influence on proliferation of lymphocyte was direct related to ratio of BMSCs. ②Under a light microscope, cytoplasm of BMScs were shrinkd, which presented typical perikaryon morphology at hour 2 after culture with method 1. The majority of BMSCs were formed neuronal-like cells without number changes at hours 3 5, which

关 键 词：骨髓间充质干细胞 免疫调节 神经元样细胞 分化 

分 类 号：R394.2[医药卫生—医学遗传学]