mdx鼠骨髓间充质干细胞体外分离培养及向肌细胞的诱导分化

背景：自体干细胞移植治疗可以克服异体干细胞移植治疗的局限性，但目前对于进行性肌营养不良模型mdx鼠骨髓间充质干细胞生物学特性及肌分化潜能研究未见报道。 目的：拟建立体外分离培养mdx鼠骨髓间充质干细胞的方法，并观察其成肌分化能力。 设计、时间及地点：细胞学体外观察，于2006-01/07在中山大学附属第一医院神经病学实验室完成。 材料：4~6周龄纯系雄性mdx鼠10只，购自美国Jackson实验室，饲养于中山大学北校区动物实验中心。碱性成纤维细胞生长因子和兔抗鼠MHC抗体为Chemicon公司产品。 方法：全骨髓法体外分离mdx鼠骨髓间充质干细胞，差速贴壁法纯化扩增，胰酶+EDTA消化传代。取传至第3代的细胞，按1×104/孔接种于24孔板，置于含两性霉素B、碱性成纤维细胞生长因子、胎牛血清的DMEM培养基中向肌细胞方向诱导分化，2周后更换为不含两性霉素B、但添加马血清的DMEM培养基继续培养2周。 主要观察指标：鼠骨髓间充质干细胞形态、表面标志、生长曲线及成肌诱导分化。 结果：原代培养24 h后，鼠骨髓间充质干细胞约80%贴壁生长，第3代细胞多呈梭形，形态比较均一。鼠骨髓间充质干细胞表达CD29和CD44等间质类细胞的表面分子，不表达CD11B和CD45等淋巴造血细胞的表面分子。接种后第一两天细胞处于潜伏期，第3天细胞增殖旺盛，进入对数生长期，至第4天细胞数增长一倍，第5天进入平台期。两性霉素B诱导后细胞有少量死亡，停用后更换马血清促进细胞成肌，7~10 d可见有肌管样细胞形成，免疫荧光染色可见肌管样细胞呈MHC阳性，DAPI复染后可见肌细胞内有多个细胞核。 结论：在两性霉素B与马血清依次诱导条件下，体外可成功分离培养基因缺陷mdx鼠骨髓间充质干细胞，并证明其具有成肌分化潜能。

Isolation, cultivation and myogenic differentiation of bone marrow mesenchymal stem cells from mdx mice in vitro

BACKGROUND: Autologous stem cell transplantation can overcome the limitations of xenologous transplantation. There are few studies on the biological characters and differentiation into myogenic cells of bone marrow mesenchymal stem cells (BMSCs) derived from mdx mice. OBJECTIVE: To establish a method of isolation and cultivation of mdx mouse BMSCs in vitro, and to study the differentiation into myogenic cells of BMSCs. DESIGN, TIME AND SETTING: The cytology in vitro experiment was performed at the Neurology Laboratory of the First Affiliated Hospital to Sun Yat-sen University from January to July 2006. MATERIALS: Ten male mdx mice aged 4-6 weeks were purchased from Jackson Laboratory, USA, and raised in the Animal Experimental Center, Sun Yat-sen University. Basic fibroblast growth factor and rabbit anti-mouse MHC antibody were purchased from Chemicon company. METHODS: BMSCs of mdx mice were isolated, cultured through the whole bone marrow method, amplified by the differential adherent method, and digested by trypsin + EDTA. BMSCs at passage 3 and a density of 1×104 were incubated in the 24-well plate, in the DMEM, supplemented with amphotericin B, basic fibroblast growth factor and fetal bovine serum, and induced differentiation into myocytes. At week 2, cells were incubated in the DMEM with horse serum and without amphotericin B for 2 weeks. MAIN OUTCOME MEASURES: Morphological changes, surface marker, growth curve and myogenic differentiation of mouse BMSCs were measured. RESULTS: Following 24 hours primary culture, about 80% mouse BMSCs adhered. Cells at passage 3 grew fast with morphology of spindle-shaped. Phenotype showed that mouse BMSCs were positive for the surface markers of mesenchymal cells such as CD29, CD44 and negative for the markers of lymphatic or hematogenic cells such as CD11B and CD45. Following induction, cells were in latency phase at 1-2 days, well amplified at day 3 and entered logarithmic phase, increased by one time at day 4, and entered platform at day 5. Some BMSCs died following amphotericin B induction. After stopping, horse serum was used to promote cell growth. At 7-10 days, myotube-like cells formed. Immunofluorescence staining showed that myotube-like cells were positive for MHC. Following DAPI staining, many nuclei were found in myocytes. CONCLUSION: BMSCs of genetic defective mdx mice were successfully isolated, cultured and can be induced to differentiate to myogenic cells in vitro under the induction of amphotericin B and horse serum.