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背景:轴突再生后髓鞘化是影响脊髓损伤后恢复的一个关键性因素,而少突胶质细胞存活的多少直接影响轴突再生后髓鞘化。目的:探讨骨髓间充质干细胞经神经营养因子诱导培养后,向少突胶质样细胞定向分化的可行性。设计、时间及地点:细胞分子生物学的体外实验,于2006-09/2007-06在同济医院骨科实验室完成。材料:选用2～4周龄SD大鼠5只,雌雄不拘,取其双侧股骨、胫骨骨髓,分离培养骨髓间充质干细胞。培养用诱导因子表皮生长因子、碱性成纤维细胞生长因子、胰岛素样生长因子为美国Invitrogen公司产品。方法:取培养至第4代的骨髓间充质干细胞,加入含有20ng/mL碱性成纤维细胞生长因子、20ng/mL表皮生长因子、N2添加剂的无血清培养基的诱导液诱导48h后,加含500ng/mL胰岛素样生长因子1、N2添加剂的分化培养液培养3d。主要观察指标:相差显微镜观察诱导过程中骨髓间充质干细胞的形态学变化。半定量RT-PCR检测少突胶质细胞特异性标志物mRNA的表达。应用神经元细胞标志物抗微管相关蛋白,星形胶质细胞标志物抗神经纤维酸性蛋白,少突胶质细胞标志物抗半乳糖脑苷脂、抗磷脂碱性蛋白抗体进行免疫细胞化学染色,检测骨髓间充质干细胞定向分化为少突胶质样细胞的阳性率。结果:①骨髓间充质干细胞向少突胶质样细胞诱导分化过程中的形态学变化:经诱导分化后,大部分骨髓间充质干细胞表现出少突胶质细胞的形态学特征,胞质向细胞核回缩,细胞突起向外延伸,折光性增强,随时间延长多个细胞突起相互连接形成典型的网状结构。②少突胶质细胞特异性标志物mRNA的表达:细胞诱导分化后可检测到磷脂碱性蛋白mRNA、半乳糖脑苷脂mRNA的特异性条带。③少突胶质细胞阳性率:在诱导分化条件下,半乳糖脑苷脂阳性率为65%,磷脂碱性蛋白阳性率为45%,微管相关蛋白2阳性率为10%。结论:碱性成纤维细胞生长因子、表皮生长因子与胰岛素样生长因子联合应用能够有效促进骨髓间充质干细胞向少突胶质样细胞定向分化。
BACKGROUND: Myelination after axonal regeneration is a key factor affecting the recovery from spinal cord injury. The survival of oligodendrocytes directly affects myelination after axonal regeneration. Objective: To investigate the feasibility of bone marrow mesenchymal stem cells differentiated into oligodendrocyte-like cells after induced by neurotrophic factor. DESIGN, TIME AND SETTING: In vitro experiments on cell molecular biology were performed at the Department of Orthopedics, Tongji Hospital from September 2006 to June 2007. MATERIALS: Five Sprague-Dawley (SD) rats aged 2 to 4 weeks were selected. Both male and female rats were randomly selected. Bone marrow and bone marrow of bilateral tibia were taken from the femur. The bone marrow mesenchymal stem cells were isolated and cultured. Cultivation of inducing factor epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor for the United States Invitrogen products. Methods: Bone marrow mesenchymal stem cells (MSCs) of passage 4 were cultured in the medium containing 20ng / mL basic fibroblast growth factor, 20ng / mL epidermal growth factor and N2 supplementation for 48h. Differentiation broth containing 500 ng / mL insulin-like growth factor 1, N2 additive was cultured for 3 days. MAIN OUTCOME MEASURES: Morphological changes of bone marrow mesenchymal stem cells during induction were observed by phase contrast microscopy. Semi-quantitative RT-PCR detection of oligodendrocyte-specific marker mRNA expression. Neuronal cell markers anti-microtubule-associated protein, astrocyte marker anti-neurofibrillary acidic protein, oligodendrocyte marker anti-galactose cerebroside, anti-phospholipid basic protein antibody were used for immunocytochemical staining , The detection of bone marrow-derived mesenchymal stem cells differentiate into oligodendrocyte-like cells positive rate. Results: (1) Morphological changes of bone marrow mesenchymal stem cells differentiate into oligodendrocyte-like cells: Most of the MSCs showed the morphological characteristics of oligodendrocytes after induced differentiation, Retraction to the nucleus, cell protrusions extending outward, refraction enhancement, with the extension of a number of cell protrusions connected to each other to form a typical network structure. ② The expression of oligodendrocyte-specific marker mRNA: Specific bands of mRNA of phospholipid basic protein and galactose cerebroside mRNA were detected after cell differentiation. ③ The positive rate of oligodendrocytes: under the condition of differentiation, the positive rate of galactocerebroside is 65%, the positive rate of phospholipid basic protein is 45%, the positive rate of microtubule-associated protein 2 is 10%. Conclusion: The combination of basic fibroblast growth factor, epidermal growth factor and insulin-like growth factor can effectively promote the differentiation of bone marrow mesenchymal stem cells into oligodendrocyte-like cells.