多种神经营养因子联合诱导培养骨髓间充质干细胞向少突胶质样细胞的定向分化(英文)

Directional differentiation of bone marrow mesenchymal stem cells into oligodendrocytes induced by the combination of various neurotrophic factors

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 following axonal regeneration is a key factor affecting the recovery of spinal cord injury. Oligodendrocyte survival directly affects the myeiination following axonal regeneration. OBJECTIVE： To investigate the feasibility of differentiation of rat bone marrow mesenehymal stem cells （BMSCs） into oligedendrocytes induced by neurotrophic factors. DESIGN, TIME AND SETTING： The cell molecular biology in vitro study was performed at the Laboratory of Department of Orthopaedics, Tongji Hospital from September 2006 to June 2007. MATERIALS： A total of 5 Sprague Dawley rats aged 2- 4 weeks, of both gender were selected. Bilateral femur and tibia bone marrow was obtained to harvest BMSCs. METHODS： At passage 4, BMSCs were incubated in serum-free medium, supplemented with N2, 20 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor for 48 hours, and incubated in medium containing 500 ng/mL insulin-like growth factor 1 and N2 for 3 days. MAIN OUTCOME MEASURES： Morphological changes were observed using an phase contrast microscope. Semiquantitative RT-PCR was utilized to detect specific marker mRNA expression of oligodendrocytes. Using neuron marker anti-microtubule-associated protein, astrocyte marker anti-glial fibrillary acidic protein, oligodendrocyte marker anti-galactocerebroside, anti-myelin basic protein antibody, immunocytochemical staining was performed to detect the positive rate of the differentiation of BMSCs into oligodendrocytes. RESULTS： Morphological changes in BMSCs during the differentiation into oligodendrocytes： After the induction, a majority of BMSCs presented the morphological characteristics of oligodendrocytes. Cytoplasm retraction towards nucleus, cell process extension towards outwards, and strong refraction were found. With the prolongation of time, several cell processes connected and formed a typical net-shape structure. Specific marker mRNA expression of oligodendrocytes： Following induction, specific strap of myelin basic protein mRNA and galactocerebroside mRNA could be detected. Positive rate of oligodendrocytes： During induction, the positive rates of galactocerebroside, myelin basic protein and microtubule-associated protein were 65%, 45% and 10%, respectively. CONCLUSION： The combination of epidermal growth factor, basic fibroblast growth factor and insulin-like growth factor can effectively promote the directional differentiation of BMSCs into oligodendrocytes.