来源于大鼠脊髓星形胶质细胞的神经干细胞诱导分化成运动神经元的研究

发布时间：2018-09-18 16:22

【摘要】：运动神经元损伤可导致躯体肌肉丧失神经支配,这种疾病一直以来被认为是无法治愈的。通过干细胞诱导分化成运动神经元来替代受损的运动神经元将是一种比较可行的治疗手段。近来,已有报道胚胎干细胞可以诱导分化成运动神经元。然而,胚胎干细胞存在免疫排斥和高成瘤等问题。理想的运动神经元来源是从患者自身细胞诱导得来的。我们以前的研究结果表明,在损伤的条件下成体的星形胶质细胞去分化成神经干细胞。本文旨在探讨星形胶质细胞来源的神经干细胞是否可以被诱导分化成为运动神经元。本文应用体外限制性培养基选择培养出生后15-20 d的大鼠脊髓星形胶质细胞。通过免疫荧光染色、流式细胞分析、蛋白免疫印迹、细胞转染及Time-lapse荧光追踪等实验方法,研究了星形胶质细胞在体外条件下去分化的机制。通过表皮生长因子(EGF)和碱性成纤维生长因子(bFGF)的神经球分析体系,研究去分化后的星形胶质细胞的干细胞特征。利用维甲酸(RA)和音猬因子(Shh)胞外诱导信号分子,诱导干细胞向运动神经元分化。本文结果显示,大鼠脊髓星形胶质细胞在体外选择培养的条件下去分化表达神经干细胞的标记物巢蛋白(Nestin)。在EGF和bFGF存在的条件下可以长出神经球。神经球细胞可以大量增殖并且可以传16代以上。神经球不仅可以自我更新(Self-renew),还可以分化成神经元和胶质细胞。结果表明,在体外培养条件下星形胶质细胞可以去分化成为神经干细胞。进一步的研究发现,这种神经干细胞在RA和Shh信号分子诱导5 d后,97.7 %±0.5 %的细胞是表达Olig2的运动神经元祖细胞;97.6 %±0.5 %的细胞是表达βIII-tubulin的神经元,且Olig2和βIII-tubulin在这种诱导分化后的细胞均为共表达。在加入脑源性神经营养因子(BDNF)、睫状神经营养因子(CNTF)、胶质源性神经营养因子(GDNF)和神经营养因子-3 (NT-3)继续分化成熟2周后,24.4 %±1.1 %的细胞是表达HB9的运动神经元,这些细胞也是βIII-tubulin和ChAT阳性的。在撤除RA和Shh信号分子后,运动神经元的分化比例显著的下降。结果表明,RA和Shh可以显著地诱导星形胶质细胞去分化来源的神经干细胞分化成为运动神经元。上述结果表明,星形胶质细胞源的神经干细胞可以被高效地诱导分化运动神经元的祖细胞,进而可以分化成运动神经元。临床通过组织活检很容易从患者自身获得星形胶质细胞,结合本研究结果,提示自体星形胶质细胞体外培养诱导分化为运动神经元再移植将可能成为运动神经元损伤治疗的一种有效方法。
[Abstract]:Motor neuron damage can lead to loss of muscle innervation, a disease that has long been considered incurable. Differentiation of motor neurons by stem cells in place of injured motor neurons will be a feasible treatment. Recently, embryonic stem cells have been reported to differentiate into motor neurons. However, embryonic stem cells have immune rejection and Gao Cheng tumor problems. The ideal source of motor neurons is derived from the patient's own cells. Our previous studies have shown that adult astrocytes dedifferentiate into neural stem cells under damaged conditions. This paper aims to investigate whether neural stem cells derived from astrocytes can be induced to differentiate into motor neurons. The spinal cord astrocytes from 15 to 20 days after birth were cultured in a restricted medium in vitro. The mechanism of dedifferentiation of astrocytes in vitro was studied by immunofluorescence staining, flow cytometry, Western blot, cell transfection and Time-lapse fluorescence tracing. The stem cell characteristics of dedifferentiated astrocytes were studied by using the neurosphere analysis system of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Retinoic acid (RA) and Hedgehog factor (Shh) were used to induce extracellular signaling molecules to induce stem cells to differentiate into motor neurons. The results showed that rat spinal astrocytes dedifferentiated and expressed nestin (Nestin)., a marker of neural stem cells, under the condition of selective culture in vitro. In the presence of EGF and bFGF, neurospheres can be grown. Neuroglobular cells can proliferate in large numbers and can be passed through more than 16 generations. Neurospheres can not only self-renew (Self-renew), but also differentiate into neurons and glial cells. The results showed that astrocytes could dedifferentiate into neural stem cells in vitro. Further studies showed that 97.7% 卤0.5% of the neural stem cells were motor neuron progenitor cells expressing Olig2 and 97.6% 卤0.5% of the cells expressing Olig2 were 尾 III-tubulin expressing neurons after 5 days of induction by RA and Shh signaling molecules. Both Olig2 and 尾 III-tubulin were co-expressed in the differentiated cells. After the addition of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial derived neurotrophic factor (GDNF) and neurotrophic factor -3 (NT-3), 24.4% 卤1.1% of the cells were motoneurons expressing HB9. These cells are also 尾 III-tubulin and ChAT positive. After the removal of RA and Shh signaling molecules, the differentiation ratio of motor neurons decreased significantly. The results showed that RA and Shh could significantly induce neural stem cells derived from dedifferentiation of astrocytes to differentiate into motor neurons. These results suggest that astrocyte-derived neural stem cells can be efficiently induced to differentiate into motor neuron progenitor cells and then to differentiate into motor neurons. It is easy to obtain astrocytes from patients themselves by clinical biopsies, combined with the results of this study. These results suggest that autologous astrocytes can be induced to differentiate into motor neurons in vitro and then transplanted into motor neurons, which may be an effective method for the treatment of motor neuron injury.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329

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