耳蜗雪旺细胞来源的Wnt1对耳蜗移植干细胞分化的影响及其分子机制研究

发布时间：2018-03-12 20:13

本文选题：神经干细胞　切入点：干细胞移植　出处：《第四军医大学》2012年博士论文　论文类型：学位论文

【摘要】：1目的神经干细胞（neural stem cells, NSC）的移植为神经系统退行性疾病和损伤的治疗提供了新的思路，但是移植入内耳的神经干细胞向神经元分化的效率很低，这成为移植的外源性神经干细胞替代治疗螺旋神经神经元（spiral ganglion neurons, SGNs）变性的主要障碍。在本研究中，我们验证了耳蜗螺旋神经元损伤后的局部微环境更有利于移植的外源性神经干细胞向神经元方向分化，并初步阐明了其分子机制。 2方法我们通过向耳蜗圆窗龛局部给予药物ouabain建立大鼠螺旋神经元损伤动物模型。然后将分离、培养的神经干细胞移植进入正常、及螺旋神经元损伤后的耳蜗鼓阶。用免疫荧光染色和实时定量RT-PCR等技术来鉴定螺旋神经元损伤后内耳局部微环境对移植的神经干细胞分化的的影响，并初步阐明其分子机制。最后我们使用transwell共培养系统通过体外实验验证我们的假设。 3结果通过将外源性干细胞植入螺旋神经元损伤的动物耳蜗内，我们发现，与对照组耳蜗相比，移植的神经干细胞在螺旋神经元损伤耳蜗中更易分化成MAP2阳性的神经元。采用实时定量PCR和免疫荧光法，我们也证明了在螺旋神经元损伤耳蜗内，，螺旋神经节内卫星细胞（雪旺细胞的一类，外周神经节内的雪旺细胞称卫星细胞）中Wnt1（Wnt信号通路配体）的表达显着增加。我们进一步证实，神经干细胞表达Wnt信号通路受体和Wnt信号通路胞内关键组件。在以上结果基础上我们提出假设，雪旺细胞所产生Wnt1可能参与了移植神经干细胞向神经元分化的调控。我们使用transwell共培养系统在体外验证这一假设。我们将感染了慢病毒载体高表达Wnt1的耳蜗雪旺氏细胞与神经干细胞共培养，结果显示：与高表达Wnt1的雪旺细胞共培养的神经干细胞，分化为MAP2阳性神经元的比例显着增加，然而这种促进分化的作用可被DKK1（Wnt信号通路抑制剂）抑制。 4结论这些结果表明，耳蜗雪旺氏细胞来源的Wnt1可激活移植神经干细胞的胞内Wnt信号通路，从而促进其向神经元的分化。详尽的阐明损伤微环境的改变对移植外源性干细胞的影响，有助于我们提出更有效的策略以突破移植神经干细胞向神经元的分化率低这一屏障。
[Abstract]:1 purpose. The transplantation of neural stem cells (NSCs) provides new ideas for the treatment of neurodegenerative diseases and injuries, but the neural stem cells transplanted into the inner ear are very inefficient in differentiating into neurons. This has become a major obstacle to the transplantation of exogenous neural stem cells in the treatment of spiral ganglion neuronal degeneration. We have proved that the local microenvironment after cochlear spiral neuron injury is more favorable to the differentiation of the transplanted neural stem cells into neurons and the molecular mechanism of the neural stem cells has been preliminarily elucidated. 2 method. We set up a rat model of spiral neuron injury by local administration of ouabain to the cochlea window niche, and then transplanted the isolated and cultured neural stem cells into normal. The effects of local microenvironment of inner ear on the differentiation of transplanted neural stem cells were evaluated by immunofluorescence staining and real-time quantitative RT-PCR. Finally, we used transwell co-culture system to verify our hypothesis through in vitro experiments. 3 results. By implanting exogenous stem cells into the cochlea of animals injured by spiral neurons, we found that, compared with the cochlea of the control group, Transplanted neural stem cells are more likely to differentiate into MAP2 positive neurons in cochlea injured by spiral neurons. Using real-time quantitative PCR and immunofluorescence, we have also demonstrated that in spiral neurons damaged cochlea, The expression of Wnt1(Wnt signal pathway ligand in satellite cells (Schwann cells, Schwann cells in peripheral ganglion) in spiral ganglion is significantly increased. Neural stem cells express key cellular components of the Wnt signaling pathway receptor and the Wnt signaling pathway. Wnt1 produced by Schwann cells may be involved in the regulation of neuronal differentiation of transplanted neural stem cells. We used the transwell co-culture system to verify this hypothesis in vitro. We will infect the cochlea Schwann infected with lentivirus vector overexpression of Wnt1. Co culture of neural stem cells, The results showed that the proportion of neural stem cells co-cultured with Schwann cells with high expression of Wnt1 increased significantly in differentiating into MAP2 positive neurons but the effect of promoting differentiation could be inhibited by DKK1(Wnt signaling pathway inhibitors. 4 conclusion. These results suggest that Wnt1 derived from Schwann's cells in the cochlea can activate the intracellular Wnt signaling pathway of transplanted neural stem cells and thus promote their differentiation into neurons. It is helpful for us to propose more effective strategies to break through the barrier of low differentiation rate of transplanted neural stem cells into neurons.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R764.9

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