螺旋神经元损伤机制及治疗方法的研究

发布时间：2018-02-14 15:42

本文关键词： 螺旋神经节毒毛旋花子甙嗅球干细胞氢气 microRNA 耳蜗　出处：《第四军医大学》2011年博士论文　论文类型：学位论文

【摘要】：神经性耳聋是人类最常见的疾病之一。长久以来,研究者不断的探求其发病机理、治疗方法。在诸多病因中,主要的病变部位在耳蜗毛细胞和初级传入的螺旋神经元(SGN)。由于哺乳动物耳蜗毛细胞和SGN损伤后难以再生,那么,如何修复损伤的SGN,如何提高SGN在损伤后微环境里的存活率,如何增强SGN在复杂的疾病状态下自我修复功能,是耳聋机制研究的主要方向之一。本研究致力于研究SGN损伤后给予干细胞、H2治疗,以及通过研究SGN发育、分化、成熟过程中microRNA表达,探讨基因调控水平下SGN可能的修复机制。本研究中,成功的采用毒毛旋花子甙制造了神经性耳聋的动物模型。通过药物的不同浓度,动物的ABR听力阈值有不同程度的改变,两者呈正相关。但是,DPOAE则没有明显改变。试验中继以免疫组织化学、透射电镜、扫描电镜、体外细胞培养等方法,观察毒毛旋花子甙对SGN的损伤。为下一步研究神经性耳聋的后续工作打下良好的基础。将动物的听力部分损伤后,我们予以以下两种治疗手段:一,干细胞的移植。我们将已鉴定的E14C57BL/6-GFP鼠的嗅球干细胞用于干细胞移植。采用两种途径:⑴内听道的听神经根移植干细胞术。⑵听力损伤侧颈内动脉干细胞移植术。移植干细胞后,给予一定的外界声刺激,观察其迁移、存活、分化现象。本实验观察:经由此两种路径移植后,在损伤的螺旋神经节附近出现零星的干细胞团。二,H2的保护作用。将听力部分损伤的动物,手术后定时放入H2治疗箱内治疗,之后通过ABR检测,听力阈值有统计学意义上的下降。免疫组织化学观察:治疗后SGN状态较治疗前,有比较明显的改善。作为一种医疗气体,H2近年来广泛用于许多领域的研究和多种疾病的治疗。但是,其对神经系统的保护和修复机制,目前仍然在探索。本实验中,虽然在损伤SGN处有移植入的干细胞团出现,但是没有预期的良好效果,考虑是否因为其他的一些因素的影响,如:SGN自身的遗传发育特性。我们通过研究SGN发育中microRNA表达的变化,将得到对其影响明确的目的microRNA。在干细胞模拟发育的过程中,观察基因调控水平下,干细胞是否向SGN“倾向性”迁移、增殖、分化。本实验我们将E14、P1、P5以及成年鼠的SGN,采用基因芯片技术筛选,得到随发育过程上调表达的17个,下调表达的miRNA 19个。这一结果提示我们,这些差异表达microRNA很可能参与了大鼠耳蜗SGN的发育过程。其后,我们对芯片筛选得到的部分microRNA进行q-PCR分析,最终确定了3种与microRNA芯片差异表达趋势相符的miRNA,即miR-21,miR-135a*和miR-205。后续的工作中,我们将进一步验证这3种microRNA在SGN发育、分化过程中的重要作用。
[Abstract]:Neurodeafness is one of the most common diseases in human beings. For a long time, researchers have been exploring the pathogenesis and treatment of neurodeafness. The main lesions are in the hair cells of the cochlea and the primary afferent spiral neurons, SGN.As the hair cells and SGN of the mammalian cochlea are difficult to regenerate after injury, how to repair the damaged SGNs, how to improve the survival rate of SGN in the microenvironment after the injury, How to enhance the self-repair function of SGN in complex disease is one of the main directions in the study of deafness mechanism. This study is devoted to the study of the treatment of stem cell H _ 2 after SGN injury and the development and differentiation of SGN. The expression of microRNA during maturation was studied to explore the possible repair mechanism of SGN at the level of gene regulation. In this study, the animal model of neurogenic deafness was successfully established by using venomous anthocyanin. The hearing threshold of ABR was changed in different concentrations of drugs. There was a positive correlation between the two methods, but DPOAE did not change significantly. The methods of immunohistochemistry, transmission electron microscope, scanning electron microscope, cell culture in vitro and so on were used in the experiment. To observe the damage of SGN induced by anthocyanin and lay a good foundation for the further study of neurodeafness. After partial hearing loss in animals, we will give the following two treatments: first, Stem cell transplantation. We used the identified olfactory bulb stem cells from E14C57BL / 6-GFP mice for stem cell transplantation. Given a certain external sound stimulation to observe its migration, survival, differentiation phenomenon. Sporadic stem cell clusters appeared near the injured spiral ganglion. The protective effect of dian2 H2. The animals with partial hearing impairment were treated regularly in the H2 treatment box after operation, and then detected by ABR. There was a statistically significant decrease in hearing threshold. Immunohistochemical observation: the state of SGN after treatment was higher than that before treatment. As a kind of medical gas, H _ 2 has been widely used in many fields of research and treatment of many diseases in recent years. However, its mechanism of protection and repair of nervous system is still being explored. In this study, although there were transplanted stem cell clusters at the site of SGN injury, there was no expected good effect. For example, the genetic and developmental characteristics of SGN itself. By studying the changes in the expression of microRNA in the development of SGN, we will get the specific purpose of microRNA. during the simulated development of stem cells, we will observe the level of gene regulation. Whether stem cells migrate, proliferate and differentiate into SGN. In this experiment, we screened E14P1P5 and SGNs of adult mice by gene chip technique, and obtained 17 up-regulated expression with developmental process. The down-regulated miRNA expression was 19. The results suggested that these differentially expressed microRNA might be involved in the development of rat cochlear SGN. Subsequently, we performed q-PCR analysis on some of the microRNA selected by microarray. Finally, three kinds of miRNAs, miR-21 miR-135a* and miR-205A *, which are consistent with the differential expression trend of microRNA microarray, were identified. In the following work, we will further verify the important role of these three microRNA in the development and differentiation of SGN.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R764.43

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