發(fā)布時(shí)間：2018-02-14 15:42

  本文關(guān)鍵詞： 螺旋神經(jīng)節(jié) 毒毛旋花子甙 嗅球干細(xì)胞 氫氣 microRNA 耳蝸　出處：《第四軍醫(yī)大學(xué)》2011年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：神經(jīng)性耳聾是人類(lèi)最常見(jiàn)的疾病之一。長(zhǎng)久以來(lái),研究者不斷的探求其發(fā)病機(jī)理、治療方法。在諸多病因中,主要的病變部位在耳蝸毛細(xì)胞和初級(jí)傳入的螺旋神經(jīng)元(SGN)。由于哺乳動(dòng)物耳蝸毛細(xì)胞和SGN損傷后難以再生,那么,如何修復(fù)損傷的SGN,如何提高SGN在損傷后微環(huán)境里的存活率,如何增強(qiáng)SGN在復(fù)雜的疾病狀態(tài)下自我修復(fù)功能,是耳聾機(jī)制研究的主要方向之一。本研究致力于研究SGN損傷后給予干細(xì)胞、H2治療,以及通過(guò)研究SGN發(fā)育、分化、成熟過(guò)程中microRNA表達(dá),探討基因調(diào)控水平下SGN可能的修復(fù)機(jī)制。 本研究中,成功的采用毒毛旋花子甙制造了神經(jīng)性耳聾的動(dòng)物模型。通過(guò)藥物的不同濃度,動(dòng)物的ABR聽(tīng)力閾值有不同程度的改變,兩者呈正相關(guān)。但是,DPOAE則沒(méi)有明顯改變。試驗(yàn)中繼以免疫組織化學(xué)、透射電鏡、掃描電鏡、體外細(xì)胞培養(yǎng)等方法,觀(guān)察毒毛旋花子甙對(duì)SGN的損傷。為下一步研究神經(jīng)性耳聾的后續(xù)工作打下良好的基礎(chǔ)。 將動(dòng)物的聽(tīng)力部分損傷后,我們予以以下兩種治療手段:一,干細(xì)胞的移植。我們將已鑒定的E14C57BL/6-GFP鼠的嗅球干細(xì)胞用于干細(xì)胞移植。采用兩種途徑:⑴內(nèi)聽(tīng)道的聽(tīng)神經(jīng)根移植干細(xì)胞術(shù)。⑵聽(tīng)力損傷側(cè)頸內(nèi)動(dòng)脈干細(xì)胞移植術(shù)。移植干細(xì)胞后,給予一定的外界聲刺激,觀(guān)察其遷移、存活、分化現(xiàn)象。本實(shí)驗(yàn)觀(guān)察:經(jīng)由此兩種路徑移植后,在損傷的螺旋神經(jīng)節(jié)附近出現(xiàn)零星的干細(xì)胞團(tuán)。二,H2的保護(hù)作用。將聽(tīng)力部分損傷的動(dòng)物,手術(shù)后定時(shí)放入H2治療箱內(nèi)治療,之后通過(guò)ABR檢測(cè),聽(tīng)力閾值有統(tǒng)計(jì)學(xué)意義上的下降。免疫組織化學(xué)觀(guān)察:治療后SGN狀態(tài)較治療前,有比較明顯的改善。作為一種醫(yī)療氣體,H2近年來(lái)廣泛用于許多領(lǐng)域的研究和多種疾病的治療。但是,其對(duì)神經(jīng)系統(tǒng)的保護(hù)和修復(fù)機(jī)制,目前仍然在探索。 本實(shí)驗(yàn)中,雖然在損傷SGN處有移植入的干細(xì)胞團(tuán)出現(xiàn),但是沒(méi)有預(yù)期的良好效果,考慮是否因?yàn)槠渌囊恍┮蛩氐挠绊?如:SGN自身的遺傳發(fā)育特性。我們通過(guò)研究SGN發(fā)育中microRNA表達(dá)的變化,將得到對(duì)其影響明確的目的microRNA。在干細(xì)胞模擬發(fā)育的過(guò)程中,觀(guān)察基因調(diào)控水平下,干細(xì)胞是否向SGN“傾向性”遷移、增殖、分化。本實(shí)驗(yàn)我們將E14、P1、P5以及成年鼠的SGN,采用基因芯片技術(shù)篩選,得到隨發(fā)育過(guò)程上調(diào)表達(dá)的17個(gè),下調(diào)表達(dá)的miRNA 19個(gè)。這一結(jié)果提示我們,這些差異表達(dá)microRNA很可能參與了大鼠耳蝸SGN的發(fā)育過(guò)程。其后,我們對(duì)芯片篩選得到的部分microRNA進(jìn)行q-PCR分析,最終確定了3種與microRNA芯片差異表達(dá)趨勢(shì)相符的miRNA,即miR-21,miR-135a*和miR-205。后續(xù)的工作中,我們將進(jìn)一步驗(yàn)證這3種microRNA在SGN發(fā)育、分化過(guò)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R764.43

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本文編號(hào)：1511041