本文選題：神經(jīng)干細(xì)胞　切入點(diǎn)：干細(xì)胞移植　出處：《第四軍醫(yī)大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：1目的 神經(jīng)干細(xì)胞（neural stem cells, NSC）的移植為神經(jīng)系統(tǒng)退行性疾病和損傷的治療提供了新的思路，但是移植入內(nèi)耳的神經(jīng)干細(xì)胞向神經(jīng)元分化的效率很低，這成為移植的外源性神經(jīng)干細(xì)胞替代治療螺旋神經(jīng)神經(jīng)元（spiral ganglion neurons, SGNs）變性的主要障礙。在本研究中，我們驗(yàn)證了耳蝸螺旋神經(jīng)元損傷后的局部微環(huán)境更有利于移植的外源性神經(jīng)干細(xì)胞向神經(jīng)元方向分化，并初步闡明了其分子機(jī)制。 2方法 我們通過向耳蝸圓窗龕局部給予藥物ouabain建立大鼠螺旋神經(jīng)元損傷動(dòng)物模型。然后將分離、培養(yǎng)的神經(jīng)干細(xì)胞移植進(jìn)入正常、及螺旋神經(jīng)元損傷后的耳蝸鼓階。用免疫熒光染色和實(shí)時(shí)定量RT-PCR等技術(shù)來鑒定螺旋神經(jīng)元損傷后內(nèi)耳局部微環(huán)境對移植的神經(jīng)干細(xì)胞分化的的影響，并初步闡明其分子機(jī)制。最后我們使用transwell共培養(yǎng)系統(tǒng)通過體外實(shí)驗(yàn)驗(yàn)證我們的假設(shè)。 3結(jié)果 通過將外源性干細(xì)胞植入螺旋神經(jīng)元損傷的動(dòng)物耳蝸內(nèi)，我們發(fā)現(xiàn)，與對照組耳蝸相比，移植的神經(jīng)干細(xì)胞在螺旋神經(jīng)元損傷耳蝸中更易分化成MAP2陽性的神經(jīng)元。采用實(shí)時(shí)定量PCR和免疫熒光法，我們也證明了在螺旋神經(jīng)元損傷耳蝸內(nèi)，，螺旋神經(jīng)節(jié)內(nèi)衛(wèi)星細(xì)胞（雪旺細(xì)胞的一類，外周神經(jīng)節(jié)內(nèi)的雪旺細(xì)胞稱衛(wèi)星細(xì)胞）中Wnt1（Wnt信號(hào)通路配體）的表達(dá)顯著增加。我們進(jìn)一步證實(shí)，神經(jīng)干細(xì)胞表達(dá)Wnt信號(hào)通路受體和Wnt信號(hào)通路胞內(nèi)關(guān)鍵組件。在以上結(jié)果基礎(chǔ)上我們提出假設(shè)，雪旺細(xì)胞所產(chǎn)生Wnt1可能參與了移植神經(jīng)干細(xì)胞向神經(jīng)元分化的調(diào)控。我們使用transwell共培養(yǎng)系統(tǒng)在體外驗(yàn)證這一假設(shè)。我們將感染了慢病毒載體高表達(dá)Wnt1的耳蝸雪旺氏細(xì)胞與神經(jīng)干細(xì)胞共培養(yǎng)，結(jié)果顯示：與高表達(dá)Wnt1的雪旺細(xì)胞共培養(yǎng)的神經(jīng)干細(xì)胞，分化為MAP2陽性神經(jīng)元的比例顯著增加，然而這種促進(jìn)分化的作用可被DKK1（Wnt信號(hào)通路抑制劑）抑制。 4結(jié)論 這些結(jié)果表明，耳蝸雪旺氏細(xì)胞來源的Wnt1可激活移植神經(jīng)干細(xì)胞的胞內(nèi)Wnt信號(hào)通路，從而促進(jìn)其向神經(jīng)元的分化。詳盡的闡明損傷微環(huán)境的改變對移植外源性干細(xì)胞的影響，有助于我們提出更有效的策略以突破移植神經(jīng)干細(xì)胞向神經(jīng)元的分化率低這一屏障。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R764.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 阮芳銘,高文元,王海明,季紅光,趙嵐,崔風(fēng)軍;全耳蝸基底膜硬鋪片法的改進(jìn)和組化觀察[J];海軍醫(yī)學(xué)雜志;2002年02期

2 ;Molecular Characterization and SYBR Green I-Based Quantitative PCR for Duck Hepatitis Virus Type 1[J];Agricultural Sciences in China;2008年09期

本文編號(hào)：1603112