本文選題：睫狀神經(jīng)營(yíng)養(yǎng)因子 + 神經(jīng)軸突導(dǎo)向因子　； 參考：《青島大學(xué)》2015年博士論文

【摘要】：目的:中國(guó)已成為全球糖尿病患者最多的國(guó)家,近一半的糖尿病患者會(huì)出現(xiàn)原發(fā)性角膜病變,臨床表現(xiàn)為角膜上皮再生延遲和神經(jīng)末梢退行性等特點(diǎn)。我們通過(guò)基因芯片檢測(cè)發(fā)現(xiàn),睫狀神經(jīng)營(yíng)養(yǎng)因子(ciliary neurotrophic factor,CNTF)和神經(jīng)軸突導(dǎo)向因子Slit2在糖尿病小鼠角膜中的表達(dá)顯著下調(diào),本研究采用體內(nèi)動(dòng)物實(shí)驗(yàn)和體外細(xì)胞實(shí)驗(yàn),探討CNTF和Slit2對(duì)糖尿病小鼠角膜上皮和神經(jīng)損傷修復(fù)的治療作用及其機(jī)制。方法:選取8周齡以上C57BL/6小鼠,采用腹腔連續(xù)注射鏈脲霉素(STZ)誘導(dǎo)糖尿病模型,采用上皮刮除法構(gòu)建小鼠角膜上皮損傷修復(fù)模型,結(jié)膜下分別注射CNTF和Slit2重組蛋白,觀察CNTF和Slit2對(duì)角膜上皮及神經(jīng)損傷修復(fù)的影響。采用小鼠角膜上皮干/祖細(xì)胞系(TKE2)為體外細(xì)胞模型,檢測(cè)CNTF對(duì)角膜上皮干細(xì)胞活性及上皮再生信號(hào)通路的激活情況。采用高糖處理的TKE2細(xì)胞為體外細(xì)胞模型,檢測(cè)Slit2對(duì)角膜上皮再生相關(guān)信號(hào)通路的激活情況。采用原代培養(yǎng)小鼠三叉神經(jīng)節(jié)細(xì)胞,觀察Slit2對(duì)于三叉神經(jīng)節(jié)細(xì)胞軸突再生能力的影響。結(jié)果:通過(guò)建立小鼠角膜上皮損傷修復(fù)模型,檢測(cè)發(fā)現(xiàn)CNTF和Slit2都可以顯著促進(jìn)正常/糖尿病小鼠角膜上皮和角膜神經(jīng)的損傷修復(fù)。高糖處理TKE2細(xì)胞,外源性添加CNTF可以顯著促進(jìn)角膜上皮干細(xì)胞的活化。通過(guò)免疫熒光和western blot檢測(cè)發(fā)現(xiàn)CNTF可以激活角膜再生上皮和TKE2細(xì)胞中Akt和STAT3信號(hào)通路,封閉CNTF-STAT3信號(hào)通路,可導(dǎo)致角膜上皮修復(fù)明顯延遲、角膜上皮干細(xì)胞活化能力下降;Slit2可以激活糖尿病小鼠角膜上皮再生相關(guān)的EGFR、ERK和β-catenin信號(hào)通路,并顯著增強(qiáng)了細(xì)胞增殖相關(guān)基因Ki67的表達(dá)。通過(guò)體外培養(yǎng)小鼠三叉神經(jīng)節(jié)細(xì)胞,檢測(cè)發(fā)現(xiàn)外源性添加Slit2可以顯著促進(jìn)小鼠三叉神經(jīng)節(jié)細(xì)胞軸突的生長(zhǎng)。結(jié)論:1、CNTF可以顯著促進(jìn)正常/糖尿病小鼠角膜上皮的再生,并能促進(jìn)糖尿病小鼠角膜上皮下神經(jīng)叢密度的增加,此過(guò)程與激活角膜上皮再生相關(guān)的STAT3信號(hào)通路,促進(jìn)上皮細(xì)胞活化有關(guān)。2、Slit2可以顯著促進(jìn)糖尿病小鼠角膜上皮的再生,此過(guò)程與激活角膜上皮再生相關(guān)的EGFR、ERK、β-catenin信號(hào)通路,促進(jìn)上皮細(xì)胞增殖有關(guān)。Slit2可以顯著促進(jìn)糖尿病小鼠角膜上皮下神經(jīng)叢密度的增加和體外三叉神經(jīng)節(jié)細(xì)胞軸突的生長(zhǎng),具有可臨床應(yīng)用的潛在的神經(jīng)保護(hù)能力。
[Abstract]:Objective: China has become the country with the largest number of diabetic patients in the world. Nearly half of the diabetic patients will have primary keratopathy. The clinical manifestations are delayed regeneration of corneal epithelium and neurodegenerative nerve endings. We found that the expression of ciliary neurotrophic factor (ciliary neurotrophic factor) and neuronal guidance factor (Slit2) was significantly down-regulated in the cornea of diabetic mice by gene chip assay. In vivo and in vitro cell experiments were used to study the expression of ciliary neurotrophic factor (ciliary neurotrophic factor) and neurotropin Slit2 in the cornea of diabetic mice. To investigate the therapeutic effect and mechanism of CNTF and Slit2 on corneal epithelial and nerve injury in diabetic mice. Methods: the diabetic model was induced by continuous intraperitoneal injection of streptozotocin (STZ) in C57BL/6 mice over 8 weeks of age. The corneal epithelial injury repair model was established by epithelial curettage. CNTF and Slit2 recombinant proteins were injected under conjunctiva, respectively. To observe the effect of CNTF and Slit2 on corneal epithelium and nerve repair. Mouse corneal epithelial stem / progenitor cell line (TKE2) was used as a model in vitro to detect the activation of CNTF on corneal epithelial stem cell activity and epithelial regeneration signal pathway. TKE2 cells treated with high glucose were used as cell model in vitro to detect the activation of Slit2 signaling pathway related to corneal epithelium regeneration. The effect of Slit2 on axon regeneration of trigeminal ganglion cells was studied by primary culture of mouse trigeminal ganglion cells. Results: by establishing the model of corneal epithelial injury repair in mice, it was found that both CNTF and Slit2 could significantly promote the repair of corneal epithelium and corneal nerve in normal / diabetic mice. High glucose treatment of TKE2 cells, exogenous addition of CNTF can significantly promote the activation of corneal epithelial stem cells. By immunofluorescence and western blot detection, it was found that CNTF could activate Akt and STAT3 signaling pathway in corneal regenerated epithelium and TKE2 cells, and block CNTF-STAT3 signal pathway, which resulted in delayed corneal epithelial repair. The activation ability of corneal epithelial stem cells decreased and Slit2 activated the EGFR ERK and 尾 -catenin signaling pathway associated with corneal epithelial regeneration in diabetic mice, and significantly enhanced the expression of proliferation-related gene Ki67 in diabetic mice. By culture of mouse trigeminal ganglion cells in vitro, it was found that exogenous addition of Slit2 could significantly promote the growth of axons of mouse trigeminal ganglion cells. Conclusion the STAT3 signaling pathway associated with activation of corneal epithelium regeneration in diabetic / normal / diabetic mice can be significantly promoted by the STAT3 signaling pathway associated with the activation of corneal epithelial regeneration in the normal / diabetic mice and the increase in the density of the subcutaneous plexus of the cornea in the diabetic mice. Promoting the activation of epithelial cells may significantly promote the regeneration of corneal epithelium in diabetic mice. This process is related to the activation of corneal epithelial regeneration through the EGFRERK, 尾 -catenin signaling pathway. Promoting the proliferation of epithelial cells. Slit2 can significantly increase the density of corneal subcutaneous plexus and the growth of axons of trigeminal ganglion cells in vitro in diabetic mice. It has the potential neuroprotective ability for clinical application.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R587.2;R772.2

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1 黃劍，，蔡愛(ài)玲，李社會(huì);風(fēng)濕熱引起角膜病變一例[J];眼科研究;1995年02期

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本文編號(hào)：1916511