睫状神经营养因子CNTF和轴突导向因子Slit2在糖尿病角膜病变中的作用及分子机制研究

发布时间：2018-05-20 22:25

本文选题：睫状神经营养因子 + 神经轴突导向因子　；参考：《青岛大学》2015年博士论文

【摘要】：目的:中国已成为全球糖尿病患者最多的国家,近一半的糖尿病患者会出现原发性角膜病变,临床表现为角膜上皮再生延迟和神经末梢退行性等特点。我们通过基因芯片检测发现,睫状神经营养因子(ciliary neurotrophic factor,CNTF)和神经轴突导向因子Slit2在糖尿病小鼠角膜中的表达显著下调,本研究采用体内动物实验和体外细胞实验,探讨CNTF和Slit2对糖尿病小鼠角膜上皮和神经损伤修复的治疗作用及其机制。方法:选取8周龄以上C57BL/6小鼠,采用腹腔连续注射链脲霉素(STZ)诱导糖尿病模型,采用上皮刮除法构建小鼠角膜上皮损伤修复模型,结膜下分别注射CNTF和Slit2重组蛋白,观察CNTF和Slit2对角膜上皮及神经损伤修复的影响。采用小鼠角膜上皮干/祖细胞系(TKE2)为体外细胞模型,检测CNTF对角膜上皮干细胞活性及上皮再生信号通路的激活情况。采用高糖处理的TKE2细胞为体外细胞模型,检测Slit2对角膜上皮再生相关信号通路的激活情况。采用原代培养小鼠三叉神经节细胞,观察Slit2对于三叉神经节细胞轴突再生能力的影响。结果:通过建立小鼠角膜上皮损伤修复模型,检测发现CNTF和Slit2都可以显著促进正常/糖尿病小鼠角膜上皮和角膜神经的损伤修复。高糖处理TKE2细胞,外源性添加CNTF可以显著促进角膜上皮干细胞的活化。通过免疫荧光和western blot检测发现CNTF可以激活角膜再生上皮和TKE2细胞中Akt和STAT3信号通路,封闭CNTF-STAT3信号通路,可导致角膜上皮修复明显延迟、角膜上皮干细胞活化能力下降;Slit2可以激活糖尿病小鼠角膜上皮再生相关的EGFR、ERK和β-catenin信号通路,并显著增强了细胞增殖相关基因Ki67的表达。通过体外培养小鼠三叉神经节细胞,检测发现外源性添加Slit2可以显著促进小鼠三叉神经节细胞轴突的生长。结论:1、CNTF可以显著促进正常/糖尿病小鼠角膜上皮的再生,并能促进糖尿病小鼠角膜上皮下神经丛密度的增加,此过程与激活角膜上皮再生相关的STAT3信号通路,促进上皮细胞活化有关。2、Slit2可以显著促进糖尿病小鼠角膜上皮的再生,此过程与激活角膜上皮再生相关的EGFR、ERK、β-catenin信号通路,促进上皮细胞增殖有关。Slit2可以显著促进糖尿病小鼠角膜上皮下神经丛密度的增加和体外三叉神经节细胞轴突的生长,具有可临床应用的潜在的神经保护能力。
[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.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2;R772.2

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