角膜上皮细胞表达神经营养因子促进角膜神经再生的作用研究

发布时间：2018-04-22 19:04

本文选题：角膜上皮 + 神经生长因子　；参考：《济南大学》2017年硕士论文

【摘要】：目的通过正常小鼠和链脲佐菌素(STZ)诱导的I糖尿病小鼠的角膜上皮损伤模型以及小鼠角膜上皮干/祖细胞三叉神经节感觉神经元(TKE2细胞 TG细胞)体外共培养模型,检测小鼠角膜上皮细胞中神经营养因子(neurotrophic factors)的表达情况,研究其对小鼠角膜神经再生的影响。方法用C57/BL6小鼠建立角膜上皮损伤模型,检测并比较小鼠正常角膜(角膜上皮损伤未损伤)、再生期(角膜上皮损伤后2天)以及再生后(角膜上皮损伤后7天)角膜上皮细胞中神经营养因子的表达。体外培养小鼠角膜上皮干/祖细胞(TKE2细胞),利用划痕模型检测正常及损伤后TKE2细胞中神经生长因子(NGF)和胶质细胞源性神经生长因子(GDNF)的表达变化。通过TKE2细胞 TG细胞体外共培养模型,检测三叉神经节感觉神经元(TG细胞)轴突生长变化。利用链脲佐菌素(STZ)诱导I型糖尿病小鼠,通过角膜上皮损伤修复模型,检测糖尿病小鼠角膜上皮中NGF和GDNF表达变化;观察糖尿病小鼠角膜上皮修复和神经再生以及外源性NGF和GDNF对糖尿小鼠角膜上皮修复、神经再生和角膜神经敏感度恢复的影响。结果在角膜上皮修复和角膜神经再生过程中,与正常角膜相比,角膜上皮中NGF和GDNF的表达在角膜上皮再生期(角膜上皮损伤后2天)显著上升,再生后(损伤后第7天)部分恢复;而NGF中和抗体或GDNF封闭多肽可显著抑制角膜上皮损伤修复及神经再生。与正常对照组相比,TKE2细胞损伤后(划痕实验48h)NGF和GDNF的表达显著上升,且划痕后的条件培养上清可以显著促进TG细胞轴突的生长;而NGF中和抗体或GDNF封闭多肽可显著抵消TKE2条件培养上清的促进TG细胞轴突生长的作用。微流体小室共培养实验结果显示,TKE2细胞及其条件培养基可以显著促进小鼠TG细胞的轴突的生长,且呈现浓度依赖性。与正常小鼠相比,糖尿病小鼠角膜上皮损伤修复及神经再生均显著延迟;糖尿病小鼠再生期的角膜上皮NGF和GDNF的表达均显著下降;而外源性给予NGF或GDNF均可显著促进糖尿病小鼠角膜上皮损伤愈合、神经再生以及角膜神经敏感度的恢复。结论角膜上皮细胞表达和分泌多种神经营养因子促进角膜神经再生,其中NGF和GDNF在角膜神经再生中起着重要作用。
[Abstract]:Objective to study the model of corneal epithelial injury induced by streptozotocin (STZ) in normal mice and streptozotocin (STZ) induced by streptozotocin (STZ) and co-culture of TKE2 cells (TG cells) in rat corneal epithelial stem / progenitor cells. To detect the expression of neurotrophic factors in mouse corneal epithelial cells and to study the effect of neurotrophic factors on corneal regeneration in mice. Methods the corneal epithelial injury model was established in C57/BL6 mice. The expression of neurotrophic factor in normal cornea (no injury of corneal epithelium), regeneration period (2 days after corneal epithelium injury) and regeneration (7 days after corneal epithelial injury) were detected and compared. The expression of nerve growth factor (NGF) and glial cell derived nerve growth factor (GDF) in normal and injured TKE2 cells were detected by scratch model. The changes of axon growth in trigeminal ganglion sensory neurons (TG cells) were detected by co-culture of TKE2 cells and TG cells in vitro. Type I diabetic mice were induced by streptozotocin (STZ). The expression of NGF and GDNF in corneal epithelium of diabetic mice was detected by corneal epithelial injury repair model. The effects of exogenous NGF and GDNF on corneal epithelial repair, nerve regeneration and corneal nerve sensitivity recovery in diabetic mice were observed. Results in the process of corneal epithelium repair and corneal nerve regeneration, the expression of NGF and GDNF in corneal epithelium increased significantly during the period of corneal epithelium regeneration (2 days after corneal epithelial injury) compared with normal cornea. NGF neutralizing antibody or GDNF blocking polypeptide could significantly inhibit corneal epithelial injury repair and nerve regeneration after regeneration (7 days after injury). Compared with the normal control group, the expression of 48h)NGF and GDNF in TKE2 cells was significantly increased after injury (scratching test), and the supernatant of conditioned culture after scratch could significantly promote the growth of axons of TG cells. NGF neutralizing antibodies or GDNF blocking peptides could significantly counteract the role of TKE2 conditioned supernatants in promoting the axonal growth of TG cells. The results of microfluid chamber co-culture showed that TKE2 cells and their conditioned medium could significantly promote the axon growth of mouse TG cells in a concentration-dependent manner. Compared with normal mice, the corneal epithelial injury repair and nerve regeneration in diabetic mice were significantly delayed, and the expression of NGF and GDNF in corneal epithelium of diabetic mice decreased significantly during regeneration. Exogenous administration of NGF or GDNF could significantly promote corneal epithelial injury healing, nerve regeneration and recovery of corneal nerve sensitivity in diabetic mice. Conclusion the corneal epithelial cells express and secrete a variety of neurotrophic factors to promote corneal nerve regeneration. NGF and GDNF play an important role in corneal nerve regeneration.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R772.2

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