神经肽Substance P调控中性粒细胞影响角膜神经再生的作用及机制研究

发布时间：2018-05-07 04:03

本文选题：神经再生 + 神经肽P物质　；参考：《济南大学》2017年硕士论文

【摘要】：目的通过C57小鼠和B6.Cg-Tac1tm1Bbm/J(SP KO)小鼠角膜神经再生模型,研究SP-NK1R信号通路介导角膜损伤神经再生的关键作用及其分子机制。方法鼠尾基因鉴定法鉴定SP敲除小鼠;裂隙灯显微镜观察SP敲除小鼠角膜表型;角膜神经染色法检测SP敲除小鼠角膜神经分布。构建小鼠角膜上皮损伤模型,角膜荧光素钠染色法统计小鼠角膜上皮损伤修复速率;通过Real-time PCR(RT-PCR)检测小鼠角膜上皮的神经营养因子的表达。建立小鼠角膜上皮损伤模型,实验分为正常对照组、正常小鼠+L733060组(结膜下注射)、SP敲除小鼠组、SP敲除小鼠+Sar-SP组(局部点眼)。通过角膜神经染色检测小鼠角膜基底膜再生神经纤维密度;通过流式细胞仪分析角膜中中性粒细胞、巨噬细胞趋化的数量变化;通过RT-PCR检测小鼠角膜损伤后神经再生相关基因的表达;通过免疫荧光共定位验证小鼠角膜中性粒细胞浸润数量的变化;通过酶联免疫吸附试验(Elisa)检测小鼠角膜中NGF的表达水平的变化。检测中性粒细胞清除及巨噬细胞清除后,小鼠角膜基底膜再生神经纤维密度以及小鼠角膜上皮损伤修复速率。结果鼠尾基因鉴定结果验证Tac-1(SP基因)完全敲除。与对照组相比,SP敲除小鼠角膜正常,未出现自发病理特征,且SP敲除小鼠角膜基底膜神经纤维和上皮末梢神经纤维分布均无显著变化(P0.05)。与正常C57小鼠相比,SP敲除小鼠的角膜上皮修复速率未见显著变化(P0.05);而且SP敲除小鼠完整角膜上皮和再生角膜上皮中神经营养因子的表达未见显著变化(P0.05)。角膜神经染色结果显示,与正常小鼠相比,SP敲除小鼠角膜基底膜再生神经密度显著减少(P0.05),而外源性补充Sar-SP可显著恢复SP敲除小鼠角膜神经再生(P0.05);正常小鼠结膜下注射L733060可显著抑制角膜神经再生(P0.05)。流式细胞仪分析结果与免疫荧光结果显示:与对照组相比,SP敲除小鼠及L733060干预组小鼠角膜中性粒细胞(CD45+/CD11b+/Ly6G+)的浸润数量显著减少(P0.05),而Sar-SP可以显著恢复中性粒细胞的浸润,但巨噬细胞(CD45+/CD11b+/F4/80+)浸润数量未受影响(P0.05)。与正常对照组相比,中性粒细胞完全清除可以显著抑制角膜神经再生以及角膜上皮愈合(P0.05),但巨噬细胞完全清除对其无显著作用(P0.05)。与正常小鼠相比,SP敲除小鼠角膜中NGF表达明显降低(P0.05),而外源性给予Sar-SP可恢复NGF表达(P0.05)。正常小鼠L733060处理或清除中性粒细胞均显著降低角膜的NGF含量(P0.05)。而巨噬细胞清除对角膜的NGF含量没有显著影响(P0.05)。结论SP-NK1R信号通路通过影响中性粒细胞的浸润及其NGF的表达调控角膜神经的再生。
[Abstract]:Objective to study the key role and molecular mechanism of SP-NK1R signaling pathway in corneal regeneration in C57 mice and B6.Cg-Tac1tm1Bbm/J(SP KOB mice. Methods SP knockout mice were identified by rat tail gene identification, corneal phenotypes of SP knockout mice were observed by slit lamp microscope and corneal nerve distribution of SP knockout mice was detected by corneal nerve staining. The corneal epithelial injury model of mice was established. The rate of corneal epithelial injury repair was measured by fluorescein sodium staining and the expression of neurotrophic factor in corneal epithelium was detected by Real-time PCRRT-PCR. The model of corneal epithelial injury in mice was established and divided into two groups: normal control group (L733060 group) (subconjunctival injection of SP-knockout mice group) and SP knockout group (Sar-SP group). The density of regenerated nerve fibers in the corneal basement membrane of mice was detected by corneal nerve staining, and the changes of neutrophil and macrophage chemotaxis in the cornea were analyzed by flow cytometry. The expression of nerve regeneration related genes after corneal injury was detected by RT-PCR and the changes of neutrophil infiltration in the cornea of mice were verified by immunofluorescence co-localization. The expression of NGF in the cornea of mice was detected by Elisa. After clearance of neutrophils and macrophages, the density of regenerated nerve fibers in the corneal basement membrane and the repair rate of corneal epithelial injury in mice were measured. Results the results of mouse tail gene identification confirmed that the Tac-1(SP gene was completely knocked out. Compared with the control group, the cornea of SP knockout mice had normal cornea, no spontaneous pathological features, and there was no significant change in the distribution of basal membrane nerve fibers and epithelial peripheral nerve fibers in SP knockout mice. Compared with the normal C57 mice, the corneal epithelial repair rate of SP knockout mice did not change significantly, and the expression of neurotrophic factor in intact corneal epithelium and regenerated corneal epithelium of SP knockout mice was not significantly changed. Corneal nerve staining showed that Compared with the normal mice, the nerve density of corneal basement membrane regeneration in SP knockout mice decreased significantly, while exogenous Sar-SP supplementation significantly restored the corneal nerve regeneration of SP knockout mice, and the subconjunctival injection of L733060 significantly inhibited the corneal nerve regeneration of SP knockout mice. The results of flow cytometry and immunofluorescence showed that the infiltration of neutrophil CD45 / CD11b / Ly6G decreased significantly in SP knockout mice and L733060 group compared with control group, while Sar-SP could significantly restore neutrophil infiltration. But the number of macrophages in CD45 / CD11b / F4 / 80 was not affected. Compared with the normal control group, complete neutrophil clearance could significantly inhibit corneal nerve regeneration and corneal epithelial healing (P0.05A), but the complete clearance of macrophages had no significant effect on it. Compared with normal mice, the expression of NGF in cornea of SP knockout mice decreased significantly, while exogenous Sar-SP could restore the expression of NGF. Normal mice L733060 treated or eliminated neutrophils significantly decreased the NGF content of cornea (P 0.05). However, macrophage clearance had no significant effect on the NGF content of cornea. Conclusion SP-NK1R signaling pathway regulates corneal nerve regeneration by affecting neutrophil infiltration and NGF expression.
【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】：R772.2

【相似文献】