【作者】 张娜；

【导师】 史伟云；

【作者基本信息】 济南大学 ， 临床医学（眼科学）， 2017， 硕士

【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(P>0.05)。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(P>0.05);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(P>0.05)。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(P<0.05),而外源性补充Sar-SP可显著恢复SP敲除小鼠角膜神经再生(P<0.05);正常小鼠结膜下注射L733060可显著抑制角膜神经再生(P<0.05)。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(P<0.05),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(P>0.05)。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(P<0.05),但巨噬细胞完全清除对其无显著作用(P>0.05)。与正常小鼠相比,SP敲除小鼠角膜中NGF表达明显降低(P<0.05),而外源性给予Sar-SP可恢复NGF表达(P<0.05)。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(P<0.05)。而巨噬细胞清除对角膜的NGF含量没有显著影响(P>0.05)。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。更多还原

【Abstract】 PurposeTo explore the role and molecular mechanism of SP-NK1 R signaling pathway in corneal nerve regeneration through C57 and B6.Cg-Tac1tm1Bbm/ J(SP KO)mouse corneal nerve injury model.MethodsSP knockout mice were identified by mouse tail gene identification.Slit lamp microscope was used to observe SP knockout mouse corneal phenotype.Corneal nerve distribution was observed by corneal nerve staining.The corneal epithelial injury model was established,the repair rate of corneal epithelial injury was observed by using corneal fluorescein sodium staining;the expression of neurotrophic factor in corneal epithelium was by real-time PCR(RT-PCR).The corneal epithelial injury model was established,the mice were randomly divided into normal control group,normal +L733060 group(subconjunctival injection),SP knockout mice group,SP knockout mice + Sar-SP group(local application).The density of regenerated nerve fibers of mice was observed by corneal nerve staining.The number of chemotaxis of neutrophils and macrophages was analyzed by flow cytometry.The expression of genes related to nerve regeneration after corneal injury in mice was detected by RT-PCR.The changes of corneal neutrophil chemotaxis in mice cornea were verified by immunofluorescence staining.The expression level of NGF in the cornea was detected by enzyme-linked immunosorbent assay(ElISA).The regenerated nerve fiber density the repair rate of corneal epithelial were detected after the neutrophil clearance and macrophage clearance.ResultsMouse tail gene identification showed that Tac-1(SP gene)was completely knocked out.Compared with the control group,the cornea of SP knockout mice was normal,with no spontaneous pathological features in the corneal or corneal innervation(P>0.05).Compared with normal C57 mice,the corneal epithelial repair rate of SP knockout mice was no significant change(P>0.05),and the expression of neurotrophic factors in corneal epithelium of SP knockout mice were no significant change(P> 0.05).Compared with normal mice,the corneal nerve regeneration of SP knockout mouse was significantly reduced(P<0.05),while exogenous Sar-SP can significantly restore the regeneration of the corneal nerve in SP knockout mouse(P<0.05);Subcutaneous injection of L733060 to normal mice could significantly inhibit corneal nerve regeneration(P <0.05).Compared with the control group,the number of chemotaxis of neutrophils(CD45+/CD11b+ / Ly6G+)in SP knockout mouse and L733060 intervention group was significantly reduced(P<0.05),while Sar-SP can significantly restore neutrophil migration,yet the number of macrophages(CD45+/CD11b+/F4/80+)chemotaxis was not affected(P>0.05).Compared with the control group,neutrophils clearence can significantly inhibit corneal nerve regeneration and corneal epithelial healing(P<0.05),yet macrophages depletion has no significant effect on them(P>0.05).Compared with normal mice,the expression of NGF in the cornea of SP knockout mouse was significantly decreased(P<0.05),while exogenous administration of Sar-SP can restore NGF expression(P<0.05).Normal mouse treated with L733060 or removal of neutrophils could significantly reduce NGF expression(P<0.05).The macrophage clearance had no significant effect on the NGF expression(P>0.05).ConclusionSP-NK1 R signaling pathway regulates the regeneration of corneal nerves by affecting the migration of neutrophils and the NGF expression.更多还原