本文選題：神經(jīng)再生 + 神經(jīng)肽P物質(zhì)　； 參考：《濟(jì)南大學(xué)》2017年碩士論文

【摘要】：目的通過(guò)C57小鼠和B6.Cg-Tac1tm1Bbm/J(SP KO)小鼠角膜神經(jīng)再生模型,研究SP-NK1R信號(hào)通路介導(dǎo)角膜損傷神經(jīng)再生的關(guān)鍵作用及其分子機(jī)制。方法鼠尾基因鑒定法鑒定SP敲除小鼠;裂隙燈顯微鏡觀察SP敲除小鼠角膜表型;角膜神經(jīng)染色法檢測(cè)SP敲除小鼠角膜神經(jīng)分布。構(gòu)建小鼠角膜上皮損傷模型,角膜熒光素鈉染色法統(tǒng)計(jì)小鼠角膜上皮損傷修復(fù)速率;通過(guò)Real-time PCR(RT-PCR)檢測(cè)小鼠角膜上皮的神經(jīng)營(yíng)養(yǎng)因子的表達(dá)。建立小鼠角膜上皮損傷模型,實(shí)驗(yàn)分為正常對(duì)照組、正常小鼠+L733060組(結(jié)膜下注射)、SP敲除小鼠組、SP敲除小鼠+Sar-SP組(局部點(diǎn)眼)。通過(guò)角膜神經(jīng)染色檢測(cè)小鼠角膜基底膜再生神經(jīng)纖維密度;通過(guò)流式細(xì)胞儀分析角膜中中性粒細(xì)胞、巨噬細(xì)胞趨化的數(shù)量變化;通過(guò)RT-PCR檢測(cè)小鼠角膜損傷后神經(jīng)再生相關(guān)基因的表達(dá);通過(guò)免疫熒光共定位驗(yàn)證小鼠角膜中性粒細(xì)胞浸潤(rùn)數(shù)量的變化;通過(guò)酶聯(lián)免疫吸附試驗(yàn)(Elisa)檢測(cè)小鼠角膜中NGF的表達(dá)水平的變化。檢測(cè)中性粒細(xì)胞清除及巨噬細(xì)胞清除后,小鼠角膜基底膜再生神經(jīng)纖維密度以及小鼠角膜上皮損傷修復(fù)速率。結(jié)果鼠尾基因鑒定結(jié)果驗(yàn)證Tac-1(SP基因)完全敲除。與對(duì)照組相比,SP敲除小鼠角膜正常,未出現(xiàn)自發(fā)病理特征,且SP敲除小鼠角膜基底膜神經(jīng)纖維和上皮末梢神經(jīng)纖維分布均無(wú)顯著變化(P0.05)。與正常C57小鼠相比,SP敲除小鼠的角膜上皮修復(fù)速率未見(jiàn)顯著變化(P0.05);而且SP敲除小鼠完整角膜上皮和再生角膜上皮中神經(jīng)營(yíng)養(yǎng)因子的表達(dá)未見(jiàn)顯著變化(P0.05)。角膜神經(jīng)染色結(jié)果顯示,與正常小鼠相比,SP敲除小鼠角膜基底膜再生神經(jīng)密度顯著減少(P0.05),而外源性補(bǔ)充Sar-SP可顯著恢復(fù)SP敲除小鼠角膜神經(jīng)再生(P0.05);正常小鼠結(jié)膜下注射L733060可顯著抑制角膜神經(jīng)再生(P0.05)。流式細(xì)胞儀分析結(jié)果與免疫熒光結(jié)果顯示:與對(duì)照組相比,SP敲除小鼠及L733060干預(yù)組小鼠角膜中性粒細(xì)胞(CD45+/CD11b+/Ly6G+)的浸潤(rùn)數(shù)量顯著減少(P0.05),而Sar-SP可以顯著恢復(fù)中性粒細(xì)胞的浸潤(rùn),但巨噬細(xì)胞(CD45+/CD11b+/F4/80+)浸潤(rùn)數(shù)量未受影響(P0.05)。與正常對(duì)照組相比,中性粒細(xì)胞完全清除可以顯著抑制角膜神經(jīng)再生以及角膜上皮愈合(P0.05),但巨噬細(xì)胞完全清除對(duì)其無(wú)顯著作用(P0.05)。與正常小鼠相比,SP敲除小鼠角膜中NGF表達(dá)明顯降低(P0.05),而外源性給予Sar-SP可恢復(fù)NGF表達(dá)(P0.05)。正常小鼠L733060處理或清除中性粒細(xì)胞均顯著降低角膜的NGF含量(P0.05)。而巨噬細(xì)胞清除對(duì)角膜的NGF含量沒(méi)有顯著影響(P0.05)。結(jié)論SP-NK1R信號(hào)通路通過(guò)影響中性粒細(xì)胞的浸潤(rùn)及其NGF的表達(dá)調(diào)控角膜神經(jīng)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.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R772.2

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