【摘要】：目的探討脂多糖(lipopolysaccharide,LPS)誘導(dǎo)小鼠急性角膜炎的臨床表現(xiàn)和炎癥因子的表達(dá)水平變化：研究消退素D1 (RvD1)在LPS誘導(dǎo)小鼠急性角膜炎癥中發(fā)揮的作用,對(duì)細(xì)胞因子及趨化因子的影響,探索消退素D1促進(jìn)炎癥消退的分子機(jī)制,初步了解其可能參與的信號(hào)通路。方法本實(shí)驗(yàn)包括兩個(gè)部分,第一部分通過建立LPS誘導(dǎo)小鼠急性角膜炎模型,觀察急性期的臨床表現(xiàn),通過病理學(xué)及免疫組織化學(xué)的方法研究其炎癥特點(diǎn),然后使用實(shí)時(shí)熒光定量PCR方法檢測LPS處理前后角膜組織內(nèi)IL-1β、IL-6、MCP-1及MIP-2四種細(xì)胞因子及趨化因子的表達(dá)。第二部分使用不同濃度RvD1(50ng/d和500ng/d)對(duì)急性期角膜組織進(jìn)行局部干預(yù),觀察1、3、5天時(shí)RvD1對(duì)角膜混濁程度及上皮愈合速度的影響；比較角膜組織炎癥反應(yīng)程度；應(yīng)用熒光定量PCR及ELISA方法檢測IL-1β、IL-6、MCP-1及MIP-2四種炎癥因子在角膜組織中mRNA及蛋白水平上的表達(dá)差異；并通過western blot方法檢測NF-κB信號(hào)途徑中內(nèi)源性NF-κB抑制因子IκBα在角膜組織中表達(dá)水平的變化,從而探索RvD1可能參與炎癥反應(yīng)的信號(hào)通路。結(jié)果1、LPS刺激后小鼠角膜混濁,HE染色及免疫組化顯示角膜基質(zhì)內(nèi)中性粒細(xì)胞浸潤,1-3天達(dá)高峰,3-5天逐漸減輕；2、LPS刺激后角膜基質(zhì)細(xì)胞合成的細(xì)胞因子IL-1β和IL-6,趨化因子MCP-1和MIP-2的mRNA表達(dá)水平較正常小鼠明顯增加(P0.05)；3、通過RvD1-50組(低劑量50ng/d)、RvD1-500(高劑量500ng/d)和對(duì)照組(PBS)進(jìn)行比較,發(fā)現(xiàn)RvD1可明顯減輕角膜炎癥后混濁程度,加快上皮愈合速度;HE染色及免疫組化顯示RvD1可減少中性粒細(xì)胞數(shù)量,防止中性粒細(xì)胞持續(xù)浸潤；4、熒光實(shí)時(shí)定量PCR和ELISA檢測發(fā)現(xiàn)RvD1可降低急性期角膜組織IL-1p、IL-6、MCP-1和MIP-2的在mRNA水平和蛋白水平的表達(dá),且和劑量呈正相關(guān)；5、western blot顯示在1天和3天時(shí)RvD1-500ng/d與對(duì)照組相比可增加LPS誘導(dǎo)后NF-κB抑制因子IKBα的表達(dá),而在5d時(shí)差異不明顯。結(jié)論LPS刺激后小鼠角膜組織內(nèi)細(xì)胞因子IL-1p和IL-6,趨化因子MCP-1和MIP-2的mRNA表達(dá)水平較正常小鼠明顯增加,提示IL-1β、IL-6、MCP-1和MIP-2可能參與了角膜炎癥反應(yīng)。RvD1減輕角膜炎癥后混濁程度,促進(jìn)角膜上皮愈合；減少中性粒細(xì)胞持續(xù)浸潤；RvD1可降低角膜組織IL-1β、IL-6、MCP-1和MIP-2的表達(dá),炎癥早期可部分逆轉(zhuǎn)LPS誘導(dǎo)后抑制因子IKBα的表達(dá)下降,提示RvDl可能是通過抑制NF-κB信號(hào)傳導(dǎo)通路起作用,促進(jìn)LPS誘導(dǎo)的小鼠角膜炎癥的消退。
[Abstract]:Objective to investigate the clinical manifestation and expression of inflammatory factors in acute keratitis induced by lipopolysaccharide (lipopolysaccharide,LPS) in mice. To study the role of RvD1 in acute keratitis induced by LPS in mice. The effects on cytokines and chemokine were studied to explore the molecular mechanism of anti-retrogression D1 in promoting inflammatory regression and to understand the signal pathway in which it might be involved. Methods this experiment consists of two parts. In the first part, the acute keratitis model of mice induced by LPS was established, the clinical manifestation of acute phase was observed, and the inflammatory characteristics were studied by means of pathology and immunohistochemistry. Then the expression of IL-1 尾, IL-6,MCP-1 and MIP- 2 cytokines and chemokines in corneal tissues before and after LPS treatment were detected by real-time fluorescence quantitative PCR. In the second part, different concentrations of RvD1 (50ng/d and 500ng/d) were used for local intervention in acute corneal tissue. The effects of RvD1 on corneal opacity and epithelial healing rate were observed at 1, 3, 5 days, and the degree of corneal inflammation was compared. The expression of IL-1 尾, IL-6,MCP-1 and MIP- 2 in corneal tissue was detected by fluorescence quantitative PCR and ELISA. Western blot was used to detect the expression of endogenous NF- 魏 B inhibitor I 魏 B 偽 in corneal tissues in the NF- 魏 B signaling pathway, so as to explore the possible involvement of RvD1 in the signal pathway of inflammatory reaction. Results 1LPs-stimulated corneal opacity, HE staining and immunohistochemistry showed that neutrophil infiltration in corneal stroma reached its peak at 3 days and decreased gradually at 3 days. 2. The mRNA expression levels of IL-1 尾 and IL-6, chemokine MCP-1 and MIP-2 in corneal stromal cells stimulated by LPS were significantly higher than those in normal mice (P0.05). (3) by comparing RvD1-50 group (low dose 50ng/d), RvD1-500 (high dose 500ng/d) and control group (PBS), it was found that RvD1 could significantly reduce the degree of opacity after corneal inflammation and accelerate the healing rate of epithelium. HE staining and immunohistochemistry showed that RvD1 could decrease the number of neutrophils and prevent the persistent infiltration of neutrophils. 4. Real-time fluorescence quantitative PCR and ELISA showed that RvD1 could decrease the expression of IL-1p,IL-6,MCP-1 and MIP-2 at mRNA level and protein level in acute corneal tissue, and it was positively correlated with the dose. 5. Western blot showed that RvD1-500ng/d increased the expression of NF- 魏 B inhibitor IKB 偽 on day 1 and day 3 compared with the control group, but there was no significant difference on day 5. Conclusion the mRNA expression levels of cytokines IL-1p, IL-6, chemokine MCP-1 and MIP-2 in corneal tissue of LPS-stimulated mice were significantly higher than those of normal mice, suggesting that IL-1 尾 and IL-6, were significantly increased. MCP-1 and MIP-2 may be involved in corneal inflammation. RvD1 may reduce the degree of opacity after corneal inflammation and promote corneal epithelial healing. Decrease the persistent neutrophil infiltration; RvD1 decreased the expression of IL-1 尾, IL-6,MCP-1 and MIP-2 in corneal tissue, and partially reversed the decreased expression of inhibitor IKB 偽 after LPS induction in the early stage of inflammation, suggesting that RvDl may play a role in inhibiting the signal transduction pathway of NF- 魏 B. To promote the regression of LPS-induced corneal inflammation in mice.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R772.21

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;NF-κB and Its Regulation on the Immune System[J];Cellular & Molecular Immunology;2004年05期

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