本文關(guān)鍵詞： 脂多糖 緊密連接 p38MAPK信號(hào)通路 JNK信號(hào)通路 基質(zhì)金屬蛋白酶　出處：《廣西醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分在脂多糖誘導(dǎo)的血腦屏障破壞中p38MAPK、JNK信號(hào)通路的作用機(jī)制研究目的：探討p38MAPK和c-Jun氨基末端激酶(JNK)信號(hào)通路在脂多糖(lipopolysaccharide, LPS)誘導(dǎo)血腦屏障(blood-brain barrier, BBB)的破壞中的作用。方法：1.培養(yǎng)人腦微血管內(nèi)皮細(xì)胞(human cerebral microvascular endothelial cells, hCMEC/D3),用不同濃度的LPS、p38MAPK和JNK信號(hào)通路抑制劑(分別為SB203580、SP600125)分別刺激細(xì)胞24h,用四甲基偶氮唑鹽(MTT法)檢測(cè)其對(duì)細(xì)胞活力的影響。2.用不同濃度的LPS刺激細(xì)胞,蛋白免疫印跡法(Western blot法)檢測(cè)緊密連接蛋白Occludin、ZO-1的變化。3.用LPS刺激細(xì)胞不同的時(shí)間后,用蛋白免疫印跡法檢測(cè)p38MAPK及JNK信號(hào)通路磷酸化水平的變化。4.用SB203580、SP600125預(yù)處理細(xì)胞1h后,再加入LPS共培養(yǎng)24h,分別用Western blot法及熒光定量PCR法(RT-PCR)檢測(cè)緊密連接Occludin、ZO-1蛋白及其mRNA表達(dá)的變化。結(jié)果：1. LPS、SB203580和SP600125濃度分別在10μg/ml、7.69μg/m1和0.22μg/ml以下時(shí)對(duì)hCMEC/D3細(xì)胞活力無(wú)明顯影響。2.LPS刺激細(xì)胞可誘導(dǎo)緊密連接Occludin、ZO-1蛋白及其mRNA的表達(dá)水平顯著下調(diào),Western blot法進(jìn)一步證實(shí)LPS刺激細(xì)胞后p38MAPK和JNK信號(hào)通路分子的磷酸化顯著增加。3.SB203580或SP600125預(yù)處理細(xì)胞1h,可以顯著上調(diào)LPS誘導(dǎo)的Occludin蛋白及其mRNA的表達(dá)水平,但對(duì)LPS誘導(dǎo)的ZO-1蛋白及mRNA表達(dá)的影響無(wú)統(tǒng)計(jì)學(xué)意義。結(jié)論：LPS可誘導(dǎo)緊密連接Occludin、ZO-1蛋白及其mRNA的下調(diào)導(dǎo)致BBB破壞；LPS可能通過(guò)激活p38MAPK和JNK信號(hào)通路磷酸化,從而調(diào)節(jié)hCMEC/D3細(xì)胞緊密連接Occludin蛋白及其mRNA的表達(dá)；然而LPS對(duì)ZO-1蛋白及其mRNA表達(dá)的影響可能通過(guò)其他信號(hào)通路實(shí)現(xiàn)。第二部分在脂多糖誘導(dǎo)的血腦屏障破壞中基質(zhì)金屬蛋白酶的作用及其信號(hào)通路機(jī)制的研究目的：探討基質(zhì)金屬蛋白酶-2和-9(MMP-2和MMP-9)在LPS誘導(dǎo)BBB破壞中的作用及其信號(hào)通路機(jī)制。方法：1.培養(yǎng)hCMEC/D3,用p38MAPK和JNK信號(hào)通路抑制劑預(yù)處理細(xì)胞1h,再與LPS共培養(yǎng)24h,分別用酶聯(lián)免疫吸附試驗(yàn)(ELISA)和RT-PCR法分別檢測(cè)MMP-2活性和MMP-9mRNA表達(dá)的變化。2.用MMPs總抑制劑、MMP-2及MMP-9抑制劑(分別為Doxycycline hyclate、SB-3CT 13.9 nmol/1及SB-3CT 600nmol/L)分別預(yù)處理細(xì)胞1h后,再與LPS共培養(yǎng),Western blot法檢測(cè)Occludin蛋白表達(dá)的變化。結(jié)果：1.LPS刺激細(xì)胞后可觀察到MMP-2活性和MMP-9 mRNA的過(guò)度表達(dá),p38MAPK和JNK信號(hào)通路抑制劑預(yù)處理后可顯著下調(diào)LPS的誘導(dǎo)作用。2.LPS刺激細(xì)胞后緊密連接Occludin表達(dá)顯著下降,MMPs總抑制劑、MMP-2及MMP-9抑制劑預(yù)處理后可明顯逆轉(zhuǎn)LPS誘導(dǎo)的緊密連接Occludin蛋白的下調(diào)。結(jié)論：LPS誘導(dǎo)緊密連接Occludin的降解可能與MMP-2和MMP-9的過(guò)度表達(dá)直接相關(guān),p38MAPK和JNK信號(hào)通路可能參與其調(diào)控過(guò)程。
[Abstract]:Part I study on the mechanism of p38 MAPK- JNK signaling pathway in lipopolysaccharide (LPS-) -induced blood-brain barrier damage objective: to investigate the role of p38MAPK and c-Jun amino-terminal kinase (c-Jun) signal pathway in the destruction of blood-brain barrier blood-brain barrier (BBBB) induced by lipopolysaccharide (LPS). Human cerebral microvascular endothelial cells, hCMEC-D3N were cultured in human microvascular endothelial cells. The cells were stimulated with different concentrations of LPS-p38MAPK and JNK signal pathway inhibitor (SB203580SP600125, respectively) for 24 hours. To stimulate cells with different concentrations of LPS, Western blotting was used to detect the changes of tight junction protein Occludingnan ZO-1. 3. After stimulated by LPS for different time, the phosphorylation level of p38 MAPK and JNK signaling pathway was detected by Western blotting. The cells were pretreated with SB203580 and SP600125 for 1 hour. After co-culture with LPS for 24 h, the expression of tight junction Occludingnan ZO-1 protein and its mRNA were detected by Western blot assay and fluorescence quantitative PCR RT-PCR.The results showed that LPSN SB203580 and SP600125 concentration below 10 渭 g / ml SB203580 and 0.22 渭 g / ml had no significant effect on the viability of hCMEC/D3 cells. Stimulated cells induced a significant downregulation of the expression of tight junctional Occludingnan ZO-1 protein and its mRNA. It was further demonstrated by Western blot that the phosphorylation of p38 MAPK and JNK signaling pathway molecules increased significantly after LPS stimulation. 3. SB203580 or SP600125 pretreated cells for 1 hour, which could significantly increase the expression of p38 MAPK and JNK signaling pathway molecules. Regulating the expression of Occludin protein and its mRNA induced by LPS, But there was no significant difference in the expression of ZO-1 protein and mRNA induced by LPS. Conclusion the down-regulation of tight junction protein ZO-1 and its mRNA may lead to the phosphorylation of BBB by activating p38 MAPK and JNK signaling pathway. Thus, the expression of Occludin protein and its mRNA were regulated in hCMEC/D3 cells. However, the effect of LPS on the expression of ZO-1 protein and its mRNA may be achieved through other signal transduction pathways. Part 2: the role of matrix metalloproteinases and the mechanism of signal pathway in the damage of blood-brain barrier induced by lipopolysaccharide:. To investigate the role of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in BBB damage induced by LPS and its signaling pathway mechanism. Methods 1: 1. HCMEC-D3 was cultured. Cells were pretreated with p38 MAPK and JNK signaling pathway inhibitor for 1 h, then co-cultured with LPS for 24 h, respectively. Enzyme linked immunosorbent assay (Elisa) was used. The activity of MMP-2 and the expression of MMP-9mRNA were detected by Elisa and RT-PCR. The cells were pretreated with Doxycycline hyclatette SB-3CT 13.9 nmol/1 and SB-3CT 600nmol / L respectively for 1 h after treatment with MMP 2 and MMP-9 inhibitor (Doxycycline hyclatette SB-3CT 13.9 nmol/1 and SB-3CT 600nmol / L, respectively). Results 1. After stimulated by LPS, the activity of MMP-2 and the overexpression of MMP-9 mRNA, p38 MAPK and JNK signal pathway inhibitor pretreated could significantly down-regulate the induction of LPS. 2. The expression of tightly-junctional Occludin decreased significantly after stimulation. MMPs master inhibitor (MMPs) and MMP-9 inhibitor pretreatment could significantly reverse the down-regulation of tight junction Occludin protein induced by LPS. Conclusion the degradation of tight junction Occludin induced by LPS may be associated with the degradation of MMP-2 and MMP-9. Overexpression may be involved in the regulation of p38 MAPK and JNK signaling pathway.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R741

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