在脂多糖诱导的血脑屏障破坏中p38MAPK、JNK信号通路与基质金属蛋白酶作用机制的研究

发布时间：2018-02-24 19:56

本文关键词： 脂多糖紧密连接 p38MAPK信号通路 JNK信号通路基质金属蛋白酶　出处：《广西医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：第一部分在脂多糖诱导的血脑屏障破坏中p38MAPK、JNK信号通路的作用机制研究目的：探讨p38MAPK和c-Jun氨基末端激酶(JNK)信号通路在脂多糖(lipopolysaccharide, LPS)诱导血脑屏障(blood-brain barrier, BBB)的破坏中的作用。方法：1.培养人脑微血管内皮细胞(human cerebral microvascular endothelial cells, hCMEC/D3),用不同浓度的LPS、p38MAPK和JNK信号通路抑制剂(分别为SB203580、SP600125)分别刺激细胞24h,用四甲基偶氮唑盐(MTT法)检测其对细胞活力的影响。2.用不同浓度的LPS刺激细胞,蛋白免疫印迹法(Western blot法)检测紧密连接蛋白Occludin、ZO-1的变化。3.用LPS刺激细胞不同的时间后,用蛋白免疫印迹法检测p38MAPK及JNK信号通路磷酸化水平的变化。4.用SB203580、SP600125预处理细胞1h后,再加入LPS共培养24h,分别用Western blot法及荧光定量PCR法(RT-PCR)检测紧密连接Occludin、ZO-1蛋白及其mRNA表达的变化。结果：1. LPS、SB203580和SP600125浓度分别在10μg/ml、7.69μg/m1和0.22μg/ml以下时对hCMEC/D3细胞活力无明显影响。2.LPS刺激细胞可诱导紧密连接Occludin、ZO-1蛋白及其mRNA的表达水平显著下调,Western blot法进一步证实LPS刺激细胞后p38MAPK和JNK信号通路分子的磷酸化显著增加。3.SB203580或SP600125预处理细胞1h,可以显著上调LPS诱导的Occludin蛋白及其mRNA的表达水平,但对LPS诱导的ZO-1蛋白及mRNA表达的影响无统计学意义。结论：LPS可诱导紧密连接Occludin、ZO-1蛋白及其mRNA的下调导致BBB破坏；LPS可能通过激活p38MAPK和JNK信号通路磷酸化,从而调节hCMEC/D3细胞紧密连接Occludin蛋白及其mRNA的表达；然而LPS对ZO-1蛋白及其mRNA表达的影响可能通过其他信号通路实现。第二部分在脂多糖诱导的血脑屏障破坏中基质金属蛋白酶的作用及其信号通路机制的研究目的：探讨基质金属蛋白酶-2和-9(MMP-2和MMP-9)在LPS诱导BBB破坏中的作用及其信号通路机制。方法：1.培养hCMEC/D3,用p38MAPK和JNK信号通路抑制剂预处理细胞1h,再与LPS共培养24h,分别用酶联免疫吸附试验(ELISA)和RT-PCR法分别检测MMP-2活性和MMP-9mRNA表达的变化。2.用MMPs总抑制剂、MMP-2及MMP-9抑制剂(分别为Doxycycline hyclate、SB-3CT 13.9 nmol/1及SB-3CT 600nmol/L)分别预处理细胞1h后,再与LPS共培养,Western blot法检测Occludin蛋白表达的变化。结果：1.LPS刺激细胞后可观察到MMP-2活性和MMP-9 mRNA的过度表达,p38MAPK和JNK信号通路抑制剂预处理后可显著下调LPS的诱导作用。2.LPS刺激细胞后紧密连接Occludin表达显著下降,MMPs总抑制剂、MMP-2及MMP-9抑制剂预处理后可明显逆转LPS诱导的紧密连接Occludin蛋白的下调。结论：LPS诱导紧密连接Occludin的降解可能与MMP-2和MMP-9的过度表达直接相关,p38MAPK和JNK信号通路可能参与其调控过程。
[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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R741

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