Src激酶调控Nrf2出核转运在内毒素血症小鼠肝肺损伤中的作用及机制

发布时间：2018-05-07 17:05

本文选题：Src激酶 + 内毒素血症　；参考：《第三军医大学》2015年硕士论文

【摘要】：背景和目的:内毒素血症是感染导致的全身性炎症反应,是感染及重症监护室(Intensive Care Unit,ICU)患者死亡的主要原因之一。尽管近年来医学发展的突飞猛进,然而内毒素血症的死亡率仍然居高不下,高达30%—60%。肝脏和肺脏是内毒素血症中较易受损的两个器官,而内毒素血症肝肺损伤发病机制复杂,目前缺乏有效的治疗措施。核因子E2相关因子2(nuclear factor-erythroid-2-related factor 2,Nrf2)是内源性抗损伤系统的关键转录因子,在肝肺组织抗损伤中具有重要作用。酪氨酸激酶Src是Nrf2信号通路上游具有调控Nrf2出核转运作用的重要激酶,在内毒素血症肝肺损伤中也同样具有重要作用。我们团队前期研究发现高浓度肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)和过氧化氢(hydrogen peroxide,H2O2)导致肺微血管内皮细胞(pulmonary microvascular endothelial cells,PMVECs)Nrf2转录活性降低。因此在前期研究的基础上推测:Src激酶催化Nrf2第568位酪氨酸发生磷酸化,继而导致Nrf2转运出核降解,最终使内源性抗损伤能力降低是内毒素肝肺损伤发生的另一重要机制。本研究拟通过LPS诱导的肝肺损伤模型和离体细胞实验研究Src激酶调控Nrf2的出核转运在内毒素血症肝肺损伤中的作用,为从内源性抗损伤途径干预内毒素血症肝肺损伤提供新思路和治疗靶点。方法:1抑制Src激酶对内毒素血症小鼠肝肺损伤的影响1.1采用经腹腔注射LPS的方式构建小鼠内毒素血症肝肺损伤的模型,注射LPS后2小时经腹腔给予Src激酶抑制剂PP2。1.2取小鼠肝肺组织切片进行HE染色观察抑制Src激酶后病理学变化。1.3采用试剂盒检测小鼠肝肺组织超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、髓过氧化物酶(myeloperoxidase,MPO)*本课题受国家自然科学基金青年科学基金项目(酪氨酸磷酸化修饰调控Nrf2出核转运的机制及其在内毒素肺损伤中的作用,基金号:81100055)资助。MPO的水平变化。1.4采用试剂盒检测小鼠血清碱性磷酸酶(alkaline phosphatase,ALP)的水平变化。1.5采用western blot检测小鼠肝肺组织Nrf2的表达变化。2 Nrf2出核转运在LPS诱导A549细胞损伤中的作用研究2.1采用免疫荧光检测LPS刺激下Nrf2在A549细胞中的分布变化。2.2采用免疫荧光检测给予Src激酶抑制剂PP2和出核转运蛋白CRM1抑制剂来普霉素B(leptomycin B,LMB)后LPS诱导Nrf2的分布变化情况。2.3采用免疫荧光检测LPS对Nrf2+/+,Nrf2Y568A腺病毒转染细胞中Nrf2分布的影响。2.4采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)检测LPS刺激后正常细胞、给予PP2和LMB的细胞、Nrf2+/+和Nrf2Y568A腺病毒转染细胞的活性。结果:1.Src激酶抑制剂PP2使内毒素血症小鼠肺肝组织SOD、Nrf2水平明显升高,MPO、MDA水平和血清ALP水平显著降低,肝肺损伤程度明显减轻。2.PP2、LMB能够阻断LPS诱导的Nrf2出核转运,Nrf2上第568位酪氨酸突变后Nrf2不能转运出核。3.PP2、LMB、Nrf2Y568A腺病毒转染能显著提高LPS刺激后的细胞活性。结论:1.经腹腔注射LPS成功构建内毒素血症小鼠肝肺损伤模型。2.Src激酶抑制剂阻断Nrf2出核转运在减轻内毒素血症小鼠肝肺损伤中有重要作用。3.阻断Nrf2的出核转运在Nrf2发挥细胞保护作用中具有重要意义,使用Src激酶抑制剂阻断Nrf2的出核转运可能是增强细胞抗损伤能力的有效措施。
[Abstract]:Background and purpose: Endotoxemia is a systemic inflammatory response caused by infection and one of the main causes of death in Intensive Care Unit (ICU) patients. Despite the rapid progress of medical development in recent years, the mortality of endotoxemia remains high, and the liver and lung are endotoxin blood as high as 30% to 60%.. The pathogenesis of endotoxemia and liver lung injury is complex, and the pathogenesis of endotoxemia and liver lung injury is complex, and there is no effective treatment. Nuclear factor E2 related factor 2 (nuclear factor-erythroid-2-related factor 2, Nrf2) is the key transcription factor of endogenous anti injury system. It plays an important role in the anti injury of liver and lung tissue. Enzyme Src is an important kinase in the upstream of the Nrf2 signaling pathway that regulates the transport of Nrf2 nuclei. It also plays an important role in the liver and lung injury of endotoxemia. Our team earlier study found that the high concentration of tumor necrosis factor - alpha (tumor necrosis factor-alpha, TNF- alpha) and hydrogen peroxide (hydrogen peroxide, H2O2) led to the intravascular microvessel of the lung The transcriptional activity of pulmonary microvascular endothelial cells (PMVECs) Nrf2 is reduced. Therefore, it is presumed that the Src kinase catalyzes the phosphorylation of Nrf2 568th tyrosine in the presence of Src kinase, which leads to the Nrf2 transport of nuclear degradation, and ultimately the decrease of endogenous anti damage ability is another important machine for the occurrence of endotoxin liver lung injury. The purpose of this study is to study the role of Src kinase in LPS induced liver lung injury model and in vitro cell experiment to regulate the role of Nrf2 in the liver and lung injury of endotoxemia in order to provide new ideas and targets for the intervention of endotoxemia and liver lung injury from endogenous anti injury pathway. 1 inhibition of Src kinase to endotoxemia mice The effect of liver and lung injury 1.1 model of liver and lung injury in mice with endotoxemia by intraperitoneal injection of LPS, 2 hours after injection of Src kinase inhibitor PP2.1.2 by intraperitoneal injection, the liver lung tissue section of mice was obtained by HE staining to observe the pathological changes of Src kinase and to detect the hyperoxidation of liver and lung tissue in mice after the inhibition of Src kinase. Superoxide dismutase (SOD), malondialdehyde (malondialdehyde, MDA) and myeloperoxidase (myeloperoxidase, MPO) * this subject is subject to the National Natural Science Fund Youth Science Fund Project (the mechanism of tyrosine phosphorylation modification regulating Nrf2 transshipment and its role in endotoxin lung injury, fund number: 81100055) funding.MPO Level changes of serum alkaline phosphatase (alkaline phosphatase, ALP) in mice using a kit to detect the changes in.1.5 using Western blot to detect the changes in the expression of Nrf2 in the liver and lung tissues of mice by Western blot and the effect of.2 Nrf2 on the nuclear transport of.2 Nrf2 in LPS inducible A549 cell damage. 2.1 Distribution and changes of.2.2 were given by immunofluorescence. Src kinase inhibitor PP2 and nuclear transporter CRM1 inhibitor were applied to the distribution of LPS induced Nrf2 after B (leptomycin B, LMB). Ll counting kit-8, CCK-8) detected the normal cells after LPS stimulation, and gave PP2 and LMB cells, Nrf2+/+ and Nrf2Y568A adenovirus transfection cell activity. Results: 1.Src kinase inhibitor PP2 makes the lung and liver tissues of the mice with endotoxemia, the level and the level of serum decrease significantly, and the degree of liver and lung injury is obviously reduced. .PP2, LMB can block the Nrf2 nuclear transfer induced by LPS, 568th tyrosine mutations on Nrf2 can not transport the nucleus.3.PP2, LMB, and Nrf2Y568A adenovirus transfection can significantly increase the activity of the cells after LPS stimulation. Conclusion: 1. the hepatotoxicity model of endotoxemia mice was successfully constructed by intraperitoneal injection. Transshipment plays an important role in alleviating liver and lung injury in mice with endotoxemia,.3. blocking the nuclear transport of Nrf2 plays an important role in Nrf2 cell protection. Blocking the nuclear transfer of Nrf2 by using Src kinase inhibitor may be an effective measure to enhance the anti injury ability of the cells.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614

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