本文選題：Src激酶 + 內毒素血癥��； 參考：《第三軍醫(yī)大學》2015年碩士論文

【摘要】：背景和目的:內毒素血癥是感染導致的全身性炎癥反應,是感染及重癥監(jiān)護室(Intensive Care Unit,ICU)患者死亡的主要原因之一。盡管近年來醫(yī)學發(fā)展的突飛猛進,然而內毒素血癥的死亡率仍然居高不下,高達30%—60%。肝臟和肺臟是內毒素血癥中較易受損的兩個器官,而內毒素血癥肝肺損傷發(fā)病機制復雜,目前缺乏有效的治療措施。核因子E2相關因子2(nuclear factor-erythroid-2-related factor 2,Nrf2)是內源性抗損傷系統的關鍵轉錄因子,在肝肺組織抗損傷中具有重要作用。酪氨酸激酶Src是Nrf2信號通路上游具有調控Nrf2出核轉運作用的重要激酶,在內毒素血癥肝肺損傷中也同樣具有重要作用。我們團隊前期研究發(fā)現高濃度腫瘤壞死因子-α(tumor necrosis factor-alpha,TNF-α)和過氧化氫(hydrogen peroxide,H2O2)導致肺微血管內皮細胞(pulmonary microvascular endothelial cells,PMVECs)Nrf2轉錄活性降低。因此在前期研究的基礎上推測:Src激酶催化Nrf2第568位酪氨酸發(fā)生磷酸化,繼而導致Nrf2轉運出核降解,最終使內源性抗損傷能力降低是內毒素肝肺損傷發(fā)生的另一重要機制。本研究擬通過LPS誘導的肝肺損傷模型和離體細胞實驗研究Src激酶調控Nrf2的出核轉運在內毒素血癥肝肺損傷中的作用,為從內源性抗損傷途徑干預內毒素血癥肝肺損傷提供新思路和治療靶點。方法:1抑制Src激酶對內毒素血癥小鼠肝肺損傷的影響1.1采用經腹腔注射LPS的方式構建小鼠內毒素血癥肝肺損傷的模型,注射LPS后2小時經腹腔給予Src激酶抑制劑PP2。1.2取小鼠肝肺組織切片進行HE染色觀察抑制Src激酶后病理學變化。1.3采用試劑盒檢測小鼠肝肺組織超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、髓過氧化物酶(myeloperoxidase,MPO)*本課題受國家自然科學基金青年科學基金項目(酪氨酸磷酸化修飾調控Nrf2出核轉運的機制及其在內毒素肺損傷中的作用,基金號:81100055)資助。MPO的水平變化。1.4采用試劑盒檢測小鼠血清堿性磷酸酶(alkaline phosphatase,ALP)的水平變化。1.5采用western blot檢測小鼠肝肺組織Nrf2的表達變化。2 Nrf2出核轉運在LPS誘導A549細胞損傷中的作用研究2.1采用免疫熒光檢測LPS刺激下Nrf2在A549細胞中的分布變化。2.2采用免疫熒光檢測給予Src激酶抑制劑PP2和出核轉運蛋白CRM1抑制劑來普霉素B(leptomycin B,LMB)后LPS誘導Nrf2的分布變化情況。2.3采用免疫熒光檢測LPS對Nrf2+/+,Nrf2Y568A腺病毒轉染細胞中Nrf2分布的影響。2.4采用細胞計數試劑盒-8(cell counting kit-8,CCK-8)檢測LPS刺激后正常細胞、給予PP2和LMB的細胞、Nrf2+/+和Nrf2Y568A腺病毒轉染細胞的活性。結果:1.Src激酶抑制劑PP2使內毒素血癥小鼠肺肝組織SOD、Nrf2水平明顯升高,MPO、MDA水平和血清ALP水平顯著降低,肝肺損傷程度明顯減輕。2.PP2、LMB能夠阻斷LPS誘導的Nrf2出核轉運,Nrf2上第568位酪氨酸突變后Nrf2不能轉運出核。3.PP2、LMB、Nrf2Y568A腺病毒轉染能顯著提高LPS刺激后的細胞活性。結論:1.經腹腔注射LPS成功構建內毒素血癥小鼠肝肺損傷模型。2.Src激酶抑制劑阻斷Nrf2出核轉運在減輕內毒素血癥小鼠肝肺損傷中有重要作用。3.阻斷Nrf2的出核轉運在Nrf2發(fā)揮細胞保護作用中具有重要意義,使用Src激酶抑制劑阻斷Nrf2的出核轉運可能是增強細胞抗損傷能力的有效措施。
[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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R614

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