缺血預(yù)處理對(duì)人肺缺血再灌注損傷的蛋白質(zhì)組學(xué)研究
發(fā)布時(shí)間：2018-08-03 20:10
【摘要】： 缺血預(yù)處理是指預(yù)先給予一次或多次短暫的缺血,以增強(qiáng)組織對(duì)隨后長(zhǎng)時(shí)間缺血再灌注損傷的耐受性或適應(yīng)性。缺血再灌注損傷常見于休克時(shí)微循環(huán)的疏通、冠狀動(dòng)脈痙攣的緩解、心腦血管栓塞再通、心肺手術(shù)后和心臟驟停后心肺腦復(fù)蘇、斷肢再植、器官移植血供恢復(fù)等,是引起隨后器官功能障礙的主要原因之一。大量的研究表明缺血預(yù)處理能顯著減輕隨后的缺血再灌注損傷。肺缺血再灌注損傷多見于體外循環(huán)心內(nèi)直視術(shù)、肺動(dòng)脈袖狀切除術(shù)、肺移植術(shù)及需要阻斷一側(cè)肺動(dòng)脈才能進(jìn)行的肺葉切除術(shù)中,對(duì)肺功能恢復(fù)不利,并可能引起術(shù)后并發(fā)癥和影響預(yù)后。肺缺血預(yù)處理是防治肺缺血再灌注損傷最有效的措施,但其確切的保護(hù)機(jī)制尚未闡明。研究顯示,給予缺血預(yù)處理的肺組織與未予缺血預(yù)處理的肺組織相比,在經(jīng)過隨后長(zhǎng)時(shí)間的缺血再灌注后,表現(xiàn)出更好的缺血耐受性和再灌注耐受性,其中性白細(xì)胞浸潤(rùn),肺水腫,局部出血和肺泡破裂均明顯減少,其細(xì)胞結(jié)構(gòu),內(nèi)環(huán)境平衡,微循環(huán)灌注,能量代謝,組織功能和遺傳重組等方面具有更好的延續(xù)性和恢復(fù)能力。廣大科學(xué)工作者從動(dòng)物實(shí)驗(yàn)和臨床實(shí)驗(yàn)對(duì)肺缺血預(yù)處理的肺保護(hù)機(jī)制進(jìn)行了卓有成效的研究,發(fā)現(xiàn)腺苷、緩激肽、花生四烯酸代謝產(chǎn)物、阿片類、兒茶酚胺類、自由基、鈣離子、一氧化氮和活性氧等與保護(hù)效應(yīng)的啟動(dòng)相關(guān);G蛋白、磷脂酶C、蛋白激酶A、酪氨酸蛋白激酶及絲裂原活化的蛋白激酶等與保護(hù)效應(yīng)的介導(dǎo)相關(guān),而保護(hù)效應(yīng)的發(fā)揮則與ATP敏感鉀通道、5'核苷酶與腺苷、轉(zhuǎn)錄因子、誘生型一氧化氮合成酶、環(huán)氧化酶2、醛糖還原酶、抗自由基酶類和熱休克蛋白等相關(guān)。雖然已有的研究揭示了一些肺缺血預(yù)處理的保護(hù)機(jī)制,但目前仍然不能完全解釋肺缺血預(yù)處理引起的保護(hù)效應(yīng)并有效逆轉(zhuǎn)缺血再灌注損傷。 肺缺血預(yù)處理的臨床研究開展較晚,但是缺血預(yù)處理作為大手術(shù)的輔助措施能夠安全而巧妙地實(shí)施。蛋白質(zhì)組學(xué)技術(shù)為研究缺血預(yù)處理的保護(hù)機(jī)制提供了新的工具。為此,本研究以臨床中的手術(shù)患者為對(duì)象,采用蛋白質(zhì)組學(xué)技術(shù)和方法研究缺血預(yù)處理抗人肺缺血再灌注損傷的分子機(jī)制。 首先選擇合適的手術(shù)患者分別歸入假手術(shù)組(Sham group,S組)、缺血再灌注組(Ischemia-reperfusion group,I/R組)和缺血預(yù)處理組(Ischemic preconditioning group IP組)。I/R組經(jīng)歷單純的缺血再灌注,IP組在缺血再灌注前予以缺血預(yù)處理,S組始終保持供血與通氣,在術(shù)后收取標(biāo)本。然后采用一步抽提法制備S組,I/R組和IP組肺組織的總蛋白質(zhì),應(yīng)用雙向凝膠電泳技術(shù)建立了重復(fù)性和分辨率均較好的S組,I/R組和IP組肺組織總蛋白質(zhì)的雙向凝膠電泳圖譜。PDQuest軟件對(duì)三組肺組織蛋白質(zhì)的雙向電泳圖譜進(jìn)行了分析比較,得出20個(gè)4倍以上差異表達(dá)的蛋白質(zhì)點(diǎn),采用MALDI-TOF-MS對(duì)差異表達(dá)的蛋白質(zhì)進(jìn)行質(zhì)譜分析,獲取MALDI-TOF-MS肽質(zhì)量指紋圖譜,數(shù)據(jù)庫(kù)搜索鑒定了17個(gè)差異表達(dá)的蛋白質(zhì)。最后,為了驗(yàn)證比較蛋白質(zhì)組學(xué)研究結(jié)果,采用Western blot技術(shù)對(duì)部分差異表達(dá)蛋白質(zhì)在IP組和I/R組肺組織中的表達(dá)水平進(jìn)行了檢測(cè),其結(jié)果與比較蛋白質(zhì)組學(xué)研究的結(jié)果一致。 本研究在建立S組、I/R組和IP組肺組織總蛋白質(zhì)雙向電泳圖譜的基礎(chǔ)上,采用質(zhì)譜分析結(jié)合數(shù)據(jù)庫(kù)搜索共鑒定出17個(gè)明顯差異表達(dá)的蛋白質(zhì),這些蛋白質(zhì)按功能主要可以分為八類,即代謝相關(guān)酶類,抗氧化劑,貯鐵蛋白,鈣結(jié)合蛋白質(zhì),分子伴侶,細(xì)胞骨架蛋白質(zhì),細(xì)胞增殖相關(guān)蛋白,以及信號(hào)傳導(dǎo)相關(guān)蛋白質(zhì)。其中一些蛋白質(zhì)與自發(fā)性抗缺血再灌注損傷有關(guān),一些蛋白質(zhì)如HSP27及Cu/ZnSOD則與缺血預(yù)處理抗缺血再灌注損傷的主動(dòng)效應(yīng)有關(guān),還有一些蛋白質(zhì)與缺血預(yù)處理抗缺血再灌注損傷的穩(wěn)定效應(yīng)有關(guān)。研究結(jié)果為揭示缺血預(yù)處理的肺保護(hù)機(jī)制,以及尋找逆轉(zhuǎn)缺血再灌注損傷的方法提供了科學(xué)依據(jù)。
[Abstract]:Ischemic preconditioning refers to the preconditioning of one or more transient ischemia to enhance the tolerance or adaptability of the tissue to the subsequent long ischemic reperfusion injury. Ischemia reperfusion injury is common to the dredging of microcirculation during shock, remission of coronary spasm, cardio cerebral vascular embolization, cardiopulmonary surgery and cardiac arrest after cardiopulmonary resuscitation. A large number of studies have shown that ischemic preconditioning can significantly reduce subsequent ischemia-reperfusion injury. Lung ischemia reperfusion injury is often seen in cardiopulmonary bypass, pulmonary sleeve resection, lung transplantation, and the need to block one. During lobectomy of the lateral pulmonary artery, it is unfavorable to the recovery of lung function and may cause postoperative complications and prognosis. Pulmonary ischemic preconditioning is the most effective measure for the prevention and treatment of pulmonary ischemia reperfusion injury, but the exact mechanism of protection has not been clarified. The study showed that the lung tissue was given ischemic preconditioning with the preconditioning without ischemic preconditioning. Compared to the lung tissue, after a long period of ischemia and reperfusion, it showed better ischemic tolerance and reperfusion tolerance, including leukocyte infiltration, pulmonary edema, local hemorrhage and alveolar rupture, and its cellular structure, internal environment balance, microcirculation perfusion, energy metabolism, tissue function and genetic recombination. There are better continuity and resilience. Many scientists have conducted fruitful studies on lung protection mechanism of lung ischemic preconditioning from animal experiments and clinical trials, and found that adenosine, bradykinin, peanut four enoic acid metabolites, opioids, catecholamines, free radicals, calcium ions, nitric oxide and reactive oxygen species and other protective effects G proteins, phospholipase C, protein kinase A, tyrosine protein kinase and mitogen activated protein kinase are related to the protective effects, while the protective effects play with the ATP sensitive potassium channel, 5'nucleoside and adenosine, transcription factors, inducible nitrogen oxide synthase, cyclooxygenase 2, aldose reductase, and anti free radical enzymes. Although some studies have revealed protective mechanisms of lung ischemic preconditioning, there is still no complete explanation of the protective effects of lung ischemic preconditioning and the effective reversal of ischemia-reperfusion injury.
The clinical study of pulmonary ischemic preconditioning is carried out later, but ischemic preconditioning can be implemented safely and skillfully. Proteomics technology provides a new tool to study the protective mechanism of ischemic preconditioning. For this reason, this study uses proteomics techniques and methods in clinical patients. Objective to study the molecular mechanism of ischemic preconditioning against human lung ischemia-reperfusion injury.
First, the appropriate surgical patients were selected for the sham operation group (Sham group, group S), the ischemic reperfusion group (Ischemia-reperfusion group, I/R group) and the ischemic preconditioning group (Ischemic preconditioning group IP group).I/R group underwent simple ischemia reperfusion. The IP group was pretreated with ischemia before reperfusion. The total protein of lung tissue in group S, I/R group and IP group was prepared by one step extraction. The two way gel electrophoresis technique was used to establish the S group with good reproducibility and resolution. The bidirectional gel electrophoresis of the total protein of lung tissue in group I/R and IP group.PDQuest software for the three groups of lung tissue proteins. In the analysis and comparison of the swimming atlas, 20 protein points with 4 times of differential expression were obtained. MALDI-TOF-MS was used to analyze the proteins expressed differently, to obtain the mass fingerprint of MALDI-TOF-MS peptide, and to identify 17 differentially expressed proteins by database search. Finally, to verify the results of comparative proteomics, use We Stern blot technique was used to detect the expression level of partially differentially expressed proteins in the lung tissue of group IP and I/R group, and the results were in agreement with the results of comparative proteomics.
In this study, on the basis of the bi-directional electrophoresis of the total protein of lung tissue in group S, group I/R and IP, 17 proteins with distinct differentially expressed proteins were identified by mass spectrometry analysis and database search. These proteins can be divided into eight categories according to their function, namely, metabolic enzymes, antioxidants, ferric proteins, calcium binding proteins, and molecules. Chaperones, cytoskeleton proteins, cell proliferation related proteins, and signal transduction related proteins. Some proteins are associated with spontaneous anti ischemia-reperfusion injury, and some proteins such as HSP27 and Cu/ZnSOD are associated with the active effect of ischemic preconditioning against ischemia-reperfusion injury, and some proteins and ischemic preconditioning. The results of the study provide a scientific basis for revealing the mechanism of ischemic preconditioning and finding a way to reverse the injury of ischemia-reperfusion.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363
本文編號(hào)：2162875
[Abstract]:Ischemic preconditioning refers to the preconditioning of one or more transient ischemia to enhance the tolerance or adaptability of the tissue to the subsequent long ischemic reperfusion injury. Ischemia reperfusion injury is common to the dredging of microcirculation during shock, remission of coronary spasm, cardio cerebral vascular embolization, cardiopulmonary surgery and cardiac arrest after cardiopulmonary resuscitation. A large number of studies have shown that ischemic preconditioning can significantly reduce subsequent ischemia-reperfusion injury. Lung ischemia reperfusion injury is often seen in cardiopulmonary bypass, pulmonary sleeve resection, lung transplantation, and the need to block one. During lobectomy of the lateral pulmonary artery, it is unfavorable to the recovery of lung function and may cause postoperative complications and prognosis. Pulmonary ischemic preconditioning is the most effective measure for the prevention and treatment of pulmonary ischemia reperfusion injury, but the exact mechanism of protection has not been clarified. The study showed that the lung tissue was given ischemic preconditioning with the preconditioning without ischemic preconditioning. Compared to the lung tissue, after a long period of ischemia and reperfusion, it showed better ischemic tolerance and reperfusion tolerance, including leukocyte infiltration, pulmonary edema, local hemorrhage and alveolar rupture, and its cellular structure, internal environment balance, microcirculation perfusion, energy metabolism, tissue function and genetic recombination. There are better continuity and resilience. Many scientists have conducted fruitful studies on lung protection mechanism of lung ischemic preconditioning from animal experiments and clinical trials, and found that adenosine, bradykinin, peanut four enoic acid metabolites, opioids, catecholamines, free radicals, calcium ions, nitric oxide and reactive oxygen species and other protective effects G proteins, phospholipase C, protein kinase A, tyrosine protein kinase and mitogen activated protein kinase are related to the protective effects, while the protective effects play with the ATP sensitive potassium channel, 5'nucleoside and adenosine, transcription factors, inducible nitrogen oxide synthase, cyclooxygenase 2, aldose reductase, and anti free radical enzymes. Although some studies have revealed protective mechanisms of lung ischemic preconditioning, there is still no complete explanation of the protective effects of lung ischemic preconditioning and the effective reversal of ischemia-reperfusion injury.
The clinical study of pulmonary ischemic preconditioning is carried out later, but ischemic preconditioning can be implemented safely and skillfully. Proteomics technology provides a new tool to study the protective mechanism of ischemic preconditioning. For this reason, this study uses proteomics techniques and methods in clinical patients. Objective to study the molecular mechanism of ischemic preconditioning against human lung ischemia-reperfusion injury.
First, the appropriate surgical patients were selected for the sham operation group (Sham group, group S), the ischemic reperfusion group (Ischemia-reperfusion group, I/R group) and the ischemic preconditioning group (Ischemic preconditioning group IP group).I/R group underwent simple ischemia reperfusion. The IP group was pretreated with ischemia before reperfusion. The total protein of lung tissue in group S, I/R group and IP group was prepared by one step extraction. The two way gel electrophoresis technique was used to establish the S group with good reproducibility and resolution. The bidirectional gel electrophoresis of the total protein of lung tissue in group I/R and IP group.PDQuest software for the three groups of lung tissue proteins. In the analysis and comparison of the swimming atlas, 20 protein points with 4 times of differential expression were obtained. MALDI-TOF-MS was used to analyze the proteins expressed differently, to obtain the mass fingerprint of MALDI-TOF-MS peptide, and to identify 17 differentially expressed proteins by database search. Finally, to verify the results of comparative proteomics, use We Stern blot technique was used to detect the expression level of partially differentially expressed proteins in the lung tissue of group IP and I/R group, and the results were in agreement with the results of comparative proteomics.
In this study, on the basis of the bi-directional electrophoresis of the total protein of lung tissue in group S, group I/R and IP, 17 proteins with distinct differentially expressed proteins were identified by mass spectrometry analysis and database search. These proteins can be divided into eight categories according to their function, namely, metabolic enzymes, antioxidants, ferric proteins, calcium binding proteins, and molecules. Chaperones, cytoskeleton proteins, cell proliferation related proteins, and signal transduction related proteins. Some proteins are associated with spontaneous anti ischemia-reperfusion injury, and some proteins such as HSP27 and Cu/ZnSOD are associated with the active effect of ischemic preconditioning against ischemia-reperfusion injury, and some proteins and ischemic preconditioning. The results of the study provide a scientific basis for revealing the mechanism of ischemic preconditioning and finding a way to reverse the injury of ischemia-reperfusion.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363
【參考文獻(xiàn)】
相關(guān)期刊論文 前8條
1 陳勝喜,李國(guó)虎,龍隆;缺血預(yù)處理對(duì)肺保護(hù)的臨床研究[J];湖南醫(yī)科大學(xué)學(xué)報(bào);1999年04期
2 楊揚(yáng),陳勝喜,張衛(wèi)星;缺血預(yù)處理對(duì)人在體肺組織細(xì)胞凋亡及調(diào)控基因蛋白bcl-2表達(dá)的影響[J];湖南醫(yī)科大學(xué)學(xué)報(bào);2002年01期
3 張春芳,陳勝喜,郭海周,羅萬(wàn)俊;缺血預(yù)處理對(duì)豬肺缺血再灌注性損傷的保護(hù)機(jī)制[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2005年01期
4 羅萬(wàn)俊,陳勝喜,蔣海河;缺血預(yù)處理對(duì)犬肺移植供肺組織腺苷酸含量的影響[J];湖南醫(yī)科大學(xué)學(xué)報(bào);1997年01期
5 羅萬(wàn)俊,陳勝喜,蔣海河,郭曲練,李國(guó)虎,周漢槎;缺血預(yù)處理對(duì)犬移植肺保存效果的研究[J];湖南醫(yī)科大學(xué)學(xué)報(bào);1997年03期
6 羅萬(wàn)俊,陳勝喜,胡佳心,蔣海河;缺血預(yù)處理對(duì)肺再灌注損傷脂質(zhì)過氧化的影響[J];中國(guó)病理生理雜志;1999年09期
7 吳成云,徐正坸,戴元榮,王萬(wàn)鐵,方周溪;左旋硝基精氨酸對(duì)肺缺血預(yù)處理的抗缺血/再灌注損傷作用的影響[J];中國(guó)應(yīng)用生理學(xué)雜志;2005年03期
8 張春芳,陳主初,郭海周,張恒,肖志強(qiáng),陳勝喜;缺血預(yù)處理對(duì)兔肺缺血再灌注性損傷的蛋白質(zhì)組學(xué)研究[J];中華實(shí)驗(yàn)外科雜志;2005年11期
，本文編號(hào)：2162875
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2162875.html
最近更新
教材專著
