本文關(guān)鍵詞：重癥失血性休克及復(fù)蘇條件下大鼠VAP-1表達(dá)規(guī)律及調(diào)控機(jī)制研究　出處：《第二軍醫(yī)大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 重癥休克 血管粘附蛋白-1 含銅胺氧化酶-3 氨基脲敏感性胺氧化酶 大鼠肝血竇內(nèi)皮細(xì)胞

【摘要】：【背景】 基于目前重度失血性休克救治中靶向治療手段的缺乏，以及創(chuàng)傷失血性休克相關(guān)基礎(chǔ)研究與臨床的脫節(jié)，急切需要我們重新審視已知對(duì)創(chuàng)傷失血性休克病理的認(rèn)識(shí)。前期，我們?cè)谝环N相對(duì)接近臨床的肝左外葉切除+2.5ml/100g（體重）的大鼠創(chuàng)傷失血性休克模型上，摸索到24小時(shí)死亡率接近50%的干預(yù)條件，之后對(duì)存活和死亡大鼠失血前肝臟進(jìn)行表達(dá)譜基因研究，篩查出約100個(gè)差異基因，隨后對(duì)這些差異基因進(jìn)行調(diào)控網(wǎng)絡(luò)分析。數(shù)據(jù)表明有18個(gè)基因參與了這些差異基因的網(wǎng)絡(luò)調(diào)控，結(jié)合專業(yè)知識(shí)，AOC3基因由于其編碼蛋白具有細(xì)胞粘附功能及SSAO活性而進(jìn)入我們視野。 AOC3基因所編碼的蛋白是一個(gè)由763個(gè)氨基酸組成的含銅胺氧化酶(CAOs)，稱為VAP-1。VAP-1是一種具有雙重功能的蛋白。一方面作為內(nèi)皮細(xì)胞粘附因子（VAP-1）可誘導(dǎo)白細(xì)胞粘附至血管內(nèi)皮；另一方面作為胺氧化酶（SSAO）可催化芳香族和脂肪族的伯胺類物質(zhì)轉(zhuǎn)化成相應(yīng)的醛類，并產(chǎn)生H2O2和NH3。通過文獻(xiàn)復(fù)習(xí)我們發(fā)現(xiàn)VAP-1蛋白的功能與重癥休克期微循環(huán)障礙及血管反應(yīng)性降低有密切聯(lián)系。因此我們認(rèn)為VAP-1在重癥休克病理過程中可能具有重要意義，有必要進(jìn)行研究闡明，以期為臨床救治重癥休克提供一種新的治療手段。 【目的】 1、明確大鼠定壓失血性休克-復(fù)蘇過程中肝臟組織AOC3基因及其編碼蛋白VAP-1表達(dá)的變化規(guī)律； 2、明確大鼠定壓失血性休克-復(fù)蘇過程中血液中sVAP-1含量及SSAO酶活性的變化規(guī)律； 3、構(gòu)建AOC3過表達(dá)腺病毒載體，為下一步體外功能實(shí)驗(yàn)奠定基礎(chǔ)； 4、檢測(cè)AOC3過表達(dá)重組腺病毒對(duì)大鼠肝血竇內(nèi)皮細(xì)胞的轉(zhuǎn)染作用； 5、明確低氧環(huán)境中大鼠肝血竇內(nèi)皮細(xì)胞AOC3基因及其編碼蛋白VAP-1的表達(dá)規(guī)律。 【方法】 1、大鼠定壓失血性休克模型的建立； 2、通過大鼠定壓失血性休克模型在休克-復(fù)蘇過程中的既定時(shí)間點(diǎn)采集大鼠肝臟組織標(biāo)本，通過Real-time PCR、Western-Blot及免疫組化等方法檢測(cè)休克-復(fù)蘇各階段AOC3基因及其編碼蛋白VAP-1的表達(dá)情況； 3、通過大鼠定壓失血性休克模型采集大鼠休克-復(fù)蘇既定時(shí)間點(diǎn)的外周血，使用ELISA法及分光光度計(jì)分別檢測(cè)休克-復(fù)蘇過程不同階段sVAP-1含量及SSAO酶活性； 4、AOC3過表達(dá)腺病毒載體的設(shè)計(jì)、構(gòu)建、病毒包裝、大量擴(kuò)增、濃縮及滴定測(cè)定，用腺病毒轉(zhuǎn)染大鼠腸微血管內(nèi)皮細(xì)胞并測(cè)定MOI值； 5、用重組腺病毒及空病毒轉(zhuǎn)染大鼠肝血竇內(nèi)皮細(xì)胞，并通過降低細(xì)胞培養(yǎng)箱氧濃度制造低氧環(huán)境。使用Real-time PCR、Western-Blot檢測(cè)大鼠肝血竇內(nèi)皮細(xì)胞在腺病毒轉(zhuǎn)染前后在常氧條件及低氧條件下其AOC3基因及編碼蛋白VAP-1的表達(dá)情況。 【結(jié)果】 1、建立了穩(wěn)定的大鼠定壓失血性休克模型； 2、在定壓失血性休克-復(fù)蘇過程中，Real-time PCR結(jié)果顯示肝臟組織AOC3基因表達(dá)量休克組顯著高于復(fù)蘇組及對(duì)照組，復(fù)蘇組顯著高于對(duì)照組； 3、在定壓失血性休克-復(fù)蘇過程中， Western-blot結(jié)果顯示在肝臟組織VAP-1/GAPDH蛋白濃度比值休克組顯著高于復(fù)蘇組及對(duì)照組，復(fù)蘇組明顯高于對(duì)照組； 4、大鼠定壓失血性休克-復(fù)蘇過程中，ELISA定量結(jié)果顯示血清中sVAP-1含量休克組顯著高于復(fù)蘇組及對(duì)照組，復(fù)蘇組顯著高于對(duì)照組。SSAO酶活性測(cè)定結(jié)果顯示血清中酶活性休克組顯著高于復(fù)蘇組及對(duì)照組，復(fù)蘇組顯著高于對(duì)照組； 5、成功建立AOC3過表達(dá)腺病毒載體（pIRES-EGFP-AOC3），重組腺病毒感染大鼠腸微血管內(nèi)皮細(xì)胞的MOI值選取5-10為最佳； 6、在常氧及低氧培養(yǎng)條件下，Real-time PCR結(jié)果均顯示大鼠肝血竇內(nèi)皮細(xì)胞經(jīng)重組腺病毒轉(zhuǎn)染后其AOC3表達(dá)量顯著高于轉(zhuǎn)染前，而該細(xì)胞經(jīng)空病毒轉(zhuǎn)染前后的AOC3表達(dá)量無顯著差異。Western-blot結(jié)果顯示相同的趨勢(shì)； 7、Real-time PCR結(jié)果顯示無論大鼠肝血竇內(nèi)皮細(xì)胞是否經(jīng)重組腺病毒或空病毒轉(zhuǎn)染，在低氧培養(yǎng)條件下其AOC3表達(dá)量均顯著高于常氧培養(yǎng)條件。Western-blot結(jié)果雖然顯示相同的趨勢(shì)，但差異并不如Real-time PCR結(jié)果顯著。 【結(jié)論】 1、體內(nèi)試驗(yàn)證實(shí)大鼠重癥休克病程中肝臟組織及血清中VAP-1的含量升高，血清中SSAO酶活性增強(qiáng)，而隨著復(fù)蘇的進(jìn)行，各組織中VAP-1含量及SSAO酶活性均有下降趨勢(shì)。 2、AOC3過表達(dá)腺病毒載體構(gòu)建成功。在常氧或厭氧條件下，大鼠肝血竇內(nèi)皮細(xì)胞經(jīng)AOC3過表達(dá)重組腺病毒轉(zhuǎn)染后其AOC3基因及編碼蛋白VAP-1表達(dá)量均有明顯上升。 3、在大鼠肝血竇內(nèi)皮細(xì)胞經(jīng)AOC3過表達(dá)重組腺病毒轉(zhuǎn)染前后，，其在低氧條件下AOC3的表達(dá)量均明顯高于常氧狀態(tài)下。其編碼蛋白VAP-1表現(xiàn)同種趨勢(shì)，然而該變化不如基因?qū)用婷黠@。
[Abstract]:[background]
At present in the treatment of severe hemorrhagic shock to treatment based on the lack of target, and the disconnection of traumatic hemorrhagic shock related basic research and clinical, we urgently need to re-examine the known on hemorrhagic shock. The pathological stage, we are in a relatively close clinical hepatic left lateral lobectomy (+2.5ml/100g body weight). Traumatic hemorrhagic shock model of rats, to explore the 24 hour mortality close to 50% after the intervention condition, the survival and death of rat liver blood before gene expression profiles of genes, screening about 100 differences with after the analysis of these differences in gene regulatory networks. Data showed that 18 genes involved in the network these differences in gene regulation, combined with professional knowledge, because of its AOC3 gene encoding protein with cell adhesion function and SSAO activity into our field of vision.
The AOC3 gene encoding protein is composed of 763 amino acids containing copper amine oxidase (CAOs), called VAP-1.VAP-1 is a protein with double functions. On the one hand as an endothelial cell adhesion factor (VAP-1) can induce leukocyte adhesion to vascular endothelium; on the other hand, as the primary amine oxidase (SSAO) amines can catalyze aromatic and aliphatic aldehydes were converted into the corresponding H2O2 and NH3., and through literature review we found that the function of VAP-1 protein and severe microcirculatory disorders and vascular hyporeactivity are closely linked. Therefore we believe that VAP-1 may play an important role in the pathological process of severe hemorrhagic shock, it is necessary to study to clarify, in order to provide a new therapeutic method for the clinical treatment of severe hemorrhagic shock.
[Objective]
1, the changes in the expression of AOC3 gene and the expression of the encoded protein VAP-1 in the liver tissue during the process of hemorrhagic shock and resuscitation in rats were determined.
2, the changes of the content of sVAP-1 and the activity of SSAO in the blood of rats were determined during the process of hemorrhagic shock and resuscitation.
3, the AOC3 overexpression adenovirus vector is constructed, which lays the foundation for the next in vitro functional experiment.
4, the effect of AOC3 overexpression of recombinant adenovirus on the transfection of rat hepatic sinusoidal endothelial cells was detected.
5, the expression of AOC3 gene and its encoded protein VAP-1 in rat hepatic sinusoidal endothelial cells in hypoxic environment was clearly defined.
[method]
1, the establishment of a rat model of constant pressure hemorrhagic shock.
2, the rat blood pressure were collected in liver tissues of rats given time point of shock - shock model in the recovery process, by Real-time PCR, the expression of Western-Blot and immunohistochemistry method to detect the shock and resuscitation in various stages of AOC3 gene and its encoding protein VAP-1;
3, we collected rat peripheral blood from shock to resuscitation time by fixed pressure hemorrhagic shock model, and detected sVAP-1 content and SSAO enzyme activity in different stages of shock resuscitation by ELISA and spectrophotometer.
4, AOC3 overexpression adenovirus vector design, construction, virus packaging, amplification, concentration and titration, adenovirus transfected rat intestinal microvascular endothelial cells and MOI value.
5, using hepatic blood recombinant adenovirus and empty vector transfected into rat sinusoidal endothelial cells, and cell culture box by reducing the oxygen concentration of the hypoxic environment. The use of Real-time manufacturing PCR, Western-Blot detection of rat hepatic sinusoidal endothelial cells before and after transfection in normoxia and hypoxia condition and the AOC3 gene encoding protein VAP-1 the expression.
[results]
1, a stable rat model of constant pressure and hemorrhagic shock was established.
2, in the process of constant pressure, hemorrhagic shock and resuscitation, the results of Real-time PCR showed that the expression of AOC3 gene in liver tissue was significantly higher in shock group than in resuscitation group and control group, and that in resuscitation group was significantly higher than that in control group.
3, in the process of constant pressure, hemorrhagic shock and resuscitation, Western-blot results showed that the ratio of VAP-1/GAPDH protein in liver tissue was significantly higher in shock group than in resuscitation group and control group, and the recovery group was significantly higher than that in control group.
4, the constant pressure of rats with hemorrhagic shock in the recovery process, quantitative ELISA results showed that the shock group serum sVAP-1 content was significantly higher than that in recovery group and control group, recovery group was significantly higher than the control group.SSAO activity determination results showed enzyme activity in serum was significantly higher than that in shock group, resuscitation group and control group, recovery group was significantly higher than that of control group;
5, the AOC3 overexpression adenovirus vector (pIRES-EGFP-AOC3) was successfully established, and the optimal MOI value of the recombinant adenovirus infected rat intestinal microvascular endothelial cells was 5-10.
6, in normoxia and hypoxia conditions, Real-time PCR showed that the expression of AOC3 in rat liver sinusoidal endothelial cells by recombinant adenovirus after transfection was significantly higher than that before transfection, the cells before and after transfection of the empty AOC3 expression had no significant difference between the results of.Western-blot showed the same trend;
7. Real-time PCR results showed that both the sinusoidal endothelial cells of rat liver blood by recombinant adenovirus or empty vector, in hypoxia under the AOC3 conditions of the expression were significantly higher than those in normal oxygen culture conditions of.Western-blot although the results showed the same trend, but the difference was not significant Real-time PCR results.
[Conclusion]
1, in vivo experiments confirmed that the content of VAP-1 in liver tissue and serum increased and the activity of SSAO enzyme increased in the course of severe shock in rats. However, the activity of VAP-1 and SSAO activity in all tissues decreased with the recovery.
2, AOC3 overexpression of adenovirus vector was successfully constructed. Under normoxic or anaerobic conditions, the expression of AOC3 gene and coding protein VAP-1 increased significantly after transfection of recombinant adenovirus with AOC3 overexpression in rat hepatic sinusoidal endothelial cells.
3, the expression of AOC3 in hypoxic condition was significantly higher than that in normoxic state before and after transfection of recombinant adenovirus with AOC3 overexpression in rat hepatic sinusoidal endothelial cells. The encoded protein VAP-1 showed the same trend, but this change was not as obvious as that of the gene level.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R605.971;R-332

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 俞曉軍;錢t$;張育tF;胡志前;;微量失血對(duì)左肝外葉切除+定容失血性休克大鼠生存率的影響[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2011年19期

2 趙惠卿,朱玲玲,趙彤,吳麗穎,李海生,丁愛石,范明;低氧對(duì)胚胎干細(xì)胞增殖的影響[J];中國(guó)應(yīng)用生理學(xué)雜志;2004年03期

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