重症失血性休克及复苏条件下大鼠VAP-1表达规律及调控机制研究

发布时间：2018-01-11 10:14

本文关键词：重症失血性休克及复苏条件下大鼠VAP-1表达规律及调控机制研究　出处：《第二军医大学》2012年博士论文　论文类型：学位论文

【摘要】：【背景】基于目前重度失血性休克救治中靶向治疗手段的缺乏，以及创伤失血性休克相关基础研究与临床的脱节，急切需要我们重新审视已知对创伤失血性休克病理的认识。前期，我们在一种相对接近临床的肝左外叶切除+2.5ml/100g（体重）的大鼠创伤失血性休克模型上，摸索到24小时死亡率接近50%的干预条件，之后对存活和死亡大鼠失血前肝脏进行表达谱基因研究，筛查出约100个差异基因，随后对这些差异基因进行调控网络分析。数据表明有18个基因参与了这些差异基因的网络调控，结合专业知识，AOC3基因由于其编码蛋白具有细胞粘附功能及SSAO活性而进入我们视野。 AOC3基因所编码的蛋白是一个由763个氨基酸组成的含铜胺氧化酶(CAOs)，称为VAP-1。VAP-1是一种具有双重功能的蛋白。一方面作为内皮细胞粘附因子（VAP-1）可诱导白细胞粘附至血管内皮；另一方面作为胺氧化酶（SSAO）可催化芳香族和脂肪族的伯胺类物质转化成相应的醛类，并产生H2O2和NH3。通过文献复习我们发现VAP-1蛋白的功能与重症休克期微循环障碍及血管反应性降低有密切联系。因此我们认为VAP-1在重症休克病理过程中可能具有重要意义，有必要进行研究阐明，以期为临床救治重症休克提供一种新的治疗手段。【目的】 1、明确大鼠定压失血性休克-复苏过程中肝脏组织AOC3基因及其编码蛋白VAP-1表达的变化规律； 2、明确大鼠定压失血性休克-复苏过程中血液中sVAP-1含量及SSAO酶活性的变化规律； 3、构建AOC3过表达腺病毒载体，为下一步体外功能实验奠定基础； 4、检测AOC3过表达重组腺病毒对大鼠肝血窦内皮细胞的转染作用； 5、明确低氧环境中大鼠肝血窦内皮细胞AOC3基因及其编码蛋白VAP-1的表达规律。【方法】 1、大鼠定压失血性休克模型的建立； 2、通过大鼠定压失血性休克模型在休克-复苏过程中的既定时间点采集大鼠肝脏组织标本，通过Real-time PCR、Western-Blot及免疫组化等方法检测休克-复苏各阶段AOC3基因及其编码蛋白VAP-1的表达情况； 3、通过大鼠定压失血性休克模型采集大鼠休克-复苏既定时间点的外周血，使用ELISA法及分光光度计分别检测休克-复苏过程不同阶段sVAP-1含量及SSAO酶活性； 4、AOC3过表达腺病毒载体的设计、构建、病毒包装、大量扩增、浓缩及滴定测定，用腺病毒转染大鼠肠微血管内皮细胞并测定MOI值； 5、用重组腺病毒及空病毒转染大鼠肝血窦内皮细胞，并通过降低细胞培养箱氧浓度制造低氧环境。使用Real-time PCR、Western-Blot检测大鼠肝血窦内皮细胞在腺病毒转染前后在常氧条件及低氧条件下其AOC3基因及编码蛋白VAP-1的表达情况。【结果】 1、建立了稳定的大鼠定压失血性休克模型； 2、在定压失血性休克-复苏过程中，Real-time PCR结果显示肝脏组织AOC3基因表达量休克组显著高于复苏组及对照组，复苏组显著高于对照组； 3、在定压失血性休克-复苏过程中， Western-blot结果显示在肝脏组织VAP-1/GAPDH蛋白浓度比值休克组显著高于复苏组及对照组，复苏组明显高于对照组； 4、大鼠定压失血性休克-复苏过程中，ELISA定量结果显示血清中sVAP-1含量休克组显著高于复苏组及对照组，复苏组显著高于对照组。SSAO酶活性测定结果显示血清中酶活性休克组显著高于复苏组及对照组，复苏组显著高于对照组； 5、成功建立AOC3过表达腺病毒载体（pIRES-EGFP-AOC3），重组腺病毒感染大鼠肠微血管内皮细胞的MOI值选取5-10为最佳； 6、在常氧及低氧培养条件下，Real-time PCR结果均显示大鼠肝血窦内皮细胞经重组腺病毒转染后其AOC3表达量显著高于转染前，而该细胞经空病毒转染前后的AOC3表达量无显著差异。Western-blot结果显示相同的趋势； 7、Real-time PCR结果显示无论大鼠肝血窦内皮细胞是否经重组腺病毒或空病毒转染，在低氧培养条件下其AOC3表达量均显著高于常氧培养条件。Western-blot结果虽然显示相同的趋势，但差异并不如Real-time PCR结果显著。【结论】 1、体内试验证实大鼠重症休克病程中肝脏组织及血清中VAP-1的含量升高，血清中SSAO酶活性增强，而随着复苏的进行，各组织中VAP-1含量及SSAO酶活性均有下降趋势。 2、AOC3过表达腺病毒载体构建成功。在常氧或厌氧条件下，大鼠肝血窦内皮细胞经AOC3过表达重组腺病毒转染后其AOC3基因及编码蛋白VAP-1表达量均有明显上升。 3、在大鼠肝血窦内皮细胞经AOC3过表达重组腺病毒转染前后，，其在低氧条件下AOC3的表达量均明显高于常氧状态下。其编码蛋白VAP-1表现同种趋势，然而该变化不如基因层面明显。
[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.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R605.971;R-332

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