【作者】 郑伟；

【导师】 周虹；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2007， 博士

【摘要】 失血性休克（hemorrhagic shock）是因急性血液或血浆的大量丢失导致循环血量骤减而引起。失血性休克是战时阵亡和伤死的首要因素，也是平时1-44岁人群死亡的第一要因。目前临床治疗失血性休克主要的措施是恢复血压、调节微循环、恢复重要脏器的血液灌流等，但对急性重度失血性休克的救治效果不是很理想。原因在于失血性休克是一种全身性、多系统、多器官参与的严重的综合症，其生理病理机制还需要不断补充和完善。各种疾病的治疗现均转向以疾病的分子机制的阐明为救治依据的研究，而蛋白质组技术的出现使高通量筛选疾病的诊断标志物和疾病的药物靶标等成为可能，利用蛋白质组学技术所获得的高通量差异蛋白质信息也成为解析疾病生理病理机制的重要基础。目前还未见有关失血性休克时血浆和肝脏蛋白质组学的研究报道。近年来的研究发现，失血性休克时体内的迷走神经及其递质Ach与免疫系统相互作用，参与抗炎，并将之命名为胆碱能抗炎通路，而ACTH是此通路的重要激活因素。目前ACTH参与胆碱能抗炎通路救治失血性休克的研究主要是在延长动物的存活时间方面，极少涉及对ACTH参与胆碱能抗炎通路发挥抗休克作用机制的研究。本研究通过蛋白质组学技术，研究在急性重度失血性休克（acute severe hemorrhagic shock，ASHS）条件下以及给予ACTH作为干涉条件治疗后，大鼠血浆蛋白质组及肝脏蛋白质组的差异表达，并初步鉴定和分析差异表达蛋白质的功能，以及它们在失血性休克发生发展过程中所起的作用，以期从蛋白质整体水平上探讨失血性休克的生理病理机制，找出失血性休克诊断的新靶标和治疗的新方法。ASHS大鼠模型的建立和ACTH抗失血性休克作用的研究ASHS大鼠模型：手术后让大鼠在手术台上静躺10 min，模型0-15min快速失血使平均动脉压降至35 mmHg，随后15 min调整失血量使血压维持在这一水平，血压35-45mmHg平台期50 min，失血量为总血量的40-45％，模型80 min时处死大鼠。通过检测正常对照组（sham hemorrhage shock，SHS）（n=10）和ASHS组（n=10）大鼠血浆中SOD、MDA、AST、ALT、GST、LDH和TNF-α等生化指标及观察肝脏组织的形态学改变和HSP70的含量变化，表明建立的ASHS模型稳定性好，符合ASHS的要求，可以作为ASHS的动物模型。在模型50 min时，ACTH组（n=10）在1 min内以3 ug·kg^-1静脉注射ACTH；NS组（n=10）静脉注射ACTH的等体积数的生理盐水。通过观察大鼠的存活时间及以上各项指标及形态学的变化表明ACTH对ASHS有一定的救治效果。在本节实验中还发现血浆中SOD活性和MDA含量的变化与目前报道认为二者总是负相关变化不一致，在ASHS时二者的变化是正相关的，即均呈逐渐上升趋势。ASHS大鼠血浆蛋白质组学差异分析由于血浆中白蛋白、IgG、α1-抗胰糖蛋白和α2-巨球蛋白等高丰度的蛋白的含量占80％，严重影响2-DE中低丰度蛋白的分离和鉴定，所以在本节实验2-DE前预先去除了这类高丰度蛋白中的大部分。实验结果显示SHS组、ASHS组和ACTH组去除高丰度蛋白的血浆的2-DE图谱分别平均识别到570±0.88、578±1.45和578±0.88个有意义的蛋白质斑点。SHS组与ASHS组两胶进行蛋白点匹配，匹配了493对，匹配率为85.4％；ASHS组与ACTH组两胶进行蛋白点匹配，匹配了493对，匹配率为85.2％。在SHS组和ASHS组去除高丰度蛋白的血浆2-DE凝胶图谱中共发现8个差异有意义的蛋白质点，通过MALDI-TOF-MS鉴定出了7个蛋白质斑点共3类蛋白（白蛋白、甲状腺激素结合蛋白-D链蛋白和信号素-3D前体）。ACTH治疗后发现8个斑点中有5个发生了显著性差异表达，鉴定出4个蛋白质斑点共2类蛋白（白蛋白和信号素-3D前体）。ASHS大鼠肝脏总蛋白质组学差异分析SHS组、ASHS组和ACTH组肝脏组织总蛋白质的2-DE图谱分别平均识别到698±11、700±13并H696±18个有意义的蛋白质斑点。SHS组与ASHS组两胶进行蛋白点匹配，匹配了641对，匹配率为91.7％；ASHS组与ACTH组两胶进行蛋白点匹配，匹配了637对，匹配率为91.3％。SHS组与ASHS组肝脏总蛋白质组学研究发现并鉴定出10个差异显著的蛋白质斑点，共8种蛋白{肿瘤抑制性抗原gp96、葡萄糖调节蛋白58（glucose regulated protein, 58 kDa, GRP58）、ATP合成酶β亚单位、二磷酸果糖酶B、三磷酸甘油醛脱氢酶、谷光苷肽转移酶、过氧还蛋白Ⅰ和细胞色素B5}。ACTH治疗后发现并鉴定出2个显著性差异表达的蛋白质斑点（肿瘤抑制性抗原gp96和GRP58）。GRP58在ASHS中的初步研究GRP58是发现的7个表达差异有显著性的蛋白质斑点中肽段匹配分值最高（224），氨基酸序列覆盖率最高（41％）的蛋白。对其进行Western-blot、MALDI-TOF-MS、免疫荧光组化及RT-PCR实验，结果显示在ASHS时，GRP58发生了150位丝氨酸残基的去磷酸化。给予ACTH治疗后，150位丝氨酸残基又向磷酸化方向变化。综上研究结果表明，本研究发现在ASHS时，血浆中SOD活性与MDA含量的变化均呈逐渐上升趋势，血浆和肝脏蛋白质中分别有白蛋白和GRP58等许多蛋白发生了显著性的差异表达，GRP58在这一过程中发生了150位丝氨酸残基的去磷酸化，提示白蛋白和GRP58可能是失血性休克诊断和治疗的新靶标。更多还原

【Abstract】 Hemorrhagic shock is one of the severe hypovolemia shocks, induced by extensive blood loss, or sudden and unexpected depletion of the circulating blood. Hemorrhagic shock is a major independent risk factor of the pathogenesis of trauma-related multiple organ failure and death. It is one of the most common causes of death all over the world today. There are various treatments on shock now. For example, the doctor can boost pressure, regulat microcirculation and re-perfuse the several important organs including heart, liver, spleen, lung, kidney and brain. They are not effective, however, hemorrhagic shock develops refractory hemorrhagic shock. The reason is that hemorrhagic shock is characterized by a loss in circulatory volume, which results in decreased venous return, decreased filling of the cardiac chambers, and hence a decreased cardiac output which leads to increase in the systemic vascular resistance. Hemorrhagic shock is one of four major categories of the syndrome of shock based on cardiovascular characteristics. It’s pathomechanism is not clear, and need to be replenished in order to make perfect. Nowadays, the therapise of many kinds of diseases are being adjusted to research their molecule mechanisms. Proteomic approach is considered to be a powerful technology in the global analysis of protein expression and has been widely used in disease proteomics fields.Some of the latest studies propose that, vagus nerve and Ach interact with immune system to resist inflammatory responses. It is called the cholinergic anti-inflammatory pathway. Adrenocorticotrophin （ACTH） is one of the important activation agents in the pathway. Nowadays, there are not any reports, which investigate plasma and liver proteomic analysis in hemorrhagic shock, and which research proteomic analysis with ACTH in hemorrhagic shock.This study was to screen the differential expression proteins by proteome analysis technology in the rat plasma and liver under acute severe hemorrhagic shock （ASHS） and post-treatment with using ACTH. The differential expression proteins were cut from the gels, analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）, and identified through searching Swiss-prot database with Mascot software. These results provided scientific datum for screening the molecular biomarker used to diagnose and treat hemorrhagic shock, as well as seek out new target to elevate the patient’s prognosis and provide new clue for the research on pathogenic mechanism of hemorrhagic shock. Establish rat model in ASHS and treatment method with using ACTH ASHS （?）t model: After lying quietly for 10 minutes, ASHS rats were induced by being withdrawn blood through the femoral arterial catheter in 15 minutes. The mean arterial blood pressure （MABP） decreased to 35-40 mmHg and stabilized MABP for 15 minutes. The MABP was maintained at this level for 1 hour. The total amount of blood withdrawn then was about 40-45% of rat blood volume. The animals were sacrificed at 80 minutes. The activity of SOD, MDA, AST, ALT, GST, LDH and TNF-αin plasma and HSP70 in liver were measured respectively by using the their test kits. And pathological changes of liver were observed by microscopy. These results suggested ASHS rat model had satisfactory stability. Then this model could be operated in ASHS research. At 50 minutes of ACTH model, we administered ACTH (3 ug·kg^-1) intravenous injection, while we administered normal saline （equality volum of ACTH） in normal saline （NS） model. The results of survival time of SHS, ASHS, NS and ACTH suggested ACTH could remedy ASHS in some extent. Furthermore, the changes of the activity of SOD and the level of MDA in ASHS plasma might be positive correlation.Plasma proteomic analysis in ASHS Albumin, IgG,α1-Antitrypsin and a2-Macroglobulin count above 80 % in all the proteins of plasma, so we depleted them before 2-DE. About 570±0.88, 578 + 1.45 and 578 + 0.88 protein spots were respectively detected by Image Master analysis software in SHS, ASHS and ACTH. It was 85.4 % （493 pairs） that the ratio of protein spots matched between SHS and ASHS. There was 85.2 % （493 pairs） between ASHS and ACTH. Eight protein spots with significant difference were found, and upregulated or downregulated after ASHS. Seven of eight differential proteins were identified, and they were albumin, chain D of rat tansthyretin and semaphoring-3D precursor. With ACTH treating, there were five significant difference protein spots. Four of five differential proteins were identified, and they were albumin and semaphoring-3D precursor.Liver proteomic analysis in ASHS The 2-DE results of livers showed about 698±11, 700±13 and 696±18 protein spots were respectively detected in SHS, ASHS and ACTH. It was 91.7 % （641 pairs） that the ratio of protein spots matched between SHS and ASHS. There was 91.3 % （637 pairs） between ASHS and ACTH. Ten protein spots with significant difference were found, and upregulated or downregulated, or appeared after ASHS. All of ten differential proteins were successfully identified. These proteins included tumor rejection antigen gp96, glucose regulated protein, 58 kDa （GRP58）, ATP synthase beta subunit, aldob protein, gapd protein, glutathione transferase, peroxiredoxin I , cytochrome B5 etc. With ACTH treating, there were two significant difference protein spots. The both of differential proteins were identified, and they were tumor rejection antigen gp96 and GRP58.Original research of GRPS8 in ASHS There were seven significant difference protein spots in plasma and liver proteomic analysis in ASHS with ACTH treating. GRP58 is the biggest （224） in paired protein score, and is the most （41 %） in sequence coverage. So GRP58 was chosen to investigate profoundly with western-blot, MALDI-TOF-MS, immunohistochemistry and RT-PCR. The results indicated that Ser150 of GRP58 was dephosphorylation after ASHS, while Ser150 was phosphorylation after treating with ACTH.In summary, we showed the changes of the activity of SOD and the level of MDA in ASHS plasma might be positive correlation. There were many of significant difference protein spots in plasma and liver proteomic analysis in ASHS. And Ser150 of GRP58 was dephosphorylation after ASHS. Therefore, all of these indicated albumin and GRP58 could be used as two new targets in diagnosing and treating hemorrhagic shock.更多还原