【作者】 王新友；

【导师】 李荣；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2010， 博士

【摘要】 失血性休克的传统复苏方法(conventional resuscitation, CR)是在控制出血的基础上,通过静脉补液快速恢复有效血容量,但是这种复苏方式过程中的顽固性低血压、复苏后全身炎性综合症(systemic inflammatory response syndrome, SIRS)及多器官功能不全综合症(multiple organ dysfunction syndrome, MODS)等后期并发症的发生率及致死率居高不下,成为失血性休克治疗的难题。临床和实验研究表明,肠道屏障功能障碍在失血性休克进程中伴有重要作用,休克时肠系膜血管痉挛缺血,肠道屏障功能受损,肠道细菌／内毒素移位(Bacteria/endotoxin translocation, BET),细菌和毒性物质进入肠淋巴循环和／或血液循环,加重了创伤及缺血所致的器官功能障碍,导致重要脏器的损伤以及SIRS和MODS的产生,因此有观点认为“肠道是休克的靶器官和SIRS和MODS的始动器官”。近年来人们对通过保护休克期肠道屏障、防止有害物质进入循环系统来阻断休克的进展做了大量研究。常用的措施有选择性肠道净化、改善内脏血流、加强肠道营养以及阻断肠系膜淋巴途径等,但是这些方法往往缺乏实用性或是效果有限,失血性休克的治疗面临困境。在CR基础上通过腹腔注入腹膜透析液来扩张痉挛肠系膜血管即腹腔复苏(peritoneal resuscitation, PR)是近年来失血性休克治疗的一个新尝试,该方法由美国Louisville军事医学中心研究人员提出,该研究目前处于初步探索阶段,PR疗效有待不同休克模型来证实,其分子作用机制有待深入研究。本研究将利用重症失血性休克大鼠模型来评价这一方法的疗效,并从肠系膜微循环途径及肠淋巴途径来探讨其作用机理,系统评价该方法对全身重要脏器的灌注及功能的影响,为这一方法的临床应用打下基础。目的评价辅助PR对重症失血性休克大鼠的治疗效果；探讨辅助PR对失血性休克大鼠重要脏器功能和组织结构的影响以及进一步从肠系膜微循环途径和肠系膜淋巴途探讨PR的作用机制。1、辅助PR救治重症大鼠的疗效观察休克模型：大鼠放血至平均动脉压(MAP) 40mmHg并维持到失血第90min,实验分四组：假休克(SS)组,仅行插管监测MAP; HS+CR组行失血回输+2倍体积林格乳酸钠静脉治疗；HS+CR+PR组CR治疗同时行2.5%腹膜透析液行PR治疗；休克不复苏(HS)组。监测大鼠MAP,测定稳定后、复苏前和复苏后2h动脉血PH、LA、GLU、双抗体夹心法测定TGF-a水平,观察大鼠存活时间。2、辅助PR对失血性休克大鼠重要脏器功能和组织形态的影响休克模型：大鼠放血至MAP50mmHg,维持60min。实验分为SS组、HS+CR组及HS+CR+PR组。复苏12h后全自动生化分析仪测定动脉血LDH、CK, ALT、AST、Urea, Cre,评价肺、心、肝、肾组织形态的变化。3、辅助PR对肠系膜微循环的影响休克模型：大鼠放血至MAP40mmHg,维持60min。实验分SS组、HS+CR组及HS+CR+PR组。复苏后2h观察大鼠活体循环,测定肠系膜微动静脉直径、微静脉白细胞附壁数和肠粘膜微区灌注量,实验结束时处死大鼠取远端回肠组织,测定肠组织含水量及评价肠粘膜损伤程度。4、辅助PR对肠系膜淋巴活性的影响休克模型：大鼠放血至MAP40mmHg,维持60min。实验分为：①SS组,②SS+CR组,③SS+CR+PR,④HS组,⑤HS+CR组,⑥HS+CR+PR组。复苏后2h剖腹收集肠系膜淋巴测定肠系膜淋巴流量、休克淋巴介导的中性粒细胞呼吸爆发水平、休克淋巴对HUVECS活力的影响,3h后处死大鼠,伊文思蓝示踪法测定肺组织微血管通透性。结果1、同HS+CR组相比,HS+CR+PR组MAP在复苏后2h显著提高(P<0.05)；复苏后2h HS+CR+PR组动脉血PH显著高于HS+CR组(P<0.05),乳酸水平显著低于HS+CR(P<0.05)组：血清TGF-α测定显示,HS+CR+PR组显著低于HS+CR组(P<0.01)；HS+CR+PR组大鼠72h存活率58.3%,显著高于HS+CR组(72h存活率8.3%)(P<0.05)2、复苏后12h, HS+CR+PR组ALT、AST、Urea、Cre, LDH及CK均较HS+CR组显著下降；组织形态学分析显示,HS+CR+PR组肺、肝、心、肾等重要脏器组织损伤较HS+CR组明显减轻。3、复苏后2h后剖腹,活体微循环观察显示：HS+CR+CR组微动、微静脉血流呈线流或线粒流,部分血管管壁有红细胞聚集,可见少数中性粒细胞附壁；HS+CR组微循环血流慢,血流多呈粒流,部分血管还可见血流停滞,中性粒细胞粘附较多；HS+CR+PR组肠系膜微动、微静脉血管管径分别是HS+CR组的1.41倍(P<0.05)和1.80倍(P<0.01),中性粒细胞附壁数较HS+CR组显著减少(P<0.01),回肠组织含水量显著少于HS+CR组(P<0.01)；肠粘膜组织损伤评分显著低于HS+CR组(P<0.01)。4、复苏后2h剖腹,引流并收集肠系膜淋巴。结果显示：复苏2hHS+CR+PR组淋巴流量是HS+CR组1.68倍(P<0.01)；流式细胞仪检测显示HS+CR+PR组MFI值为HS+CR组的69.7%(P<0.01)；MTT方法检测HUVECs活力显示,HS+CR+PR组治疗后HUVECs活力较HS+CR组提高25%,(P<0.01),肺微血管通透性显示HS+CR+PR组伊文思蓝漏出率为HS+CR组的55.50(P<0.01)。结论1、在CR基础上行2.5%腹膜透析液PR治疗能提高重症失血性休克大鼠的长期存活率；2、辅助PR治疗能较好改善休克大鼠心、肝、肾等重要脏器功能,减轻肺、心、肝、肾等重要脏器组织损伤；3、辅助PR作用机制可能与PR改善休克大鼠肠系膜微循环、较好保护肠道屏障完整性以及降低休克肠系膜淋巴活性有关。更多还原

【Abstract】 Background Today the major clinical therapeutic methods for trauma-hemorrhagic shock are controlling of bleeding and rapid correcting of intravascular volume by intravenous infusion. However, refractory hypotension during the process of resuscitation and the complications after resuscitation such as systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) are still tough problems for the treatment of trauma-hemorrhagic shock.Studies have already confirmed that gut barrier dysfunction plays an important role during the progress of hemorrhagic shock. During shock mesenteric blood vessel convulsion and hypoperfusion and its sequale of ischemia result in impairment of gut barrier. And as a consequence the bacteria/endotoxin translocated to circulation. They aggravate injury caused by ischemia and lead to SIRS and MODS. Some therapeutic strategies to prevent gut injury or dysfunction including selective gut decontamination, enteral nutritional regimens, improving blood flow of visceral and preventing toxic mesenteric lymph entering into circulation have been attempted. However, the methods are not well clinically applied. Recently researchers of Louisville veterans affairs medical center introduced a new resuscition method, adjunct peritoneal resuscitation (PR) for hemorrhagic shock.This technique uses a clinical peritoneal dialysis solution injecting to abdominal cavity at the time of conventional resuscitation(CR) to dilate mesenteric mircocirculation. To date this technique is on the way of its initial exploration and its effects and molecular mechanisms need further inv(?)stigation. In this study severe trauma-hemorrhagic rat shock model was used to evaluate its effect. And its mechanisms were explored from mesenteric microcirculation and mesenteric lymph route. And its influences to some important organs were also evaluated.Objective To evaluate the therapeutic effects of adjunct PR on severe hemorrhagic shock in rats and its influence on the functions and structure of some important organs of shock rats and for the further to explore its mechanism from mesenteric microcirculation and lymph routes.Methods1. Severe hemorrhagic shock rats model was established. Male SD rats were divided into 4 groups:Sham group (SS),②hemorrhagic shock with CR (HS+CR),③hemorrhagic shock with CR (HS+CR+PR),④emorrhagic shock with no resuscitation (HS). Arterial PH、LA、GLU and TGF-αwere determined at equilibrium,before resuscitation and 2h after resuscitation. The survival time were recorded.2. Hemorrhagic shock model was established. The rats were divided into 3 groups:HS、HS+CR and HS+CR+PR group. Blood concentrations of LD、CK、ALT、AST、Urea、Cre were determined by automatic biochemical analyzer 2h after resuscitation. Tissue structural changes of lung, liver, heart and kidney were analysed.3. Hemorrhagic shock model was established. Mesenteric circulation in vivo were observed 2h after resuscitation. Mesenteric microvascular diameter and blood flow in intestinal wall was measured.The numbers of neutrophils adherent to veinules were accounted. A segment of the terminal ileum was harvested at death and processed for evaluation of mucosal injury and water content.4. Hemorrhagic shock model was established. The rats were divided into six groups as follows:①SS group②SS+CR group③SS+CR+PR group④HS group⑤HS+CR group⑥HS+CR+PR group.Two hours after resuscitation mesenteric lymph was collected by laparotomy. Bioactivity of mesenteric lymph was tested by lymph mediated neutrophils burst activity and HUVECs injury. Three hours aftrer resuscitation pulmonary microvascular permeability was investigated.Results1. Compared with HS+CR group, MAP in HS+CR+PR group was significantly improved 2h after resuscitation. And blood concentrations of LA and TGF-αin HS+CR+PR group were significantly decreased. Arterial PH in HS+CR+PR group was significantly improved. Survival to 72h in HS+CR+PR group was 58.3%, significantly higher than that in HS+CR group (8.3%) (P<0.05)2. Compared with HS+CR group, Blood concentration of ALT. AST、Urea、Cre. LDH and CK in HS+CR+PR group were all significantly decreased 12h after resuscitation. Histological evaluation revealed tissue injury of lung、live、heart and kidney in HS+CR+PR group were significantly decreased.3. Mesenteric microcirculation revealed that blood flow of arterioles and veinules in HS+CR+PR was slower than that in HS+CR group. And there were less neutrophils adherent to veinules in HS+CR+PR group. The diameters of arterioles and venules in HS+CR+PR group were 1.41 fold and 1.80 fold to those in HS+CR group respectively. Water content and ileum injury in HS+CR+IR group were both greatly decreased.4. Compared with HS+CR group, mesenteric lymph flow in HS+CR+PR group was greatly impoved. Shock lymph mediated neutrophils respiratory burst activity and HUVECs injury in HS+CR+PR group was significantly decreased and pulmonary microvascular permeability in HS+CR+PR group was also decreased.Conclusion1. Adjunct PR with 2.5% peritoneal dialysis solution can better improved MAP,decrease concentrations of LA,TGF-αand attenuate acidosis. Adjunct PR can also achieve a better long time survival of severe hemorrhagic shock in rats.2. Compared with CR, adjunct PR can usefully decrease LDH,CK, ALT, AST, Urea, Cre concentrations in blood of shock rats,ameliorate hear、liver and kidney function and relieve tissue injury of lung、heart、liver and kidney.3. The mechanism of adjunct PR may be that it ameliorates mesenteric microcirculation, proctects gut barrier and decrease bioactivity of mesenteric lymph of shock rats.更多还原