【作者】 刘骏峰；

【导师】 顾勇； 丁峰； 薛骏；

【作者基本信息】 复旦大学 ， 内科学， 2008， 博士

【摘要】 第一部分内毒素致多脏器功能障碍综合征动物模型的研究背景脓毒血症（sepsis）是一种常见而且致命的疾病,其实质是一种被恶性扩大的炎症反应,结果容易导致多脏器功能障碍综合征（Multiple Organ DysfunctionSyndrome,MODS）,这是当今创伤外科和危重病医学领域中研究的热点课题。MODS是一类与休克和感染等应激打击关系密切,很可能是由失控的全身炎症和细胞氧代谢障碍所致的急性器官损伤。因此脓毒血症和MODS密不可分,长久以来其发病率和死亡率一直居高不,尤其是在重症监护室,脓毒血症、MODS是引起死亡主要的原因之一,因此有关于脓毒血症的研究成了医学界一个挑战。目前缺少非常有效治疗脓毒症尤其是伴有MODS的方法,主要是对脓毒症发病机制还没有完全了解,因此建立一个合理的动物脓毒血症模型,有助于了解脓毒血症的发生过程,以及发展成MODS的过程,并为临床上解决脓毒血症提供有力的帮助。目前建立脓毒血症伴MODS的动物模型有很多种,本文的目的就是初步探讨内毒素致脓毒血症、并发展成MODS的特点。方法选取5头健康杂交家猪,雌雄随机,年龄3个月左右,体重在16—21kg。用标准内毒素(0.25mg·kg^-1)由右侧颈内静脉缓慢注入,分别于注入内毒素前及注入后1、2、3、4、5、6h检测血常规、血气分析、血生化、凝血酶原时间,同时记录动脉压及平均动脉压（MAP）、心率（HR）等血流动力学指标。同时留取血清标本用于检测白介素-1（IL-1）、白介素-10（IL-10）、可溶性血栓调节蛋白（sTM）、内皮素-1（ET-1）等指标。结果经脉输注内毒素后,平均动脉压（MAP）较前显著下降（p＜0.01）,但肺动脉压（PA）则持续升高。血清肌酐（SCr）和丙氨酸转氨酶（ALT）升高,凝血酶原时间（PT）较前延长。细胞因子中白介素-1、白介素-10和内皮素-1的变化一致,先有轻度升高,随后开始下降,而可溶性血栓调节蛋白的变化相反。结论内毒素输注能够诱发猪脓毒血症,并在短期内成功造成多脏器功能障碍综合征。第二部分内皮细胞生物反应器在猪脓毒血症伴多脏器功能障碍综合征中的运用背景由于本文的第一部分已经成功构建了猪脓毒血症伴MODS的动物模型,而我们实验室已经建立内皮细胞生物反应器（endothelial bioreactor device,EBD）,并对反应器中的内皮细胞进行鉴定。由于内皮细胞生物反应器中内皮细胞是生长在反应器的外腔,那么血液通过反应器时并不和内皮细胞直接接触,有效避免了急性排异反应。反应器的内外腔可以进行物质交换,因此外腔中的内皮细胞可以通过这部分物质交换而对整个血循环进行调节。本研究的目的为初步证明内皮细胞生物反应器能在猪脓毒血症伴MODS中的作用。方法将健康杂交家猪10头随机分为内皮细胞生物反应器组（EBD组）和假性循环组（对照组）,每组各5头。静滴内毒素（LPS,0.25 mg·kg-1,30min内滴完）制造脓毒症模型后进行体外循环。在实验的0h、1h、3h和6h分别观察实验动物的血流动力学指标、血常规、血生化指标和炎症、凝血指标。血流动力学指标主要包括平均动脉压（MAP）、血生化指标主要包括血清肌酐（SCr）和血钾（K）。炎症、凝血指标主要包括白介素-1（IL-1）、白介素-10（IL-10）、内皮素-1（ET-1）、一氧化氮合酶（NOs）和蛋白C（PC）,最后记录实验动物的生存时间。结果在注射LPS后30min到2h,两组的平均动脉压（MAP）都开始显著下降,但内皮细胞反应器组此后MAP开始缓慢升高,并能维持一段时间,而假性循环组的血压则持续下降,没有升高趋势,两组有明显差异（p＜0.01）。实验开始后,两组的肾功能开始有所变化,内皮细胞反应器组在实验的结束时,肌酐也没有明显升高,但假性循环组的血清肌酐则明显升高,两组有显著差异（p=0.001）。血钾的变化和血清肌酐相似,在试验6h,假性循环组的血钾明显高于内皮细胞反应器组,p=0.002。IL-1和IL-10在输注LPS后都开始升高,2小时后到达高峰,随后浓度开始下降,两组之间没有差异。内皮细胞生物反应器组的内皮素-1（ET-1）开始缓慢下降,到3小时后有所回升,一氧化氮合酶（NOs）则开始升高,2小时后有所下降,而假性循环组的变化恰好相反,ET-1先是升高,NOs则是下降,到了2小时后,ET-1急剧下降,而NOs则升高,两组相比有显著差异。实验结束时治疗组和对照组的生存时间分别为5.5±0.3天和6.7±0.4天（p＜0.05）。结论内皮细胞生物反应器不仅能够改善猪脓毒血症的血流动力学,而且能够维持重要脏器的功能,使得实验动物的存活时间延长。第三部分内皮细胞生物反应器对猪内毒素诱导急性肺损伤中炎症因子的影响背景在脓毒血症的发生和发展过程中,由于炎症因子调节失控,容易导致多脏器功能不全（MODS）,从而增加死亡率。而在器官损伤中,肺是最早也是最容易出现的。在肺损伤的发病机制中,炎症因子的参与起到了至关重要,尤其是IL-1、IL-10和TNF等促进了炎症反应的进一步扩大,引起肺组织中性粒细胞浸润,毁损肺上皮细胞,导致肺泡毛细管屏障功能减弱,通透性增加,最终出现急性呼吸窘迫综合征（ARDS）。由于本文的第二部分已经成功证实内皮细胞生物反应器对猪脓毒血症的治疗作用,并且这种治疗作用是通过调节细胞因子来实现的。而细胞因子在内毒素诱导的急性肺损伤（ALI）中起到关键作用,因此本文目的就是初步探讨内皮细胞生物反应器对内毒素诱导的ALI中炎症因子的影响。方法将健康杂交家猪10头随机分为内皮细胞生物反应器组（治疗组）和假性循环组（对照组）,每组各5头。静滴内毒素（LPS,0.25 mg·kg-1,30min内滴完）制造脓毒症模型后进行体外循环。在实验的0h、1h、3h和6h分别留取血清标本测定IL-1和IL-10的浓度,同时取动物肺组织进行HE染色,并由病理专科医师进行读片和肺损伤分数的计算。同时利用western blot检测肺组织中TNF-α、组织因子（TF）等蛋白含量。结果在注射LPS后30min到2h,两组的IL-1和IL-10都开始升高,2小时后到达高峰,随后浓度开始下降,两组之间没有差异,但在治疗组IL-1和IL-10的下降幅度小于对照组。对照组的肺组织病理中出现较多的呼吸道上皮增生、肺泡出血和血管血栓形成。而治疗组肺组织病理也可见有炎症细胞浸润,但肺泡出血、血栓明显减少。而且对照组的肺损伤分数为8.2±1.0,显著高于治疗组6.1±0.9（p=0.0447）。肺组织中肿瘤坏死因子-α（TNF-α）在治疗组的蛋白含量低于对照组（p＜0.05）,但TF的蛋白含量没有显著差异。结论内皮细胞生物反应器不仅能够改善猪脓毒血症的血流动力学,而且能够减少TNF-α等在肺组织中表达,从而减轻肺组织炎症,延缓肺的损伤。更多还原

【Abstract】 PARTⅠStudy of animal model of Multiple Organ Dysfunction Syndrome induced by endotoxin in porcinesBackgroundSepsis is a common and fatal disease,and is regarded to be a vicious expansion of the inflammatory response.Multiple organ dysfunction syndrome（MODS） is the hot topics in the field of surgical trauma and critical care medicine.MODS is a kind of acute organ injury which has the close association with stress such as shock and infection,and with uncontrolled systemic inflammatory and disorders of cell oxygen metabolism.Therefore sepsis and MODS are inseparable,its morbidity and mortality is always high for a long-term.Especially in the intensive care unit,sepsis and MODS is the main cause for death,so the research of sepsis is still a challenge in medical domain.At present the puzzled pathogenesis of sepsis leads to the lack of effective treatment of sepsis especially with MODS.So how to establish a reasonable animal model of sepsis may help to understand the development of sepsis and MODS,and to provide a strong evidence for resolving sepsis.At present there are many ways to establish the animal models of sepsis with MODS,and the purpose of this paper is to provide a simple and useful MODS animal model which was mediated by endotoxin.MethodsTen white cross pigs were anesthetized and mechanically ventilated.All the animals received intravenous lipopolysaccharide（0.25mg·kg-1） slowly through right internal jugular vein.Hemodynamics、blood routine、blood biochemistry and cytokines were examined at basement and at 1,2,3,4,5 and 6 h after the injection respectively.ResultsAfter the infusion of LPS,MAP was significantly lower at 6h than it before the infusion（p＜0.01）,but the PA was raised continuously.Compared with the basement,the serum creatinine（SCr） and alanine aminotransferase（ALT） rised at 6h,and the prothrombin time（PT） prolonged 3s.Changes of cytokines such as IL-1、IL-10 and ET-1 were first raised slightly,then began to fall.However,the changes of sTM was inversely.ConclusionInfusion of LPS can induce porcine sepsis and lead to MODS in short term. PARTⅡThe role of endothelial bioreactor device in porcine sepsis with Multiple Organ Dysfunction SyndromeBackgroundIn the first part of this paper we have successfully constructed the animal model of sepsis with MODS in porcine.In our laboratory endothelial bioreactor device（BAD） has been established and the endothelial cells in this bioreactor device have been identified.Because endothelial cells in this bioreactor device exsist in the reactor’s external cavity,then the blood through the reactor does not directly contact the endothelial cells,which effectively leads to the prevention of acute rejection.Much substance can be exchanged between inside and outside of the reactor cavity,and this exchange can be used by the endothelial cells in the bioreactor device to regulate the entire blood circulation.The purpose of this study is to prove the effectiveness of endothelial bioreactor device in porcine sepsis with MODS.MethodsWe picked up 10 white cross pigs and divided them into 2 groups randomly: sham group and endothelial bioreactor device（EBD） group.All the animals were anesthetized and mechanically ventilated.After the infusion of endotoxin(LPS,0.25 mg·kg^-1) to establish the sepsis model extracorporeal circulation began to run. Hemodynamics、blood routine、blood biochemistry and inflammatory parameters were examined at basement and at 1,3,6 h after the injection respectively.Hemodynamic parameters included mean arterial pressure（MAP）,and serum biochemical parameters included serum creatinine（SCr） and potassium（K）.Inflammation parameters included interleukin-1（IL-1） and 10（IL-10）,endothelin-1（ET-1）,nitric oxide synthase（NOs）,and protein C（PC）.The survival time of experimental animals was recorded at the last.ResultsAt 30 min after injection of LPS to 2 h,the drop of MAP of two groups was obvious,but MAP of EBD group then began to slowly increase,and could be maintained for a long time,and MAP of the sham group continued to decline.The differences between the two groups was significant（p＜0.01）.After the start of the experiment,kidney function of the two group began to change,but it changed less in EBD group than in sham group.Even at the end of experiment SCr of EBD group has not significantly increased,while it was significantly increased in sham group（p=0.001）.Changes in serum potassium was similar to SCr.At 6 h,serum potassium was significantly higher than that of EBD group,p=0.002.After the LPS infusion the concentration of IL-1 and IL-10 rised and arrived at the peak in two hours,and then began to decrease,and there was no difference between the two groups.The endothelin-1（ET-1） of EBD group decreased slowly first and began to rise after three hours,but nitric oxide synthase（NOs） began to increase first and decrease after two hours,while the changes of ET-1 and NOs in sham group was on the contrary.The survival time of EBD group and sham group was 5.5±0.3 and 6.7±0.4 d（p＜0.05）.ConclusionEndothelial bioreactor device can not only improve hemodynamics of porcine sepsis,but also maintain the function of key organs,which lead to the prolongation of survival time of experimental animals. PARTⅢRole of endothelial bioreactor device in regulation of inflammatory cytokines of acute lung injury induced by endotoxin in porcineBackgroundIn the occurrence and development of sepsis,the uncontrol of inflammatory cytokines leads to multiple organ dysfunction syndrome（MODS）,which thereby increases mortality.Among the injury organs,the lung usually happens the first and the easiest.In the pathogenesis of lung injury,the involvement of inflammatory cytokines plays a vital role,particularly including IL-1,IL-10 and TNF.These cytokines can promote the further injury of the lung tissue and cause the infiltration of neutrophil and the damage of lung epithelial cells,resulting in decreased function of alveolar capillary barrier and the increase of permeability,which culminates in acute respiratory distress syndrome（ARDS）.In the second part of this research we have been successfully confirmed the effectiveness of endothelial cell bioreactor（BAD） in the treatment of porcine sepsis,and this effectiveness maybe achieve through the adjustment of cytokines.While cytokines are important in acute lung injury（ALI） induced by endotoxin,the initial purpose of this paper is to investigate the role of endothelial bioreactor device in regulating the inflammatory cytokines of acute lung injury induced by endotoxin.MethodsWe picked up 10 white cross pigs and divided them into 2 groups randomly: sham group and endothelial bioreactor device（EBD） group.All the animals were anesthetized and mechanically ventilated.After the infusion of endotoxin(LPS,0.25 mg·kg^-1) to establish the sepsis model extracorporeal circulation began to run. Interleukin-1 and 10 were examined at basement and at 1,3,6 h after the injection respectively.At the same time lung tissue from animals was obtained for HE staining, and the pathology specialist calculated the Lung Injury Score.Western blot was used to detect the protein content of TNF-αand tissue factor（TF） in the lung tissue.ResultsAfter injection of LPS,the concentration of IL-1 and IL-10 rised and arrived at the peak in two hours,and then began to decrease,and there was no difference between the two groups,but the decline was less in EBD group than in sham group.Compared with the EBD group in the lung pathology,there were more respiratory epithelial proliferation（REP）,alveolar hemorrhage（AH） and vascular thrombosis（VT） in sham group.The lung injury score in sham group was much higher than in EBD group（8.2±1.0 vs 6.1±0.9,p=0.0447）.In the lung tissue,protein content of tissue TNF-αin the treatment group was lower than in sham group（p＜0.05）,but there was no significant difference between two group in the protein content of TF.ConclusionEndothelial bioreactor device can not only improve the hemodynamics of porcine sepsis,but also can reduce the expression of TNF-αin the lung tissue,which extenuates inflammation response in the lung tissue inflammation and postpones lung injury.更多还原