【作者】 毛岸荣；

【导师】 方国恩；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 多器官功能障碍综合征(multiple organ dysfunction syndrome MODS)是临床危重患者死亡的主要原因之一。目前其发病机制尚不明确,临床治疗上缺乏有效的手段。1997年,Asahara等首先从外周血单个核细胞(PBMCs)里分离出一种被称为内皮祖细胞(EPC)的祖细胞亚群,这些细胞具有在体内外增殖、迁徙和分化为成熟内皮细胞的能力以来,干/祖细胞技术和理论得到了迅猛发展,多器官功能障碍综合征的发病机制研究和临床治疗也面临着新的契机。有研究表明,机体损伤后,血循环中具有多向分化潜能的祖细胞一方面可以在VEGF等生长因子作用下动员、增殖、迁徙入损伤器官,分化为相应的内皮细胞进行损伤修复;另一方面内皮细胞不仅是炎症反应的参与者,还是首先受损的靶细胞,并进而造成微血管损伤、微循环障碍,这可能是器官功能障碍的始发环节。大量的动物和临床实验都已经证明:当组织受到损伤时,尤其是缺血性损伤时,循环及组织中EPC动员和增殖能力增强,并可以在组织内分化为内皮细胞替换功能障碍的内皮细胞、修补裸露的血管内皮损伤区,并参与缺血或损伤组织内新血管生成,从而改善缺血器官的功能;而如果创伤后EPC的分化、迁徙功能发生严重障碍,则会导致损伤微循环严重损害而无法得到修复,甚至发生器官功能衰竭。近年来,人们已逐渐认识到MODS是机体对于多种严重损伤的一种过度的、全身性炎症反应的最终结果,以大量促炎因子(例如:TNF-α、IL-1β等)的过度释放或者抗炎因子(如IL-4、IL-10等)出现释放延迟为标志的过度炎症反应综合征或抗炎反应综合征,最终导致机体免疫失衡和MODS的发生,和各种免疫细胞一样,EPC也受到了细胞因子网络的调控。在MODS中,内毒素与缺血等可以使外周血单核细胞内的p38丝裂原活化蛋白激酶磷酸化,活化转录因子AP-1,进而引起TNF-α、IL-1β基因转录增强,TNFα、IL-1β产生增多,进而导致EPC下降,MODS加重。本实验拟在建立猪MODS动物模型和EPC体外培养和鉴定实验体系基础上,研究观察猪各主要脏器功能变化与外周血单核细胞p38MAPK磷酸化变化与TNF-α、IL-1βmRNA的表达变化和外周血血浆TNF-α、IL-1β浓度及EPC数量与功能的变化,以及在体外用不同浓度的TNF-α与IL-1β培养EPC,观察其数量与功能的变化与EPC内的p38MAPK的变化;并探讨在MODS中p38MAPK介导的信号传导通路对EPC的调控作用,从内皮祖细胞分化障碍的角度进一步研究创伤后MODS的发病机制。全文共分三部分:第一部分EPC的体外培养与生物学特性研究。方法:抽取猪骨髓20ml,用密度梯度离心法得到单核细胞,将单核细胞悬液按2×10~6/ml密度接种于直径10厘米的含有各种细胞因子的培养液的培养皿中培养,培养2天可出现梭形细胞,6天后梭形样细胞数明显增多,部分成鹅卵石状贴壁生长,进而形成细胞网或者管腔样结构。培养后的细胞传至第3代后,在免疫组化鉴定:CD133(+),CD34(+),CD31(++),KDR(++),FITC-UEA-I和Dil-acLDL双吞噬实验为阳性,血管生成实验为阳性,FCM检测贴壁细胞CD133的阳性率:18.23±7.12%;CD34的阳性率:47.71±14.85%;CD31的阳性率:71.61±13.51%;KDR的阳性率:87.24±11.40%,鉴定为EPC。在体外将TNF-α、IL-1β的标准品分别加入第3代EPC,观察其对EPC的数量、增殖、迁移、贴壁、血管新生功能的影响。结果:发现其数量与功能与TNF-α、IL-1β有浓度与时间依赖性下降,并且有显著的差异(P＜0.01),并且与p38MAPK的磷酸化密切相关,用p38MAPK的特异性抑制剂SB203580(1umol/L)能减轻EPC的数量与功能下降。结论:体外TNF-α、IL-1β通过p38MAPK的磷酸化使EPC数量与功能下降,SB203580能防止EPC数量与功能下降。第二部分利用改良Wiggers方法复制失血性休克模型,首次打击(失血性休克、复苏再灌注)和二次打击(内毒素)复合因素成功建立猪MODS模型。方法:20头猪随机分为:正常对照组(C组,n=10),失血性休克+复苏再灌注+内毒素组(M组,n=10);通过给猪放血致平均动脉压50±5mmHg,维持1.5-2h,然后回输60%所失血液和两倍平衡液复苏,12h后由静脉持续滴入内毒素(sigma,剂量0.5mg/Kg),24小时滴完。连续动态监测心、肺、肾、肝、胃肠等功能,七天后处死存活动物。用自动分析仪检测各时间点ALT、AST、Cr、BUN、CO、PaO2等指标,观察主要器官病理形态学变化。结果:M组外周血ALT、AST、Cr、BUN均明显升高,动物死亡前显著高于正常值(P＜0.01),CO,PaO2明显下降(P＜0.01);病理学改变主要以非特异炎症改变为主。M组各器官的衰竭率:肺80.0%(8例),胃肠道70.0%(7例),肝50.0%(5例),肾30.0%(3例),心功能障碍(2例)20.0%,MODS发生率达90.0%(9例),死亡率达80.0%(8例),显著高于对照组。结论:利用二次打击方法,可以成功复制出动物MODS模型,且模型MODS发生率高,死亡率高,临床典型的双相迟发MODS相似重复性好。第三部分MODS体内单核细胞内p38MAPK的磷酸化对TNF-α、IL-1β合成、分泌及外周血EPC的数量与功能的影响。方法:在体内Western-blot法检测外周血单核细胞p38MAPK磷酸化变化与Realtime-PCR法测定TNF-αmRNA、IL-1βmRNA的表达变化和ELISA法测定外周血血浆TNF-α与IL-1β浓度变化及FCM检测外周血EPC数量的变化。结果:MODS中外周血单核细胞p38MAPK的磷酸化明显增强(P＜0.01),TNF-α与IL-1β合成与分泌明显增加(P＜0.01)。外周血EPC数量与功能下降(P＜0.05);体外TNF-α与IL-1β通过EPC内的p38MAPK的磷酸化使EPC的数量与功能下降。结论:在MODS的发病机制中,外周血单核细胞p38MAPK的磷酸化使TNF-α与IL-1β等炎性因子的转录、合成增加,血浆TNF-α与IL-1β升高,再次通过EPC内p38MAPK的磷酸化使其数量与功能下降,MODS的炎症病理反应加重。全文结论:机体在失血再灌注、LPS等因素刺激下,引起外周血单核细胞内p38MAPK磷酸化增强,TNF-α、IL-1β等细胞因子的合成与分泌增加,引起EPC内的p38MAPK磷酸化增强,导致EPC数量与功能下降,各器官组织的微血管损伤修复能力减弱,使MODS加重。更多还原

【Abstract】 The multiple organ dysfunction syndrome(MODS) or multiple organ failuer(MOF) is the most common cause of death in the clinical dangerous population.At present the pathogenesis of MODS is not clear and there are not good thrapical methods in clinic.In 1997,Asahara separated progenitor cells called endothelial progenitor cells from the peripheral blood mononuclear cell first of all.The endothelial progenitor cells have the ability of proliferation,migration,differentiation and could turn into endothelial cells.With the development of clinical technologies of progenitor cells,the moment of MODS emerges in pathogenesis and clinical therapy.Some studies that indicate the progenitor cells have the ability of proliferation,migration,differentiation into damaged organs with VEGF in the peripheral blood and the differentiated endothelial cells can repaire damaged organs at the first place;The endothelial cells are not only the damaged target cells but also causes of blood capillary damage and dysfunction or the first ring-join of MODS.The massive animal and clinical experiments have already certificated that when tissues are damaged especially ischemia,the proliferation,migration,differentiation of EPC are reinforced in the peripheral blood and could turn into endothelial cells and replace the damaged endothelial cells in tissue,repaire denuded damaged endothelial cells in blood vessel,participate neovascularity in ischemia or damaged tissue and improve ischemia organs’ function.As the serious disturbance of proliferation,migration,differentiation of EPC after trauma,they could make the microcirculation unrepaired in the damaged part and MODS will take place.This study is based on animal mode of MODS of "two-hit" injury and EPC’s culture and identification in vitro,and all principal organs’ metergasis,p38MAPK’s phosphorylation,TNF-αmRNA and IL-1βmRNA’s expression in the peripheral blood mononuclear cell,TNF-αand IL-1β’s concentration and the quantity and function of the EPC with FCM are observed in the peripheral blood plasma in vivo;.and EPCs are cultured with TNF-αand IL-1β;the quantity and function of the EPC and p38MAPK’s phosphorylation in EPC are monitored in vitro and the EPC were mediated by the TNF-αand IL-1βby the p38 mitogen activated protein kinase and the pathogenesis from cell dysdifferentiation are discussed in multiple organ dysfunction syndrome.The study is divided into 3 parts.PartⅠThe culture in vitro and bionomics of EPC.Methods:drew-off 20ml bone marrow in porcines and got mononuclear cells with density gradient centrifugation.The mononuclear cell suspension was inoculated in plate(diameter=10cm) with the density of 2×10~6/ml.Spindle-shaped cell populations after 2 days and adherent cobblestone-appearance cells and cytoreticulum or lumens’ structures after 6 days were obsvered.The inoculated cells were propagated to P3.Theywere identificated with monoclonal antibody of CD133,CD34,CD31 and VEGFR-2 and they were positive.They were positive in double swallows with FITC-UEA-I and Dil-acLDL and they were positive in angiogenesis experiment.The adherent cells expressed VEGFR-2(87.24±11.40%), CD34(47.71±14.85%),CD133(18.23±7.12%) and CD31(71.61±13.51%).So they were the endothelial progenitor cells.After the endothelial progenitor cells of P3 inoculated with TNF-αand IL-1βstandard cytokine in vitro,the quantity,proliferation, migration,adherence and angiogenesis of EPC were observed.Result:the quantity and function of the EPC were dependent on TNF-αand IL-1βstandard cytokine concentration(P＜0.01) and they were correlated with p38MAPK’s phosphorylation.The SB203580(1umol/L) that was p38MAPK’s specific inhibitor could prevent the descent of the quantity and function.Conclusion:In vitro,TNF-αand IL-1βmake the quantity and function of the EPC descend through p38MAPK’s phosphorylation,The SB203580 could prevent them.PartⅡThe porcine model of MODS with Wiggers’ method that was characterized by the development of delayed two-phase process was firstly replicated.Method:Pigs were randomized into two groups:group C(n=10) as a control group,receiving anesthesia and sham operation only,group M(n=10) as a "Two-hit" injury model group,induced by hemorrhagic shock(50±5mmHg for 1.5-2hours) and resuscitation followed byⅳadministration of lipopolysaccharide(sigma,0.5mg/kg) within 24 hours.The function of hearts,lungs,kidneys,livers and gastrointestinal tracts were monitored successively and dynamically.The pigs were executed after 7 days and the principal organs’ pathomorphology were observed.Blood specimens were collected every 24 hours during the seven-day observation for the detection of serum ALT、AST、Cr、BUN、CO and arterial blood gas analysis.Result:ALT、AST、Cr、BUN obviously ascended particularly before death(P＜0.01);CO,PaO2 obviously decreased particularly before death(P＜0.01). The idio-inflammation was the principal pathematology.Compared with control group,the significant difference in incidence of dysfunction of lung(80.0%),gastrointestinal tract (70.0%),liver(50.0%),kidney(30.0%),heart dysfunction(20.0%) were observed.The incidence of MODS and mortality in group M was 90.0%and 80.0%respectively,was higher significantly compared with group C.This "Two-hit" porcine model was characterized by the pathogenesis and manifestation analogous to clinical situation and fit for futher study.PartⅢThe p38MAPK’s phosphorylation and TNF-αmRNA and IL-1βmRNA’s transcription of the peripheral blood mononuclear cell and TNF-αand IL-1βand the quantity and function of EPC of the peripheral blood were observed in MODS.Method: In vivo,p38MAPK’s phosphorylation with Western-blot,TNF-αmRNA and IL-1βmRNA with RT-PCR were monitored in the peripheral blood mononuclear cell,TNF-αand IL-1βwith ELISA and EPC with FCM were monitored in the peripheral blood plasma.Result:In vivo p38MAPK’s phosphorylation of mononuclear cell was much more(P＜0.01),the synthesis and secretion of TNF-αand IL-1βrised in the peripheral blood mononuclear cell(P＜0.01).The quantity and function of EPC conspicuously decreased in the peripheral blood(P＜0.05).In vitro,the TNF-αand IL-1βcan make the quantity and function of the EPC descend through the p38MAPK’s phosphorylation.Conclusions:phosphorylation is important to pathogenesis in MODS,the p38MAPK’s phosphorylation makes TNF-αmRNA and IL-1βmRNA’s transcription TNF-αand IL-1βof the peripheral blood mononuclear cell increased,The increased TNF-αand IL-1βof the peripheral blood plasma could make the quantity and function of EPC descend through p38MAPK’s phosphorylation.So the inflammatory reation of MODS aggravated.Conclusion The p38MAPK’s phosphorylation is important to pathogenesis in MODS, the p38MAPK’s phosphorylation makes TNF-αmRNA and IL-1βmRNA’s transcription of TNF-αand IL-1βof the peripheral blood mononuclear cell increase,The increased TNF-αand IL-1βof the peripheral blood plasma can make the quantity and function of EPC descend through p38MAPK’s phosphorylation.更多还原