自体移植内皮祖细胞防治多器官功能障碍的研究
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摘要
创伤会导致机体的靶细胞严重损伤,同时由于感染所致的脓毒症及非感染所致的全身炎症反应综合征(systemic inflammatory response syndrome,SIRS)相互作用使得自体修复功能障碍,并进一步造成微血管损伤、微循环障碍,从而引起重要脏器功能障碍,这可能就是器官功能障碍(multiple organ dysfunction syndrome,MODS)的始发环节。而MODS是重症监护病房(intensive care unit,ICU)患者死亡的最常见原因。
近年来研究表明:骨髓中有一群能够在生理性或病理性因素的刺激下,动员到外周血并分化为成熟内皮细胞(Endothelium cell,EC)以促进血管新生,并可以分化为相应的组织细胞进行损伤修复的祖细胞,这些细胞被称为内皮祖细胞(Endothelialprogenitor cell,EPC)。EPC过去被认为是胚胎时期血管新生最主要的细胞,而越来越多的证据显示EPC也是出生后生理性及病理性血管形成最主要的细胞,并参与心、肝、肺、肾等单个器官障碍时的修复;同时迁徙到损伤部位的EPC还可以对不同的局部刺激(包括缺氧或缺血)作出生理反应,依次释放血管活性物质、生长因子以及参与免疫调节的细胞因子和趋化因子,参与创伤后的炎症反应。
目前研究认为EPC主要有2类:一类是来源于骨髓和刚被动员进入外周循环的早期EPC,它们都表达三种祖细胞分子标志:CD133(AC133)、CD34和血管内皮生长因子受体-2(VEGFR-2、KDR或Flk-1),而不表达VE-钙黏素(VE-cadherin)和血小板内皮细胞粘附分子-1(CD31);另一类是晚期EPC:即早期EPC释放入外周血循环或经体外培养后则逐渐失去CD133和CD34表达等祖细胞特性,并开始表达内皮系的特征性分子标志:CD31和KDR等,同时可吞噬乙酰化低密度脂蛋白(Ac-LDL)和荆豆凝集素(UEA-1)。CD133和KDR是造血干细胞和内皮细胞的重要标志,在胚胎早期血管也有表达,因而通常被用作为识别EPC的标志之一。
目前国内外对EPC的分离和体外培养方法尚未达成一致,本实验根据国内外文献的比较以及实验中的归纳总结,以密度梯度法分离小型猪骨髓中单个核细胞,并通过体外经VEGF等生长因子诱导培养后得到EPC;并对培养出的EPC进行细胞形态学;细胞表型;细胞增殖特征;体外血管形成功能及细胞吞噬Ac-LDL和UEA-1来进行鉴定。
本研究显示,EPC可以在机体发生MODS后向不同组织迁徙或归巢,发挥组织修复作用,并通过毛细血管的新生以改善创伤后缺血缺氧引起的脏器功能障碍,自体移植EPC可以降低MODS的发生率,并改善MODS的预后。为此,本文从EPC对自体修复的角度出发,探讨MODS发病机理及自体移植EPC预防和治疗MODS的理论依据。
本研究共分为四部分,首先通过内毒素+失血性休克制造出MODS的动物模型,并在MODS的各个阶段进行外周血中EPC的监测,同时在体外建立起EPC培养和鉴定体系,将体外培养增殖的自体EPC移植入MODS的动物,观察移植后MODS的发生率和重要脏器的功能改善情况,评价自体移植EPC防治MODS的效果,并对其作用机制进行初步探讨。
第一部分多器官功能障碍动物模型的建立
在本研究的第一部分我们首先成功地复制了双相迟发型的猪MODS模型,将为研究自体移植内皮祖细胞防治创伤后多器官功能障碍的作用提供了实验的基础。
将体重25～30 Kg健康雄性家猪随机分为2组:实验组(MODS)10只,施行失血性休克+内毒素血症复合因素;对照组(C)9只,施行假手术,予以股动静脉置管,不实施失血及内毒素注射。用自动分析仪检测WBC、GRAN、SALT、SAST、Cr、BUN、动脉血氧分压(PaO2),以上各指标采用自身对照,以判断器官功能,主要器官病理形态学检查(大体、光镜)。结果显示实验组WBC、GRAN、SALT、SAST、Cr、BUN均明显升高,动物死亡前显著高于正常值,PaO_2明显下降。病理学改变主要表现为衰竭器官呈以炎症为主的非特异性改变。实验组MODS发生率为90%,死亡率为80%,显著高于对照组。本实验采用二次打击,与临床实际相符,且MODS的发生率及死亡率均高,操作简单,容易复制,是一个较成功的动物模型。
第二部分外周循环中内皮祖细胞在MODS各个阶段的数量和功能变化及意义
本部分通过监测MODS各个阶段,外周循环中内皮祖细胞的数量和功能变化,旨在为自体移植内皮祖细胞防治MODS奠定理论的基础同时确定最佳的自体移植时间。
按照上述方法造模后,分别在正常状态下、手术后、失血后2小时、血液回输后2小时、输注内毒素后1小时、12小时、24小时、48小时、96小时取外周血。以密度梯度法分离出单个核细胞(PMC,peripheral mononuclear cells),对其进行CD133和CD34双重标记,CD133~+和KDR~+双标记阳性者被认为是外周血中的EPC,并通过流式细胞仪技术对EPC进行计数。同时取外周血中以密度梯度法分离出的PMC按照1×10~6/cm2的密度接种于培养皿内,培养96小时后,进行细胞增殖、贴壁、迁徙和血管形成功能的测定,比较不同时间点外周血中EPC的功能变化。结果显示:在MODS的形成过程中,外周循环中EPC较正常组外周循环中EPC数量先增加随后出现明显的下降,并且其增殖、黏附、迁移和血管形成功能较正常组明显减退,说明MODS的发生和发展过程中的炎症的不断加重对EPC数量及功能具有明显的影响作用,同时由于EPC数量及功能出现明显的下降,导致重要的脏器功能损伤。
第三部分小型猪骨髓内皮祖细胞的体外培养、鉴定和功能检测
本部分旨在建立起小型猪骨髓EPCs的标准化分离、培养、扩增和鉴定的方法,为自体移植内皮祖细胞防止MODS提供合理的技术平台。
用密度梯度离心法从猪骨髓中分离出BMMC,按照1×10~5/cm~2的密度接种于培养皿内,使用添加了细胞因子和胎牛血清的内皮祖细胞专用培养液进行诱导分化培养,在固定时间进行消化、传代和扩增。同时,观察培养27天时P6代EPC的生长情况,并对其进行细胞形态学特征、细胞的超微结构、免疫组化、流式细胞仪技术、乙酰化的低密度脂蛋白(Dil-Ac-LDL)和荆豆凝集素-1(FITC-UEA-1)的吞噬功能、体外血管生成功能鉴定,为后文的自体移植做准备。
结果显示:培养48小时后逐渐出现梭形贴壁细胞(attaching cells,AT cells),并出现成簇现象,培养第6天时的EPC,已经开始出现成集落的贴壁细胞。在电镜下观察细胞;细胞内可检测到典型的Weibel-Palade小体。超过85%体外培养的贴壁细胞都特异性地摄取了Dil-Ac-LDL和FITC-UEA-1。在免疫组化鉴定:CD133(+),CD34(+),CD31(++),KDR(++)。流式细胞仪技术鉴定: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%。体外血管生成功能提示:细胞在特殊的细胞培养环境中可以生成新生的血管。
第四部分自体移植内皮祖细胞防治创伤后多器官功能障碍的研究
本部分旨在探讨自体移植骨髓EPC对小型猪创伤后多器官功能障碍治疗的有效性和安全性的研究,同时比较不同数量EPC进行移植治疗的疗效差异。
预先抽取实验动物骨髓,按照前述的方法进行EPC的分离、培养和扩增。后按照前述方法造模,将达到MODS的动物随机分为三组,分别按照1×10~6个细胞/Kg体重(低剂量移植组、LT组,8只)和1×10~7个细胞/Kg体重(高剂量移植组、HT组,8只)进行自体EPC移植治疗,同时以未行任何干预的MODS(M组,10只)作为对照组。
结果显示:低剂量移植组(LT)实验动物MODS的发生率和死亡率分别为(75%;6/8、75%;6/8)明显高于高剂量移植组(HT)实验动物(50%;4/8、37.5%;3/8),但低于单纯MODS组(90%;9/10、80%;8/10)(P＜0.01);同时LT组实验动物的生存时间(78.47±44.12小时)也较HT组实验动物(156.18±72.87小时)明显缩短。LT组实验动物在MODS各个阶段的WBC、GRAN、SALT、SAST、Cr、BUN均高于HT组,但较单纯MODS组略有降低。提示自体移植内皮祖细胞可以有效的促进创伤后的修复,防止MODS的发生和发展,同时可改善MODS动物的预后以及延长生存时间。
结论:体内EPC的数量减少及功能障碍可能是MODS发生发展的重要原因之一,自体移植内皮祖细胞可以在机体发生MODS后向不同组织迁徙或归巢,发挥组织修复作用,并通过毛细血管的新生以改善创伤后缺血缺氧引起的脏器功能障碍,防止MODS的发生和发展,同时可改善MODS动物的预后以及延长生存时间。
Trauma can cause severe damage of the target cells.Infectious sepsis and noninfectious systemic inflammatory response syndrome(SIRS) can interact eachother which will cause the dysfunction of self-repairing.What is more,the injury of blood capillary and microcirculation disturbance caused by dysfunction of self-repairing can result in multiple organ dysfunctions, which is deemed to be the beginning of the multiple organ dysfunction syndromes(MODS). Nowadays MODS turn to be the most frequent cause of death in patients admitted to intensive care units.
Recent research shows that EPC is one kind of progenitor cells from bone marrow which can be mobilized by physio-stimulation and patho-stimulation to peripheral blood to differentiate into mature endothelial cells(EC) to encourage angiogenesis and differentiate to different kind of cells to process repairing.EPCs were originally thought to be present only during embryonic development.However,accumulating evidence in the past several years suggested that EPCs were the major progenitor cells to participate inphysio-angiogenesis and patho-angiogenesis in adult lives.EPCs could particate in reparing of dysfunctions of heart、renal、liver and pulmonary and the EPCs which metastasized to pars affecta could be reacted to release the vasoactive substance、growth factors、immunoregulatory cytokines and chemotatic factors.
Two different EPCs subpopulations have been described,denoted as early and late EPCs with distinct cell markers.Early EPCs come from bone marrow or be mobilized to peripheral blood shortly,they all express progenitor-specific markers,including CD133(AC133)、CD34 and KDR;but they don't express VE-cadherin and vWF.Late EPCs are the early cells which mobilized into peripheral blood or cultured in vitro;they can express the endothelial-specific markers,including CD31 and KDR instead of CD133 and CD34.The late EPC can take up acetylated LDL and UEA-1.CD133 and KDR which are deemed to be the key markers to identify the EPCs are the cell markers of haemopoietic stem cells and ECs and they can also express in embryo-vessels.
The method of isolation and culture in vitro of EPC had not come to an agreement in recently studies.The method in this research was based on the summary of the national and international studies.EPCs were isolated from bone marrow monocular cells(BMMC) by density gradient centrifugation,and were cultured in vitro by some specific growth factors such as VEGF and so on.Then EPCs were identified by cell morphology、phenotype、cell proliferation、function of angiopoiesis and taking up acetylated LDL and UEA-1.
Our research shows that EPC can metastasize to pars affecta for neovascularization of ischemic or injured tissue and amelioration of functional impairment caused by trauma.So autologous transplantation of EPC could step down the incidence of MODS and improve the prognosis of MODS.This research investigated the pathogenesis of MODS from the aspects of EPC repairing function,and the theory of autologous transplantation of EPC to prevent and treat MODS in porcine.
This study was divided into four parts.The first part of our study was to replicate a porcine model of MODS,and then we monitored the number and function of EPC in peripheral blood in the stages of MODS.We used the standard method of isolation、culture and identification of EPC to proliferate EPC in vitro,and then did autologous transplantation of EPC to prevent and treat MODS.
Part 1 Establishment a porcine model of MODS
The first part of our study was to replicate a porcine model of MODS which was characterized by the development of delayed two-phase process and it was the foundation of our investigation of autologous transplantation of EPC to prevent and treat MODS.Nineteen healthy male minipigs weighing 22～30Kg were randomly divided into two groups.One group was subjected to hemorrhagic shock plus endotoxiemia(group M,n=10).Another group was normal control only with anesthesia and sham operation(group C,n=9).Blood specimens were collected every 24 hours during the seven-day observation for the detection of serum GPT,GOT,Cr,BUN and arterial blood gas analysis,which were used to judge if MODS occured by compared with the initial value of itself.Histological changes of the main organs were observed under light microscope(LM).The mobidity and mortality of MODS in group M were 88.9%and 77.8%respectively,both much higher than group C.The two-hit model of MODS was a successful animal model which conform to clinical course, also with high mobidity and mortality.And the model was easy to duplicate.
Part 2 The change of number and function of EPC in peripheral blood in steps of MODS
In the second part,we monitored the number and function of EPC in peripheral blood in the various stages of MODS to supply the theory of autologous transplantation of EPC to prevent and treat MODS.
Ten microliters of whole blood which were got at different stages(normal、postoperation、2 hours after hemorrhagic shock、2 hours after blood retransfusion、1 hour、 12 hours、24 hours、48 hours、96 hours after endotoxin transfusion) treated by density gradient centrifugation to isolate the PMC;and then incubated the PMC for 30 min in the dark with PE-labeled monoclonal antibody KDR and the FITC-labeled monoclonal antibody CD 133. The cells which were double positive were deemed as EPC in peripheral blood.The number of double positive cells was counted by flow cytometry.The PMC were processed the function detection including:proliferation、adherence、metabasis and angiopoiesis after been culture for 96 hours.The number of EPC in peripheral blood at various MODS stages was reduced sharply and the functions of EPCs were also impaired than normal.
Part 3 Standardization of Isolation,Culture,Identification and Function of Endothelial Progenitor Cells from Porcine Bone Marrow
We investigate to get the standard method of isolation,culture and identification of endothelial progenitor cell from porcine bone marrow for EPC transplantation.
BMMCs were isolated by the method of density gradient centrifugation from bone marrow and were cultured by 1×10~6/cm~2 original density with specific culture solution for EPC.After 27 days of culture,the P6-EPC were identificated by taking up Dil-ac-LDL and FITC-UEA-1、flow cytometry testing、immunohistochemistry testing、ultrastructural organization testing and functoin of angiogenesis testing.
The attaching cells would appear after 48h culture,and the cells would be clustering after 6 days culture.The Weibel-Palade body would appear in EPCs.And more than 85%EPC could take up Dil-Ac-LDL and FITC-UEA-1.CD133(+),CD34(+),CD31(++),KDR (++) would appear in the EPC by immunohistochemistry testing and the positivity of CD133 would be 18.23±7.12%;the positivity of would be 47.71±14.85%;the positivity of CD31 would be 71.61±13.51%;he positivity of KDR would be:87.24±11.40%by flow cytometry testing.EPC could be positive by angiogenesis testing.
Part Four Autologous transplantation of endothelial progenitor cells to prevent and treat MODS
This section aimed to investigate the effect of autologous transplantation EPC from bone marrow for post-traumatic multiple organ dysfunction treatment,and compare effect of treatment with different dose of EPC transplantation.
Experimental animal bone marrow was taken in advance in accordance with the aforementioned method of EPC isolation,culture and amplification.The MODS animals were randomly divided into three groups,in accordance with the 1×10~6 cells/Kg body weight (low-dose transplant group,LT group,9),and 1×10~7 cells/Kg body weight(High-dose transplantation group,HT group,9) autologous transplantation in the treatment of EPC,and the group with no transplantation as the control group(M group,10).
The results showed that:the mobidity and mortality of the experimental animals of LT group transplantation(75%;6/8、75%;6/8) was significantly higher than those of HT transplantation group(50%;4/8、37.5%;3/8),but lower than those of MODS group(90%; 9/10、80%;8/10)(P＜0.01).And the survival time of experimental animal of LT group(78.47±44.12 hours) was significantly shorer than that of the HT group(156.18±72.87 hours).And the WBC,GRAN,SALT,SAST,Cr,BUN of LT group was much worse than HT group,but slightly better than the control group.The results would suggest that autologous endothelial progenitor cell transplantation could improve the post-traumatic rehabilitation and reduce the mobidity and mortality of MODS.
Conclusion:The reduction and dysfunction of EPC may be the key to the development of MODS,and autologous transplantation of endothelial progenitor cells in vivo can migrate to different organizations and repaire the lesions.Autologous transplantation of endothelial progenitor cells can improve the function of organs with post-traumatic ischemia and hypoxia. So it can step down the mobidity and mortality of MODS and prevent the development of the MODS.
近年来研究表明:骨髓中有一群能够在生理性或病理性因素的刺激下,动员到外周血并分化为成熟内皮细胞(Endothelium cell,EC)以促进血管新生,并可以分化为相应的组织细胞进行损伤修复的祖细胞,这些细胞被称为内皮祖细胞(Endothelialprogenitor cell,EPC)。EPC过去被认为是胚胎时期血管新生最主要的细胞,而越来越多的证据显示EPC也是出生后生理性及病理性血管形成最主要的细胞,并参与心、肝、肺、肾等单个器官障碍时的修复;同时迁徙到损伤部位的EPC还可以对不同的局部刺激(包括缺氧或缺血)作出生理反应,依次释放血管活性物质、生长因子以及参与免疫调节的细胞因子和趋化因子,参与创伤后的炎症反应。
目前研究认为EPC主要有2类:一类是来源于骨髓和刚被动员进入外周循环的早期EPC,它们都表达三种祖细胞分子标志:CD133(AC133)、CD34和血管内皮生长因子受体-2(VEGFR-2、KDR或Flk-1),而不表达VE-钙黏素(VE-cadherin)和血小板内皮细胞粘附分子-1(CD31);另一类是晚期EPC:即早期EPC释放入外周血循环或经体外培养后则逐渐失去CD133和CD34表达等祖细胞特性,并开始表达内皮系的特征性分子标志:CD31和KDR等,同时可吞噬乙酰化低密度脂蛋白(Ac-LDL)和荆豆凝集素(UEA-1)。CD133和KDR是造血干细胞和内皮细胞的重要标志,在胚胎早期血管也有表达,因而通常被用作为识别EPC的标志之一。
目前国内外对EPC的分离和体外培养方法尚未达成一致,本实验根据国内外文献的比较以及实验中的归纳总结,以密度梯度法分离小型猪骨髓中单个核细胞,并通过体外经VEGF等生长因子诱导培养后得到EPC;并对培养出的EPC进行细胞形态学;细胞表型;细胞增殖特征;体外血管形成功能及细胞吞噬Ac-LDL和UEA-1来进行鉴定。
本研究显示,EPC可以在机体发生MODS后向不同组织迁徙或归巢,发挥组织修复作用,并通过毛细血管的新生以改善创伤后缺血缺氧引起的脏器功能障碍,自体移植EPC可以降低MODS的发生率,并改善MODS的预后。为此,本文从EPC对自体修复的角度出发,探讨MODS发病机理及自体移植EPC预防和治疗MODS的理论依据。
本研究共分为四部分,首先通过内毒素+失血性休克制造出MODS的动物模型,并在MODS的各个阶段进行外周血中EPC的监测,同时在体外建立起EPC培养和鉴定体系,将体外培养增殖的自体EPC移植入MODS的动物,观察移植后MODS的发生率和重要脏器的功能改善情况,评价自体移植EPC防治MODS的效果,并对其作用机制进行初步探讨。
第一部分多器官功能障碍动物模型的建立
在本研究的第一部分我们首先成功地复制了双相迟发型的猪MODS模型,将为研究自体移植内皮祖细胞防治创伤后多器官功能障碍的作用提供了实验的基础。
将体重25～30 Kg健康雄性家猪随机分为2组:实验组(MODS)10只,施行失血性休克+内毒素血症复合因素;对照组(C)9只,施行假手术,予以股动静脉置管,不实施失血及内毒素注射。用自动分析仪检测WBC、GRAN、SALT、SAST、Cr、BUN、动脉血氧分压(PaO2),以上各指标采用自身对照,以判断器官功能,主要器官病理形态学检查(大体、光镜)。结果显示实验组WBC、GRAN、SALT、SAST、Cr、BUN均明显升高,动物死亡前显著高于正常值,PaO_2明显下降。病理学改变主要表现为衰竭器官呈以炎症为主的非特异性改变。实验组MODS发生率为90%,死亡率为80%,显著高于对照组。本实验采用二次打击,与临床实际相符,且MODS的发生率及死亡率均高,操作简单,容易复制,是一个较成功的动物模型。
第二部分外周循环中内皮祖细胞在MODS各个阶段的数量和功能变化及意义
本部分通过监测MODS各个阶段,外周循环中内皮祖细胞的数量和功能变化,旨在为自体移植内皮祖细胞防治MODS奠定理论的基础同时确定最佳的自体移植时间。
按照上述方法造模后,分别在正常状态下、手术后、失血后2小时、血液回输后2小时、输注内毒素后1小时、12小时、24小时、48小时、96小时取外周血。以密度梯度法分离出单个核细胞(PMC,peripheral mononuclear cells),对其进行CD133和CD34双重标记,CD133~+和KDR~+双标记阳性者被认为是外周血中的EPC,并通过流式细胞仪技术对EPC进行计数。同时取外周血中以密度梯度法分离出的PMC按照1×10~6/cm2的密度接种于培养皿内,培养96小时后,进行细胞增殖、贴壁、迁徙和血管形成功能的测定,比较不同时间点外周血中EPC的功能变化。结果显示:在MODS的形成过程中,外周循环中EPC较正常组外周循环中EPC数量先增加随后出现明显的下降,并且其增殖、黏附、迁移和血管形成功能较正常组明显减退,说明MODS的发生和发展过程中的炎症的不断加重对EPC数量及功能具有明显的影响作用,同时由于EPC数量及功能出现明显的下降,导致重要的脏器功能损伤。
第三部分小型猪骨髓内皮祖细胞的体外培养、鉴定和功能检测
本部分旨在建立起小型猪骨髓EPCs的标准化分离、培养、扩增和鉴定的方法,为自体移植内皮祖细胞防止MODS提供合理的技术平台。
用密度梯度离心法从猪骨髓中分离出BMMC,按照1×10~5/cm~2的密度接种于培养皿内,使用添加了细胞因子和胎牛血清的内皮祖细胞专用培养液进行诱导分化培养,在固定时间进行消化、传代和扩增。同时,观察培养27天时P6代EPC的生长情况,并对其进行细胞形态学特征、细胞的超微结构、免疫组化、流式细胞仪技术、乙酰化的低密度脂蛋白(Dil-Ac-LDL)和荆豆凝集素-1(FITC-UEA-1)的吞噬功能、体外血管生成功能鉴定,为后文的自体移植做准备。
结果显示:培养48小时后逐渐出现梭形贴壁细胞(attaching cells,AT cells),并出现成簇现象,培养第6天时的EPC,已经开始出现成集落的贴壁细胞。在电镜下观察细胞;细胞内可检测到典型的Weibel-Palade小体。超过85%体外培养的贴壁细胞都特异性地摄取了Dil-Ac-LDL和FITC-UEA-1。在免疫组化鉴定:CD133(+),CD34(+),CD31(++),KDR(++)。流式细胞仪技术鉴定: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%。体外血管生成功能提示:细胞在特殊的细胞培养环境中可以生成新生的血管。
第四部分自体移植内皮祖细胞防治创伤后多器官功能障碍的研究
本部分旨在探讨自体移植骨髓EPC对小型猪创伤后多器官功能障碍治疗的有效性和安全性的研究,同时比较不同数量EPC进行移植治疗的疗效差异。
预先抽取实验动物骨髓,按照前述的方法进行EPC的分离、培养和扩增。后按照前述方法造模,将达到MODS的动物随机分为三组,分别按照1×10~6个细胞/Kg体重(低剂量移植组、LT组,8只)和1×10~7个细胞/Kg体重(高剂量移植组、HT组,8只)进行自体EPC移植治疗,同时以未行任何干预的MODS(M组,10只)作为对照组。
结果显示:低剂量移植组(LT)实验动物MODS的发生率和死亡率分别为(75%;6/8、75%;6/8)明显高于高剂量移植组(HT)实验动物(50%;4/8、37.5%;3/8),但低于单纯MODS组(90%;9/10、80%;8/10)(P＜0.01);同时LT组实验动物的生存时间(78.47±44.12小时)也较HT组实验动物(156.18±72.87小时)明显缩短。LT组实验动物在MODS各个阶段的WBC、GRAN、SALT、SAST、Cr、BUN均高于HT组,但较单纯MODS组略有降低。提示自体移植内皮祖细胞可以有效的促进创伤后的修复,防止MODS的发生和发展,同时可改善MODS动物的预后以及延长生存时间。
结论:体内EPC的数量减少及功能障碍可能是MODS发生发展的重要原因之一,自体移植内皮祖细胞可以在机体发生MODS后向不同组织迁徙或归巢,发挥组织修复作用,并通过毛细血管的新生以改善创伤后缺血缺氧引起的脏器功能障碍,防止MODS的发生和发展,同时可改善MODS动物的预后以及延长生存时间。
Trauma can cause severe damage of the target cells.Infectious sepsis and noninfectious systemic inflammatory response syndrome(SIRS) can interact eachother which will cause the dysfunction of self-repairing.What is more,the injury of blood capillary and microcirculation disturbance caused by dysfunction of self-repairing can result in multiple organ dysfunctions, which is deemed to be the beginning of the multiple organ dysfunction syndromes(MODS). Nowadays MODS turn to be the most frequent cause of death in patients admitted to intensive care units.
Recent research shows that EPC is one kind of progenitor cells from bone marrow which can be mobilized by physio-stimulation and patho-stimulation to peripheral blood to differentiate into mature endothelial cells(EC) to encourage angiogenesis and differentiate to different kind of cells to process repairing.EPCs were originally thought to be present only during embryonic development.However,accumulating evidence in the past several years suggested that EPCs were the major progenitor cells to participate inphysio-angiogenesis and patho-angiogenesis in adult lives.EPCs could particate in reparing of dysfunctions of heart、renal、liver and pulmonary and the EPCs which metastasized to pars affecta could be reacted to release the vasoactive substance、growth factors、immunoregulatory cytokines and chemotatic factors.
Two different EPCs subpopulations have been described,denoted as early and late EPCs with distinct cell markers.Early EPCs come from bone marrow or be mobilized to peripheral blood shortly,they all express progenitor-specific markers,including CD133(AC133)、CD34 and KDR;but they don't express VE-cadherin and vWF.Late EPCs are the early cells which mobilized into peripheral blood or cultured in vitro;they can express the endothelial-specific markers,including CD31 and KDR instead of CD133 and CD34.The late EPC can take up acetylated LDL and UEA-1.CD133 and KDR which are deemed to be the key markers to identify the EPCs are the cell markers of haemopoietic stem cells and ECs and they can also express in embryo-vessels.
The method of isolation and culture in vitro of EPC had not come to an agreement in recently studies.The method in this research was based on the summary of the national and international studies.EPCs were isolated from bone marrow monocular cells(BMMC) by density gradient centrifugation,and were cultured in vitro by some specific growth factors such as VEGF and so on.Then EPCs were identified by cell morphology、phenotype、cell proliferation、function of angiopoiesis and taking up acetylated LDL and UEA-1.
Our research shows that EPC can metastasize to pars affecta for neovascularization of ischemic or injured tissue and amelioration of functional impairment caused by trauma.So autologous transplantation of EPC could step down the incidence of MODS and improve the prognosis of MODS.This research investigated the pathogenesis of MODS from the aspects of EPC repairing function,and the theory of autologous transplantation of EPC to prevent and treat MODS in porcine.
This study was divided into four parts.The first part of our study was to replicate a porcine model of MODS,and then we monitored the number and function of EPC in peripheral blood in the stages of MODS.We used the standard method of isolation、culture and identification of EPC to proliferate EPC in vitro,and then did autologous transplantation of EPC to prevent and treat MODS.
Part 1 Establishment a porcine model of MODS
The first part of our study was to replicate a porcine model of MODS which was characterized by the development of delayed two-phase process and it was the foundation of our investigation of autologous transplantation of EPC to prevent and treat MODS.Nineteen healthy male minipigs weighing 22～30Kg were randomly divided into two groups.One group was subjected to hemorrhagic shock plus endotoxiemia(group M,n=10).Another group was normal control only with anesthesia and sham operation(group C,n=9).Blood specimens were collected every 24 hours during the seven-day observation for the detection of serum GPT,GOT,Cr,BUN and arterial blood gas analysis,which were used to judge if MODS occured by compared with the initial value of itself.Histological changes of the main organs were observed under light microscope(LM).The mobidity and mortality of MODS in group M were 88.9%and 77.8%respectively,both much higher than group C.The two-hit model of MODS was a successful animal model which conform to clinical course, also with high mobidity and mortality.And the model was easy to duplicate.
Part 2 The change of number and function of EPC in peripheral blood in steps of MODS
In the second part,we monitored the number and function of EPC in peripheral blood in the various stages of MODS to supply the theory of autologous transplantation of EPC to prevent and treat MODS.
Ten microliters of whole blood which were got at different stages(normal、postoperation、2 hours after hemorrhagic shock、2 hours after blood retransfusion、1 hour、 12 hours、24 hours、48 hours、96 hours after endotoxin transfusion) treated by density gradient centrifugation to isolate the PMC;and then incubated the PMC for 30 min in the dark with PE-labeled monoclonal antibody KDR and the FITC-labeled monoclonal antibody CD 133. The cells which were double positive were deemed as EPC in peripheral blood.The number of double positive cells was counted by flow cytometry.The PMC were processed the function detection including:proliferation、adherence、metabasis and angiopoiesis after been culture for 96 hours.The number of EPC in peripheral blood at various MODS stages was reduced sharply and the functions of EPCs were also impaired than normal.
Part 3 Standardization of Isolation,Culture,Identification and Function of Endothelial Progenitor Cells from Porcine Bone Marrow
We investigate to get the standard method of isolation,culture and identification of endothelial progenitor cell from porcine bone marrow for EPC transplantation.
BMMCs were isolated by the method of density gradient centrifugation from bone marrow and were cultured by 1×10~6/cm~2 original density with specific culture solution for EPC.After 27 days of culture,the P6-EPC were identificated by taking up Dil-ac-LDL and FITC-UEA-1、flow cytometry testing、immunohistochemistry testing、ultrastructural organization testing and functoin of angiogenesis testing.
The attaching cells would appear after 48h culture,and the cells would be clustering after 6 days culture.The Weibel-Palade body would appear in EPCs.And more than 85%EPC could take up Dil-Ac-LDL and FITC-UEA-1.CD133(+),CD34(+),CD31(++),KDR (++) would appear in the EPC by immunohistochemistry testing and the positivity of CD133 would be 18.23±7.12%;the positivity of would be 47.71±14.85%;the positivity of CD31 would be 71.61±13.51%;he positivity of KDR would be:87.24±11.40%by flow cytometry testing.EPC could be positive by angiogenesis testing.
Part Four Autologous transplantation of endothelial progenitor cells to prevent and treat MODS
This section aimed to investigate the effect of autologous transplantation EPC from bone marrow for post-traumatic multiple organ dysfunction treatment,and compare effect of treatment with different dose of EPC transplantation.
Experimental animal bone marrow was taken in advance in accordance with the aforementioned method of EPC isolation,culture and amplification.The MODS animals were randomly divided into three groups,in accordance with the 1×10~6 cells/Kg body weight (low-dose transplant group,LT group,9),and 1×10~7 cells/Kg body weight(High-dose transplantation group,HT group,9) autologous transplantation in the treatment of EPC,and the group with no transplantation as the control group(M group,10).
The results showed that:the mobidity and mortality of the experimental animals of LT group transplantation(75%;6/8、75%;6/8) was significantly higher than those of HT transplantation group(50%;4/8、37.5%;3/8),but lower than those of MODS group(90%; 9/10、80%;8/10)(P＜0.01).And the survival time of experimental animal of LT group(78.47±44.12 hours) was significantly shorer than that of the HT group(156.18±72.87 hours).And the WBC,GRAN,SALT,SAST,Cr,BUN of LT group was much worse than HT group,but slightly better than the control group.The results would suggest that autologous endothelial progenitor cell transplantation could improve the post-traumatic rehabilitation and reduce the mobidity and mortality of MODS.
Conclusion:The reduction and dysfunction of EPC may be the key to the development of MODS,and autologous transplantation of endothelial progenitor cells in vivo can migrate to different organizations and repaire the lesions.Autologous transplantation of endothelial progenitor cells can improve the function of organs with post-traumatic ischemia and hypoxia. So it can step down the mobidity and mortality of MODS and prevent the development of the MODS.
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