慢病毒介导hVEGF165-GFP基因转染内皮祖细胞移植治疗MODS后器官微血管密度变化的研究
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摘要
多器官功能障碍综合征(multiple organ dysfunction syndrome MODS)是临床重症患者死亡的主要原因之一。其发病机制尚未明确,最新研究认为全身炎症反应综合征(systemic inflammatory response syndrome, SIRS)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MODS,同时这可能就是MODS的始发环节。
内皮祖细胞(Endothelial progenitor cell, EPC)是成熟血管内皮细胞的前体细胞,属于干细胞群体,既可不断增殖又能定向分化为成熟的内皮细胞。EPC不仅参与人胚胎血管生成,同时也参与出生后血管新生和血管内皮损伤后的修复过程,是目前研究损伤血管修复的热点之一。
大量的动物和临床实验都已经证明：当组织受到一定程度的损伤时,循环及组织中EPC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；然而,当机体受到过度的损伤刺激时,EPC的分化、迁徙功能也会发生严重障碍,则会导致损伤部位微循环严重损害而无法得到修复,从而发生器官功能障碍。
研究显示,VEGF基因修饰的EPC移植治疗创伤后缺血性疾病,将可能最大程度的发挥两者促血管再生作用,移植VEGF基因修饰的EPC可以显著降低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,探讨携带VEGF-绿荧光蛋白(Green Fluorescent Portein, GFP)双表达基因慢病毒转染EPC后的细胞特性及移植慢病毒介导hVEGF165-GFP基因转染的内皮祖细胞预防和治疗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的家兔动物模型,同时在体外建立起EPC培养和鉴定体系,由慢病毒(LV)转染VEGF-GFP双表达基因的EPC,将体外培养增殖转染EPC移植入MODS的动物,观察移植后MODS的发生率和重要脏器的微血管密度改变情况,评价移植转染VEGF-GFP双表达基因的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家兔MODS模型,为创伤后多器官功能障碍的相关研究提供了实验基础。
将体重2.52±0.27Kg健康雄性家兔随机分为2组：实验组(MODS)12只,施行失血性休克+内毒素血症复合因素；对照组(C)12只,施行假手术,予以颈动静脉置管,不实施失血及内毒素注射。监测各重要器官功能变化,并行主要器官病理形态学检查(大体、光镜)。结果显示各重要器官功能下降或衰竭。病理学改变主要表现为炎症为主的非特异性改变。实验组MODS发生率为83.3%,死亡率为75.0%,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。第二部分携带VEGF-GFP双表达基因慢病毒载体的建立及体外转染内皮祖细胞
本部分通过建立可稳定携带VEGF+GFP的双表达基因的慢病毒载体,并转染体外培养的内皮祖细胞,为下一步的回输治疗提供充足的细胞源。
hVEGF165的PCR引物并扩增,经酶切、连接转化至含GFP的慢病毒表达载体：pWPXL-MOD后挑取阳性克隆,利用限制性内切酶BamH I、Mlu I双酶切后经电泳鉴定并测序。含VDGF-GFP的表达载体pWPXL-MOD与慢病毒包装质粒:pRsv-REV, pMDlg-pRRE,及包膜质粒pMD2G共同组成四质粒系统,通过磷酸钙法共转染293T细胞后获得慢病毒颗粒,经浓缩后可获得高滴度的LV-VEGF-GFP.在MOI值=50的条件下与EPC共培养以感染EPC。对LV/hVEGF165-GFP-EPC进行增殖功能进行检测。结果表明,目的基因插入慢病毒过表达载体pWPXL-MOD后经PCR扩增,证实载体片段与目的片段均可表达,并且基因测序报告显示：重组质粒中的插入片段序列与目的基因VEGF的CDS序列一致,成功构建并包装了慢病毒载体,并证实EPC经LV/hVEGF165-GFP转染后其增殖功能较未转染EPC提高。
第三部分慢病毒介导hVEGF165-GFP基因转染内皮祖细胞移植治疗MODS后器官微血管密度变化及意义
本部分旨在探讨移植慢病毒介导hVEGF165-GFP基因转染的内皮祖细胞对家兔创伤后多器官功能障碍治疗的有效性和安全性的研究,同时比较单纯移植EPC治疗MODS的疗效差异。
构建动物模型,将达到MODS的动物随机分为三组,单纯EPC移植组(ET)12只和转染内皮祖细胞移植组(VT)12只,在固定时间点,分别以1×107个细胞／Kg(体重)的剂量进行移植治疗。同时以未行任何干预的MODS(M组,12只)作为对照组。
结果显示：慢病毒介导hVEGF165-GFP基因转染内皮祖细胞移植组(VT)的MODS发生率和动物死亡率均低于单纯移植EPC组(ET)和对照组,各重要器官的免疫组化检测显示微血管密度明显高于单纯移植EPC组(ET)和对照组,提示移植VEGF修饰的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。
Multiple organ dysfunction syndrome (MODS) is one of the most common causes of death in the clinical treatment for severe cases. The newest research shows that, event it is unclear for its true mechanism of pathogenesis, systemic inflammatory response syndrome (SIRS) would make the auto-repair of body disorder, and induce the capillary vascular injury and microcirculation disturbance. This may be not only the cause of MODS but also the first step of MODS.
Endothelial progenitor cell (EPC) is a kind of stem cell that is the precursor cell of the matured endothelial cells. These cells both could unceasingly proliferate themselves and induced differentiate to matured endothelial cells. EPC would not only take part in the embryonic angiogenesis but also play a great role in the postnatal vasculogenesis and the repair process for the injured vascular endothelium. It is one of hotspots in the research to repair injured vascular endothelium.
A lot of animal and clinical experiments have showed that, when the body gets hurt, EPC could differentiate into matured endothelial cell to take the place of the handicapped endothelial cells and repair the area of injured vascular endothelium. Further more, it also takes part in the process of neoangiogenesis for the ischemic or injured area and then improves the ischemic condition of organs. However, if the body was excessively injured, the differentiation、migration and proliferation of EPC would turn to disorder severely, and induce the local microcirculation too badly hurt to repair that result in MODS.
Our research shew that transplanting EPC containing VEGF to treat the ischemic diseases can improve the regeneration of blood vessels. The incidence rate of MODS would significantly decrease by transplanting these kinds of EPC and the prognosis of MODS would get better. For these reasons, we used the lentiviral vector carrying VEGF-GFP to transfect EPC and investigate the cell characteristics of these kinds of EPC. In a word, our research would pave a way for the prevention and treatment of MODS.
This study was divided into three parts. The first part was to replicate a rabbit model of MODS.The second part was to transfect the VEGF-GFP gene into EPC by lentiviral vector. And then the last part was to transplant the transfected EPCs into the MODS animal model to investigate the incidence rate of MODS and the changing of microvessel density in the important organs. Make an assessment for the effect of treatment and putative mechanism of MODS.
Part 1 Establishment of a rabbit model of MODS
In the first part, we replicated a rabbit model of MODS which was characterized by the development of delayed two-phase process and it was the foundation of our investigation to prevent and treat MODS.
24 healthy male rabbits weighing2.52±0.27Kg were randomly divided into two groups. One group of rabbits were subjected to hemorrhagic shock plus endotoxiemia (group M, n=12). Another group of rabbits were normal control only with anesthesia and sham operation (group C, n=12). The change of main organs'function was observed carefully by the monitor and the pathological changes of the main organs were judged by light microscope (LM). The mobidity and mortality of MODS in group M were 83.3% and 75.0% respectively, both much higher than group C. The two-hit model of MODS was a successful animal model conforms to clinical course, also with high mobidity and mortality. And the model was easy to duplicate.
Part 2:Construction of a lentiviral vector that can express both VEGF and GFP and Transfection of EPCs in vitro
In this part we constructed the lentivital vectors that could stably express both VEGF and GFP, and transfected EPC high effectively. And then made the identification of cell function for these acquired cells, it would be the foundation for the treatment of transplantation.
The first step was design and synthesis of the VEGF PCR primers and the amplification. Combination and transformation was made after cutting these PCR products, they were inserted into the lentiviral vector--pWPXL-MODcontaining GFP gene. When the positive clones formation were ready, picked up and cut them with the restriction enzymes of BamH I、Mlu I, at last the sequence was identified by electrophoresis checking
We use the expression plasmids--pWPXL-MOD containing VEGF-GFP gene; the package plasmids-- pRsv-REV and pMDlg-pRRE; the envelope plasmids--pMD2G to construct a four plasmids system of lentivrial vectors from the calcium phosphate precipitation. We could get high concentrated solution of LV-VEGF-GFP by concentrating these vectors. Co-culture these lentivirus and EPC in the MOI for 50, the EPC could be transfected very well. Identify the function of proliferation. The results shew that when the objective gene of VEGF-GEP was inserted into the over-expression carrier of pWPXL-MOD, both the carrier fragment and objective fragment could express. Further more the report of gene sequencing shew that the sequence of insertion element in the recombinant plasmids is accordant with the sequence of objective gene of VEGF. In a word, we constructed and packaged the lentiviral vetctors successfully, and had proved that the abilities, comparing to normal EPCs, of the post-transfected EPCs in proliferation were significantly enhanced.
Part 3 Study of The Change of Organ's Microvessel Density in The Treatment of MODS after Transplantation of Endothelial Progenitor Cells Carrying the Gene of hVEGF165-GFP Transfected with Lentiviral Vector
This section aimed to investigate the effect and safety of transplantation EPC containing VEGF-GFP gene for post-traumatic multiple organ dysfunction treatment, and then to compare the effect of treatment with different teams of EPC transplantation.
MODS animals' model were constructed and randomly divided into three groups,①transplantation with EPCs (ET group,12).②transplantation with LV-VEGF-GFP-EPCs (VT group,12). And the amount of transplantated EPCs was 1×107 cells/Kg.③the group that there is no EPC transplantation would be control group (M group,12).
The results shew that the mobidity and mortality of the experimental animals of VT was significantly lower than those of ET and M groups (P<0.05). The microvessel density of vital organs was significantly higher than ET group and M group.The results would suggest that transplantation of EPC containing VEGF-GFP gene could improve the post-traumatic rehabilitation, extend the survivor time and reduce the mobidity and mortality of MODS.
内皮祖细胞(Endothelial progenitor cell, EPC)是成熟血管内皮细胞的前体细胞,属于干细胞群体,既可不断增殖又能定向分化为成熟的内皮细胞。EPC不仅参与人胚胎血管生成,同时也参与出生后血管新生和血管内皮损伤后的修复过程,是目前研究损伤血管修复的热点之一。
大量的动物和临床实验都已经证明：当组织受到一定程度的损伤时,循环及组织中EPC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；然而,当机体受到过度的损伤刺激时,EPC的分化、迁徙功能也会发生严重障碍,则会导致损伤部位微循环严重损害而无法得到修复,从而发生器官功能障碍。
研究显示,VEGF基因修饰的EPC移植治疗创伤后缺血性疾病,将可能最大程度的发挥两者促血管再生作用,移植VEGF基因修饰的EPC可以显著降低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,探讨携带VEGF-绿荧光蛋白(Green Fluorescent Portein, GFP)双表达基因慢病毒转染EPC后的细胞特性及移植慢病毒介导hVEGF165-GFP基因转染的内皮祖细胞预防和治疗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的家兔动物模型,同时在体外建立起EPC培养和鉴定体系,由慢病毒(LV)转染VEGF-GFP双表达基因的EPC,将体外培养增殖转染EPC移植入MODS的动物,观察移植后MODS的发生率和重要脏器的微血管密度改变情况,评价移植转染VEGF-GFP双表达基因的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家兔MODS模型,为创伤后多器官功能障碍的相关研究提供了实验基础。
将体重2.52±0.27Kg健康雄性家兔随机分为2组：实验组(MODS)12只,施行失血性休克+内毒素血症复合因素；对照组(C)12只,施行假手术,予以颈动静脉置管,不实施失血及内毒素注射。监测各重要器官功能变化,并行主要器官病理形态学检查(大体、光镜)。结果显示各重要器官功能下降或衰竭。病理学改变主要表现为炎症为主的非特异性改变。实验组MODS发生率为83.3%,死亡率为75.0%,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。第二部分携带VEGF-GFP双表达基因慢病毒载体的建立及体外转染内皮祖细胞
本部分通过建立可稳定携带VEGF+GFP的双表达基因的慢病毒载体,并转染体外培养的内皮祖细胞,为下一步的回输治疗提供充足的细胞源。
hVEGF165的PCR引物并扩增,经酶切、连接转化至含GFP的慢病毒表达载体：pWPXL-MOD后挑取阳性克隆,利用限制性内切酶BamH I、Mlu I双酶切后经电泳鉴定并测序。含VDGF-GFP的表达载体pWPXL-MOD与慢病毒包装质粒:pRsv-REV, pMDlg-pRRE,及包膜质粒pMD2G共同组成四质粒系统,通过磷酸钙法共转染293T细胞后获得慢病毒颗粒,经浓缩后可获得高滴度的LV-VEGF-GFP.在MOI值=50的条件下与EPC共培养以感染EPC。对LV/hVEGF165-GFP-EPC进行增殖功能进行检测。结果表明,目的基因插入慢病毒过表达载体pWPXL-MOD后经PCR扩增,证实载体片段与目的片段均可表达,并且基因测序报告显示：重组质粒中的插入片段序列与目的基因VEGF的CDS序列一致,成功构建并包装了慢病毒载体,并证实EPC经LV/hVEGF165-GFP转染后其增殖功能较未转染EPC提高。
第三部分慢病毒介导hVEGF165-GFP基因转染内皮祖细胞移植治疗MODS后器官微血管密度变化及意义
本部分旨在探讨移植慢病毒介导hVEGF165-GFP基因转染的内皮祖细胞对家兔创伤后多器官功能障碍治疗的有效性和安全性的研究,同时比较单纯移植EPC治疗MODS的疗效差异。
构建动物模型,将达到MODS的动物随机分为三组,单纯EPC移植组(ET)12只和转染内皮祖细胞移植组(VT)12只,在固定时间点,分别以1×107个细胞／Kg(体重)的剂量进行移植治疗。同时以未行任何干预的MODS(M组,12只)作为对照组。
结果显示：慢病毒介导hVEGF165-GFP基因转染内皮祖细胞移植组(VT)的MODS发生率和动物死亡率均低于单纯移植EPC组(ET)和对照组,各重要器官的免疫组化检测显示微血管密度明显高于单纯移植EPC组(ET)和对照组,提示移植VEGF修饰的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。
Multiple organ dysfunction syndrome (MODS) is one of the most common causes of death in the clinical treatment for severe cases. The newest research shows that, event it is unclear for its true mechanism of pathogenesis, systemic inflammatory response syndrome (SIRS) would make the auto-repair of body disorder, and induce the capillary vascular injury and microcirculation disturbance. This may be not only the cause of MODS but also the first step of MODS.
Endothelial progenitor cell (EPC) is a kind of stem cell that is the precursor cell of the matured endothelial cells. These cells both could unceasingly proliferate themselves and induced differentiate to matured endothelial cells. EPC would not only take part in the embryonic angiogenesis but also play a great role in the postnatal vasculogenesis and the repair process for the injured vascular endothelium. It is one of hotspots in the research to repair injured vascular endothelium.
A lot of animal and clinical experiments have showed that, when the body gets hurt, EPC could differentiate into matured endothelial cell to take the place of the handicapped endothelial cells and repair the area of injured vascular endothelium. Further more, it also takes part in the process of neoangiogenesis for the ischemic or injured area and then improves the ischemic condition of organs. However, if the body was excessively injured, the differentiation、migration and proliferation of EPC would turn to disorder severely, and induce the local microcirculation too badly hurt to repair that result in MODS.
Our research shew that transplanting EPC containing VEGF to treat the ischemic diseases can improve the regeneration of blood vessels. The incidence rate of MODS would significantly decrease by transplanting these kinds of EPC and the prognosis of MODS would get better. For these reasons, we used the lentiviral vector carrying VEGF-GFP to transfect EPC and investigate the cell characteristics of these kinds of EPC. In a word, our research would pave a way for the prevention and treatment of MODS.
This study was divided into three parts. The first part was to replicate a rabbit model of MODS.The second part was to transfect the VEGF-GFP gene into EPC by lentiviral vector. And then the last part was to transplant the transfected EPCs into the MODS animal model to investigate the incidence rate of MODS and the changing of microvessel density in the important organs. Make an assessment for the effect of treatment and putative mechanism of MODS.
Part 1 Establishment of a rabbit model of MODS
In the first part, we replicated a rabbit model of MODS which was characterized by the development of delayed two-phase process and it was the foundation of our investigation to prevent and treat MODS.
24 healthy male rabbits weighing2.52±0.27Kg were randomly divided into two groups. One group of rabbits were subjected to hemorrhagic shock plus endotoxiemia (group M, n=12). Another group of rabbits were normal control only with anesthesia and sham operation (group C, n=12). The change of main organs'function was observed carefully by the monitor and the pathological changes of the main organs were judged by light microscope (LM). The mobidity and mortality of MODS in group M were 83.3% and 75.0% respectively, both much higher than group C. The two-hit model of MODS was a successful animal model conforms to clinical course, also with high mobidity and mortality. And the model was easy to duplicate.
Part 2:Construction of a lentiviral vector that can express both VEGF and GFP and Transfection of EPCs in vitro
In this part we constructed the lentivital vectors that could stably express both VEGF and GFP, and transfected EPC high effectively. And then made the identification of cell function for these acquired cells, it would be the foundation for the treatment of transplantation.
The first step was design and synthesis of the VEGF PCR primers and the amplification. Combination and transformation was made after cutting these PCR products, they were inserted into the lentiviral vector--pWPXL-MODcontaining GFP gene. When the positive clones formation were ready, picked up and cut them with the restriction enzymes of BamH I、Mlu I, at last the sequence was identified by electrophoresis checking
We use the expression plasmids--pWPXL-MOD containing VEGF-GFP gene; the package plasmids-- pRsv-REV and pMDlg-pRRE; the envelope plasmids--pMD2G to construct a four plasmids system of lentivrial vectors from the calcium phosphate precipitation. We could get high concentrated solution of LV-VEGF-GFP by concentrating these vectors. Co-culture these lentivirus and EPC in the MOI for 50, the EPC could be transfected very well. Identify the function of proliferation. The results shew that when the objective gene of VEGF-GEP was inserted into the over-expression carrier of pWPXL-MOD, both the carrier fragment and objective fragment could express. Further more the report of gene sequencing shew that the sequence of insertion element in the recombinant plasmids is accordant with the sequence of objective gene of VEGF. In a word, we constructed and packaged the lentiviral vetctors successfully, and had proved that the abilities, comparing to normal EPCs, of the post-transfected EPCs in proliferation were significantly enhanced.
Part 3 Study of The Change of Organ's Microvessel Density in The Treatment of MODS after Transplantation of Endothelial Progenitor Cells Carrying the Gene of hVEGF165-GFP Transfected with Lentiviral Vector
This section aimed to investigate the effect and safety of transplantation EPC containing VEGF-GFP gene for post-traumatic multiple organ dysfunction treatment, and then to compare the effect of treatment with different teams of EPC transplantation.
MODS animals' model were constructed and randomly divided into three groups,①transplantation with EPCs (ET group,12).②transplantation with LV-VEGF-GFP-EPCs (VT group,12). And the amount of transplantated EPCs was 1×107 cells/Kg.③the group that there is no EPC transplantation would be control group (M group,12).
The results shew that the mobidity and mortality of the experimental animals of VT was significantly lower than those of ET and M groups (P<0.05). The microvessel density of vital organs was significantly higher than ET group and M group.The results would suggest that transplantation of EPC containing VEGF-GFP gene could improve the post-traumatic rehabilitation, extend the survivor time and reduce the mobidity and mortality of MODS.
引文
1. Okajima K. Multiple organ failure associated with severe infection--the molecular mechanism (s) and new therapeutic strategies. Nippon Rinsho 2007 Mar 28, 65:619-626.
2. Hennessy M, Korbling Z. Circulating stem cells and tissue repair. Panminerva Med 2004,46:1-11
3. Smadja DM, Bieche I, Helley D, et al. Increased VEGFR2 expression during human late endothelial progenitor cells expansion enhances in vitro angiogenesis with up-regulation of integrin alpha (6). J Cell Mol Med 2007, Sep-Oct,11:1149-61.
4. de Nigris F, Balestrieri ML, Williams-Ignarro S, et al. Therapeutic effects of autologous bone marrow cells and metabolic intervention in the ischemic hindlimb of spontaneously hypertensive rats involve reduced cell senescence and CXCR4/Akt/eNOS pathways. J Cardiovasc Pharmacol 2007 Oct,50:424-33.
5. Deiteh EA. Spontaneous lymphocyte activity:an important but neglected component of the immunologic profile of the thermally injured patient [J]. Surgery 1985,98:587.
6. Murphey ED, Herndon DN, Sherwood ER. Gamma interferon does not enhance clearance of Pseudomonas aeruginosa but does amplify a proinflammatory response in a murine model of postseptic immunosuppression. [J] Infect Immun 2004,72: 6892-6901
7. Hu S, Sheng ZY, Li JY, et al. A small dose of intraperitoneal injection of zymosan induces systemic inflammatory response and multiple organ dysfunction following gut ischemia-reperfusion injury. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue.2003 Jan, 15:11-14.
8. Botha AJ, Moore FA, Guleserian KJ et al.Early neutophil sequestration after injury:A pathogenic mechanism for multiple organ failure.J Trauma 1995,39:411-417
9.胡森,盛志勇,周宝桐.创伤后多器官衰竭发病过程的“双相预激”学说.解放军医学杂志,1995,20：325.
10. Peitzman AB, Ubekwu AO, Juan Ochoa. Bacterial translocation in trauma patients. [J] Trauma.1991,31:1083.
11.中国医学科学院.多器官功能衰竭综合征的诊断和发病机理探讨.中华医学杂志,1988,68：226-232.
12. BurdonD, Tiedje T, Pfeffer K, et al. The role of tumor necrosis factor in the development of multiple organ failure in a murine model. Crit Care Med.2000, 28:1962-1967.
13. Thomas NJ,Carcillo JA,Herzer WA, et al. Chronic type IV Phosphodiesterase inhibition protects glomer μ lar filtration rate and renal and mesenteric blood flow in a zymosan-induced model of multiple organ dysfunction syndrome treated with norepinephrine. J Pharmacol Exp Ther.2001,296:168-174.
14. Taniguchi T, Yamamoto K, Ohmoto N, et al. Effects of Propofol on hemodynamic and inflammatory responses to endotoxemia in rats. Crit Care Med.2000,28:1101-1106.
15. Cirioni O, Giacometti A, Ghiselli R, et al. Single-dose intraperitoneal magainins improve survival in a gram-negative-pathogen septic shock rat model. Antimicrob Agents Chemother.2002,46:101-104.
16. Chaudry I H. Rat and mouse of hypovolemic-traumatic shock. In:Schlag G, Redl H(ed):pathophysiology of shock, sepsis and organ failure. Germany:Springer-Verlag Berlin Heidelberg,1993,371.
1. Luttun A, Carmeliet G, Carmeliet P. Vascular progenitors:from biology to treatment. Trends Cardiovasc Med 2002,12:88-96.
2.陈建国,罗兴才,梁小卫.内皮祖细胞在血管新生中的作用.中国误诊学杂志,2006,21：4127-4128.
3. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003,9:669-676.
4. Sinn PL, Sauter SL, Mccrayjrpb. Gene therapy progress and prospects:development and improvement of lentiviral and retroviral vectors-design, biosafetyandproduction. Gene Therapy 2005,12:1089-1098.
5. Matthew J, Callaghan, Daniel J, et al. Hyperglycemiainduced reactive oxygen species and impaired endothelial progenitor cell function. Antioxid Redox Signal 2005, 7:1476-1482.
6. Asahara T, Murohara T, Alison S, et al. Isolation of putative prognitor endothelial progenitor endothelial cells of angiogenesis. Science 1997,275:964-967.
7. Choi K, Kennedy M, Kazarov A, Papadimitriou J.C. A common precursor for hematopoietic and endothelial cells. Development 1998,125:725-732.
8. Peichev M, Naiyer A.J, Pereira D. Expression of VEGFR-2 and AC133 by circulating CD34+ cells identifies a population of functional endothelial precursors. Blood 2000, 95:952-958.
9.王志伟,马秀现,李天晓.bFGF联合骨髓干细胞动员剂促进兔缺血后肢血管新生的研究.中国普通外科杂志.2007,16：136-139.
10. Connolly DT, Olander JV, Heuvelman D. Human vascular permeability factor: Isolation from U937 cells. Biol Chem 1989,264:20017-20024.
11. De Nigris F, Balestrieri ML, Williams-Ignarro S, et al. Therapeutic effects of autologous bone marrow cells and metabolic intervention in the ischemic hindlimb of spontaneously hypertensive rats involve reduced cell senescence and CXCR4/Akt/eNOS pathways. Cardiovasc Pharmacol 2007 Oct,50:424-433.
12. Mancuso P, Peccatori F, Rocca A. Circulating endothelial cell number and viability are reduced by exposure to high altitude. Endothelium 2008,15:53-58.
13. You D, Cochain C, Loinard C. Hypertension impairs postnatal vasculogenesis:role of antihypertensive agents. Hypertension 2008,51:1537-1544.
14. Chang EI, Loh SA, Ceradini DJ. Age decreases endothelial progenitor cell recruitment through decreases in hypoxia-inducible factor 1 alpha stabilization during ischemia Circulation 2007,116:2818-2829.
15. Martin K, Stanchina M, Kouttab N. Circulating endothelial cells and endothelial progenitor cells in obstructive sleep apnea. Lung 2008,186:145-150.
16. Salinas I, Meseguer J, Esteban MA. Antiproliferative effects and apoptosis induction by probiotic cytoplasmic extracts in fish cell lines. Vet Microbiol 2008,126:287-294.
17. Wilhelm C, Bal L, Smirnov P, et al. Magnetic control of vascular network formation with magnetically labeled endothelial progenitor cells. Biomaterials 2007, 28:3797-3806.
1. Boyle AJ, Schuster M, Witkowski P, et al. Additive effects of endothelial progenitor cells combined with ACE inhibition and beta-blockade on left ventricular function following acute myocardial infarction. Renin Angiotensin Aldosterone Syst 2005, 6:33-37.
2.罗天航,方国恩.自体移植内皮祖细胞防治多器官功能障碍的研究.优秀硕博论文库.2007,87-88.
3. Bone RC. Immunologic dissonance:a continuing evolution in our understanding of the systemic inflammatory response syndrome (SIRS) and the multiple organ dysfunction syndrome (MODS) [J].Ann Intern Med 1996,125:680-687.
4. Okajima K. Multiple organ failure associated with severe infection-the molecular mechanism (s) and new therapeutic strategies. Nippon Rinsho 2007,65:619-626.
5. Hill JM, Zalos G, Halcox JP, et al. Circulating Endothelial Progenitor Cells, Vascular Function, and Cardiovascular Risk. N Engl J Med 2003,348:593-600.
6. Vasa M, Fichtlscherer S, Aicher A, et al. Number and Migratory Activity of Circulating Endothelial Progenitor Cells Inversely Correlate With Risk Factors for Coronary Artery Disease. Circ Res 2001,89:E1-7.
7. Kawamoto A, Gwon HC, Iwaguro H, et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 2001,103:634-637.
8. Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis reduces remodeling and improves cardiac function. Nat Med 2001,7: 430-436.
9. Kawamoto A, Tkebuchava T, Yamaguchi J, et al.Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 2003,107:461-468.
10. Badorff C, Brandes RP, Popp R, et al. Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes. Circulation 2003,107:1024-1032.
11. Assmus B, Schachinger V, Teupe C, et al.Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation 2002,106:3009-3017.
12. Britten MB, Abolmaali ND, Assmus B, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI):mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation 2003,108:2212-2218.
13. Kalka C, Masuda H, Takahashi T, et al.Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A 2000,97:3422-3427.
14. Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells:a pilot study and a randomised controlled trial. Lancet 2002,360:427-435.
15. Nagaya N, Kangawa K, Kanda M,et al. Hybrid cell-gene therapy for pulmonary hypertension based on phagocytosing action of endothelial progenitor cells. Circulation 2003,108:889-895.
16. Zhao YD, Deng Y, Zhang Q, et al. Bone marrow derived endothelial progenitor cells involved in vascular regeneration and have a protective role in experimental pulmonary arterial hypertension. Circulation 2003,108:IV-295.
17. Takahashi M, Nakamura T, Toba T, et al. Transplantation of endothelial progenitor cells into the lung to alleviate pulmonary hypertension in dogs. Tissue Eng 2004, 10:771-779.
18. Werner N, Junk S, Laufs U, et al. Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury. Circ Res 2003,93:17-24.
19. Iwaguro H, Yamaguchi J, Kalka C, et al. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation 2002, 105:732-738.
20. Kong D, Melo LG, Mangi AA, et al.Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells. Circulation 2004,109:1769-1775.
21. Chapel A, Bertho JM, Hartley RS, et al. Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiation-induced multi-organ failure syndrome. Gene Med 2003,5:1028-1038.
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6. Peitzman AB, Ubekwu AO, Juan Ochoa. Bacterial translocation in trauma patients. [J] Trauma.1991,31:1083.
7.中国医学科学院.多器官功能衰竭综合征的诊断和发病机理探讨.中华医学杂志,1988,68：226-232.
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44.罗天航,方国恩.自体移植内皮祖细胞防治多器官功能障碍的研究.优秀硕博论文库.2007,87-88.
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2. Hennessy M, Korbling Z. Circulating stem cells and tissue repair. Panminerva Med 2004,46:1-11
3. Smadja DM, Bieche I, Helley D, et al. Increased VEGFR2 expression during human late endothelial progenitor cells expansion enhances in vitro angiogenesis with up-regulation of integrin alpha (6). J Cell Mol Med 2007, Sep-Oct,11:1149-61.
4. de Nigris F, Balestrieri ML, Williams-Ignarro S, et al. Therapeutic effects of autologous bone marrow cells and metabolic intervention in the ischemic hindlimb of spontaneously hypertensive rats involve reduced cell senescence and CXCR4/Akt/eNOS pathways. J Cardiovasc Pharmacol 2007 Oct,50:424-33.
5. Deiteh EA. Spontaneous lymphocyte activity:an important but neglected component of the immunologic profile of the thermally injured patient [J]. Surgery 1985,98:587.
6. Murphey ED, Herndon DN, Sherwood ER. Gamma interferon does not enhance clearance of Pseudomonas aeruginosa but does amplify a proinflammatory response in a murine model of postseptic immunosuppression. [J] Infect Immun 2004,72: 6892-6901
7. Hu S, Sheng ZY, Li JY, et al. A small dose of intraperitoneal injection of zymosan induces systemic inflammatory response and multiple organ dysfunction following gut ischemia-reperfusion injury. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue.2003 Jan, 15:11-14.
8. Botha AJ, Moore FA, Guleserian KJ et al.Early neutophil sequestration after injury:A pathogenic mechanism for multiple organ failure.J Trauma 1995,39:411-417
9.胡森,盛志勇,周宝桐.创伤后多器官衰竭发病过程的“双相预激”学说.解放军医学杂志,1995,20：325.
10. Peitzman AB, Ubekwu AO, Juan Ochoa. Bacterial translocation in trauma patients. [J] Trauma.1991,31:1083.
11.中国医学科学院.多器官功能衰竭综合征的诊断和发病机理探讨.中华医学杂志,1988,68：226-232.
12. BurdonD, Tiedje T, Pfeffer K, et al. The role of tumor necrosis factor in the development of multiple organ failure in a murine model. Crit Care Med.2000, 28:1962-1967.
13. Thomas NJ,Carcillo JA,Herzer WA, et al. Chronic type IV Phosphodiesterase inhibition protects glomer μ lar filtration rate and renal and mesenteric blood flow in a zymosan-induced model of multiple organ dysfunction syndrome treated with norepinephrine. J Pharmacol Exp Ther.2001,296:168-174.
14. Taniguchi T, Yamamoto K, Ohmoto N, et al. Effects of Propofol on hemodynamic and inflammatory responses to endotoxemia in rats. Crit Care Med.2000,28:1101-1106.
15. Cirioni O, Giacometti A, Ghiselli R, et al. Single-dose intraperitoneal magainins improve survival in a gram-negative-pathogen septic shock rat model. Antimicrob Agents Chemother.2002,46:101-104.
16. Chaudry I H. Rat and mouse of hypovolemic-traumatic shock. In:Schlag G, Redl H(ed):pathophysiology of shock, sepsis and organ failure. Germany:Springer-Verlag Berlin Heidelberg,1993,371.
1. Luttun A, Carmeliet G, Carmeliet P. Vascular progenitors:from biology to treatment. Trends Cardiovasc Med 2002,12:88-96.
2.陈建国,罗兴才,梁小卫.内皮祖细胞在血管新生中的作用.中国误诊学杂志,2006,21：4127-4128.
3. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003,9:669-676.
4. Sinn PL, Sauter SL, Mccrayjrpb. Gene therapy progress and prospects:development and improvement of lentiviral and retroviral vectors-design, biosafetyandproduction. Gene Therapy 2005,12:1089-1098.
5. Matthew J, Callaghan, Daniel J, et al. Hyperglycemiainduced reactive oxygen species and impaired endothelial progenitor cell function. Antioxid Redox Signal 2005, 7:1476-1482.
6. Asahara T, Murohara T, Alison S, et al. Isolation of putative prognitor endothelial progenitor endothelial cells of angiogenesis. Science 1997,275:964-967.
7. Choi K, Kennedy M, Kazarov A, Papadimitriou J.C. A common precursor for hematopoietic and endothelial cells. Development 1998,125:725-732.
8. Peichev M, Naiyer A.J, Pereira D. Expression of VEGFR-2 and AC133 by circulating CD34+ cells identifies a population of functional endothelial precursors. Blood 2000, 95:952-958.
9.王志伟,马秀现,李天晓.bFGF联合骨髓干细胞动员剂促进兔缺血后肢血管新生的研究.中国普通外科杂志.2007,16：136-139.
10. Connolly DT, Olander JV, Heuvelman D. Human vascular permeability factor: Isolation from U937 cells. Biol Chem 1989,264:20017-20024.
11. De Nigris F, Balestrieri ML, Williams-Ignarro S, et al. Therapeutic effects of autologous bone marrow cells and metabolic intervention in the ischemic hindlimb of spontaneously hypertensive rats involve reduced cell senescence and CXCR4/Akt/eNOS pathways. Cardiovasc Pharmacol 2007 Oct,50:424-433.
12. Mancuso P, Peccatori F, Rocca A. Circulating endothelial cell number and viability are reduced by exposure to high altitude. Endothelium 2008,15:53-58.
13. You D, Cochain C, Loinard C. Hypertension impairs postnatal vasculogenesis:role of antihypertensive agents. Hypertension 2008,51:1537-1544.
14. Chang EI, Loh SA, Ceradini DJ. Age decreases endothelial progenitor cell recruitment through decreases in hypoxia-inducible factor 1 alpha stabilization during ischemia Circulation 2007,116:2818-2829.
15. Martin K, Stanchina M, Kouttab N. Circulating endothelial cells and endothelial progenitor cells in obstructive sleep apnea. Lung 2008,186:145-150.
16. Salinas I, Meseguer J, Esteban MA. Antiproliferative effects and apoptosis induction by probiotic cytoplasmic extracts in fish cell lines. Vet Microbiol 2008,126:287-294.
17. Wilhelm C, Bal L, Smirnov P, et al. Magnetic control of vascular network formation with magnetically labeled endothelial progenitor cells. Biomaterials 2007, 28:3797-3806.
1. Boyle AJ, Schuster M, Witkowski P, et al. Additive effects of endothelial progenitor cells combined with ACE inhibition and beta-blockade on left ventricular function following acute myocardial infarction. Renin Angiotensin Aldosterone Syst 2005, 6:33-37.
2.罗天航,方国恩.自体移植内皮祖细胞防治多器官功能障碍的研究.优秀硕博论文库.2007,87-88.
3. Bone RC. Immunologic dissonance:a continuing evolution in our understanding of the systemic inflammatory response syndrome (SIRS) and the multiple organ dysfunction syndrome (MODS) [J].Ann Intern Med 1996,125:680-687.
4. Okajima K. Multiple organ failure associated with severe infection-the molecular mechanism (s) and new therapeutic strategies. Nippon Rinsho 2007,65:619-626.
5. Hill JM, Zalos G, Halcox JP, et al. Circulating Endothelial Progenitor Cells, Vascular Function, and Cardiovascular Risk. N Engl J Med 2003,348:593-600.
6. Vasa M, Fichtlscherer S, Aicher A, et al. Number and Migratory Activity of Circulating Endothelial Progenitor Cells Inversely Correlate With Risk Factors for Coronary Artery Disease. Circ Res 2001,89:E1-7.
7. Kawamoto A, Gwon HC, Iwaguro H, et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 2001,103:634-637.
8. Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis reduces remodeling and improves cardiac function. Nat Med 2001,7: 430-436.
9. Kawamoto A, Tkebuchava T, Yamaguchi J, et al.Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 2003,107:461-468.
10. Badorff C, Brandes RP, Popp R, et al. Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes. Circulation 2003,107:1024-1032.
11. Assmus B, Schachinger V, Teupe C, et al.Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation 2002,106:3009-3017.
12. Britten MB, Abolmaali ND, Assmus B, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI):mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation 2003,108:2212-2218.
13. Kalka C, Masuda H, Takahashi T, et al.Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A 2000,97:3422-3427.
14. Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells:a pilot study and a randomised controlled trial. Lancet 2002,360:427-435.
15. Nagaya N, Kangawa K, Kanda M,et al. Hybrid cell-gene therapy for pulmonary hypertension based on phagocytosing action of endothelial progenitor cells. Circulation 2003,108:889-895.
16. Zhao YD, Deng Y, Zhang Q, et al. Bone marrow derived endothelial progenitor cells involved in vascular regeneration and have a protective role in experimental pulmonary arterial hypertension. Circulation 2003,108:IV-295.
17. Takahashi M, Nakamura T, Toba T, et al. Transplantation of endothelial progenitor cells into the lung to alleviate pulmonary hypertension in dogs. Tissue Eng 2004, 10:771-779.
18. Werner N, Junk S, Laufs U, et al. Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury. Circ Res 2003,93:17-24.
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