【作者】 卢正茂；

【导师】 方国恩； 毕建威；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 多器官功能障碍综合症(Multiple organ dysfunction syndrome, MODS)是重症监护病房内患者死亡的最常见原因。经过对MODS的深入研究,目前多数学者已认可MODS发生的重要机制之一为：当机体发生严重创伤后,因创伤本身及各种炎症反应的刺激,导致机体全身微血管内皮的损伤与修复失衡以及微循环障碍,从而引发多个器官功能的障碍。自1997年,Asahara等首先从外周血单个核细胞(Peripheral mononuclear cells, PMCs)里分离出内皮祖细胞(Endothelial progenitor cells,EPCs)以来。这类在生理性或病理性因素的刺激下能够从骨髓中动员到外周血并分化为成熟内皮细胞(Endothelium cells, ECs)以促进内皮修复及血管新生的细胞已受到人们的广泛重视,大量的体外实验及动物实验表明EPCs是个体出生后生理性及病理性促血管修复及新生的最主要的细胞,并参与心、肝、肺、肾等单个器官内微循环的修复。VEGF (Vascular endothelial growth factor, VEGF)是血管内皮特异丝裂原,能保持内皮细胞的稳定性,诱导血管生成、增加血管通透性及维持血管功能,是目前发现的最强效的促血管生成因子和促内皮祖细胞向内皮细胞分化的重要细胞因子。慢病毒(Lentivital vector, LV)载体系统具有高效而稳定的基因转移效率且毒副作用小,现已广泛的用于各种疾病的转基因治疗中。同时,可表达荧光信号的报告基因-绿色荧光蛋白(Green fluorescent protein GFP)已普遍的应用于观察活细胞内基因表达及移植细胞在体内定位的情况,使得在分子及细胞水平上实现示踪生物学的发生及变化过程成为可能。本研究以慢病毒为载体,将VEGF+GFP双表达基因转染至EPCs内,研究转染后的EPCs在体外的增殖、迁移、分化及对严重创伤后主要炎性因子的抗杀伤能力,在移植EPCs治疗后观察其在活体内的分布情况以及对严重创伤所引发的MODS的防治作用。本研究共分为三部分,首先在体外建立起EPCs的培养和鉴定体系；其次构建LV-VEGF-GFP载体系统并转染EPCs,体外鉴定其增殖、迁移、分化及对炎性因子的抗杀伤能力；最后将经转染的EPCs移植治疗MODS的动物,观察EPCs在体内的分布情况及移植后MODS的发生率和重要脏器的功能改善情况,评价移植EPCs防治MODS的效果,并对其作用机制进行初步探讨。第一部分兔骨髓内皮祖细胞的体外培养、鉴定和功能检测目的：优化兔骨髓EPCs的分离、培养、扩增和鉴定的方法,为移植EPCs防治MODS提供合理的技术平台。方法：用密度梯度离心法从兔骨髓中分离出单个核细胞,按照l×105/cm2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14天时P3代EPCs的生长情况,并通过细胞形态学特征、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、体外血管生成功能等方法对其进行鉴定。结果：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,培养至第6天时的EPCs,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。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：EPCs在特殊的细胞培养环境中可以生成新生的血管。结论：利用密度梯度离心法可稳定的获得单个核细胞,经体外诱导培养后可获得纯度较高的,具有正常生理功能的EPCs。第二部分：含hVEGF165+GFP双表达基因的慢病毒载体的建立及感染EPCs后的功能鉴定目的：构建可稳定携带hVEGF165+GFP双表达基因的慢病毒载体,高效转染EPCs,提高EPCs增殖、迁移、分化、成血管以及对缺血缺氧和炎症因子的抵抗力。方法：设计并合成：hVEGF165的PCR引物：hVEGF165-F:5’-CGG GAT CCA TGA ACT TTC TGC TG-3’; hVEGF165-R:5’-CGA CGC GTC CGC CTC GGC TTG TCT-3’。以pDC316-hVEGF165质粒为模板进行PCR扩增。酶切回收产物经pWPXL-MOD质粒连接、转化,挑取阳性克隆并抽提质粒后利用限制性内切酶BamH I、Mlu I双酶切,电泳鉴定重组质粒后测序。采用四质粒系统的慢病毒包装质粒,其组成为pRsv-REV, pMDlg-pRRE, pMD2G以及含hVEGF165-GFP的pWPXL-MOD。其中pRsv-REV, pMDlg-pRRE, pMD2G含有病毒包装所必须的元件。质粒载体以磷酸钙法共转染293T细胞,转染后8h更换为完全培养基,培养48h后,收集富含慢病毒颗粒的细胞上清液,经浓缩后得到高滴度的慢病毒浓缩液,采用倍比稀释法检测病毒滴度。解冻经培养纯化的EPCs以1×106/cm2接种于培养瓶中,观察EPCs铺满60%皿底后更换培养液。稀释浓缩病毒,以MOI值为50感染EPCs,共孵育24h,细胞铺满80%-85%皿底后传代,经数次传代后可扩增细胞。对感染后的EPCs细胞进行增值、迁移、分化、成血管能力及凋亡实验检测。结果：经纯化浓缩后的慢病毒的最终滴度为5.0×108 TU/m。以MOI值为50转染EPCs其平均感染效率为87.9%±5.6%。经慢病毒感染后的EPCs的功能鉴定提示：迁移及成血管能力较未感染的EPCs略有增强；增值、分化及抗凋亡能力明显增强。结论：EPCs经慢病毒转染后可将hVEGF165-GFP双表达基因整合入细胞基因组中。EPCs的增值,迁移,分化,成血管及抗炎性杀伤能力得到增强,为在体外短时间能获得具有较强的抗炎性因子杀伤作用及较高靶向迁移能力的EPCs奠定基础。EPCs的分化能力的提高使得EPCs在体外向成熟内皮细胞转化的速度加快,降低了EPCs自我更新及体外传代的能力。第三部分移植未转染与经转染的EPCs防治创伤后多器官功能障碍的研究目的：旨在探讨移植经慢病毒转染的骨髓来源的EPCs对兔创伤后多器官功能障碍治疗的有效性和安全性的研究,同时比较经转染与未转染的EPCs进行移植治疗后的疗效差异。方法：预先抽取实验动物骨髓,按照前述的方法进行EPCs的分离、培养和扩增。利用二次打击建立双相迟发型家兔MODS动物模型,将达到MODS的动物随机分为五组,以1×107个细胞/Kg体重为回输剂量,按照：A组：转染LV-hVEGF165-GFP的EPCs实验组(12只)；B组：转染LV-GFP的EPCs实验组(12只)；C组：未经转染的EPCs实验组(12只)；同时以D组：肌注hVEGF165治疗组及E组：未行任何干预的MODS(12只)及作为对照组。观察不同实验组的EPCs进行移植治疗后EPCs在体内的分布态势对机体各个重要脏器的改善情况。结果：E组：单纯MODS实验组动物死亡率为75%(12只死亡9只)；而A组：移植LV/hVEGF165-GFP-EPCs组的死亡率为25%(12只死亡3只)；B组：移植LV-GFP-EPCs组动物的死亡率为58.3%(12只死亡7只)；C组：普通EPCs组动物的死亡率50%(12只死亡6只)；D组：肌注hVEGF165组动物的死亡率为83.3%(12只死亡10只)。同时A组实验动物的生存时间(136.48±42.27小时)也较其他各组(B组：87.79±24.12小时；C组90.34±23.92小时；D组：59.21±20.35小时；E组：63.45±23.55小时)明显延长(P<0.01)。结论：移植经慢病毒介导的hVEGF165-GFP双表达基因转染的内皮祖细胞可以有效的抵抗严重创伤后机体内环境炎性因子对EPCs的杀伤作用并促进创伤后的微循环修复,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。小结：机体发生炎症的创伤时,体内EPCs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,移植的内皮祖细胞可以在机体发生MODS后向不同组织迁徙或归巢,对微循环具有重要的修复及血管新生作用,以改善创伤后缺血缺氧引起的脏器功能障碍,而经慢病毒介导的hVEGF165-GFP基因转染的EPCs可以对创伤后的全身炎症反应所产生的主要炎性因子具有较好的抗杀伤能力,且针对损伤器官及循环具有更好的靶向治疗作用,可较好的防止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。更多还原

【Abstract】 Multiple organ dysfunction syndrome (MODS) has been the most frequent cause of death in patients admitted to intensive care units. In recent years, most of researchers have made a common agreement that one of the most important mechanisms of MODS is the unbalance between the injuries and repair to systemic capillary vascular endothelium, when the body gets severe injury.Since Ashara firstly isolated the endothelial progenitor cells (EPCs) from peripheral mononuclear cells in 1997, researchers had paid more and more attention on EPCs because these cells have a pivotal potency that they could differentiate into matured endothelial cells and then repair the endothelium. Most of the EPCs circulating in the peripheral blood come from bone marrow in the stimulation of all kinds of physiogenic or pathological factors. Numerous experiments in vitro or in vivo have demonstrated that, in the physiogenic or pathological conditions, EPCs are the most important cells that can repair vascular endothelium and promote the angiogenesis of organs. Furthermore the injuries of microcircu-lation in the single organ such as heart, liver, lung and kidney could be repaired by EPCs too.Vascular endothelial growth factor (VEGF)is a special mitogen for vascular endothelium. It can maintain the homeostasis of endothelial cells, induce the vascularization, enhance vascular permeability and maintain normal function of vessels. It is deem to the most superactive factor that can promote vascularization happen and make EPCs defferentiate into endothelial cells (ECs). Since the carrier system of lentivital vector (LV) has high and stable effectiveness for gene transfer and low adverse effect, it has been used more and more comprehensively in transgenes therapy for all kinds of diseases. In addition, the green fluorescent protein (GFP),which can express the fluorescent signal, has been used to observe gene expression and site-specific of transplantation cells in vivo. The discovery of GFP makes the development and variation of biological tracing come true in molecular and cellular level. In our research, we used the lentivral vectors to transfect the double express gene-hVEGF165-GFP with EPCs. Secondly, we studied the function of post-transfected EPCs in proliferation, immigration, differentiation and antiapoptotic ability for inflammatory environment. At length, we transplanted either untransfected or post-transfected EPCs into the MODS animal in order to figure out their distribution in vivo and observe the preventive and therapeutic effect of MODS.There were three parts in our study. In the first part, we constructed and optimized the system for the isolation、cultivation and identification of EPCs in order to proliferate EPCs fastly in vitro. In the second part, we constructed the carrier system of LV-hVEGF165-GFP to transfect with EPCs and identified the function of post-transfected EPCs in proliferation, immigration, differentiation and anti-apoptosis ability to inflammatory environment. In the third part, we transplanted the untransplanted or post-transplanted EPCs into animal model respectively to observe these cells’distribution in vivo and then investigated the mobidity and mortality of MODS animals. In the last part, we would assess the value of EPCs’ transplantation and make an approach to the putative mechanism of MODS.Part 1 Isolation, Cultivation, Identification of endothelial progenitor cells from rabbit bone marrow in vitroObjective:to optimize the system for isolation, cultivation and identification of endothelial progenitor cells from rabbit bone marrow for transplantation.Methods:BMMCs were isolated by the method of density gradient centrifugation from bone marrow and cultured as 1×106/cm2 original density with specific culture medium for EPCs. After14 days of cultivation, the P3-EPCs were identificated by taking up Dil-ac-LDL and FITC-UEA-1、flow cytometry testing、immunohistochemistry testing、ultrastructural organization testing and the functoin of angiogenesis testing.Result:The attaching cells appeared after 48h culture, and these cells got clustering after 6 days culture. The Weibel-Palade body, which is the special characteristics for EPCs, appeared in our cultured cells. And more than 80% EPCs could take up Dil-Ac-LDL and FITC-UEA-1. CD133 (+),CD34 (+),CD31 (++),KDR (++) appeared in these cells by immunohistochemistry testing. Our cultured cells also were positive in angiogenesis testing.Conclusion:Density gradient centrifugation is a stable method to isolate BMMCs from the peripheral blood. It could provide very pure EPCs by this specific cell-culture way in vitro. Further more, these EPCs have all the normal function that they should have!Part 2:Construct a lentiviral vectors that carry both hVEGF165 and GFP Gene, Identify the function of those post-transfected EPCsObjective:to construct the lentivital vectors that can stably carry the double express gene of hVEGF165-GFP and can transfect with EPCs high effectively. To improve the EPCs’ ability of proliferation、migration、differentiation, angiogenesis and the resistance for ischemia、anoxia、inflammatory factors.Methods:Firstly, we designed the hVEGF165 PCR primers:hVEGF165-F:5’-CGG GAT CCA TGA ACT TTC TGC TG-3’; hVEGF165-R:5’-CGA CGC GTC CGC CTC GGC TTG TCT-3’. Secondly, the plasmid of pDC316-hVEGF165 was used to be the template to amplify hVEGF165 by PCR. Combination and transformation would be performed when both the products of PCR and the plasmid of pWPXL-MOD had been cut by the enzyme of BamH I、Mlu I. This new acquired plasmid of pWPXL-MOD, which contains the exogenous gene of hVEGF165-GFP, was spreaded on the LB plate and incubated at 37℃overnight. As soon as the monoclones appeared on the plate 12-16 hours later, they would be picked up and expanded to extract the plasmids DNA. Thirdly, these acquired plasmids DNA were cut by the restriction enzyme of BamH I、Mlu I, and then the fragments of these recombinant plasmids DNA were identifed by electrophoresis. When the size of fragment had been confirmed, the sequence of targeting gene would be checked to make asure that it was definitely correct.We used the four plasmids system to package the lentivrial vector, including pRsv-REV, pMDlg-pRRE, pMD2G and the recombinant plasmid of pWPXL-MOD which contained the exogeneous gene of hVEGF165-GFP. The three orther plasmids of pRsv-REV、pMDlg-pRRE and pMD2G had all the imperative components for viral packaging. After these four plasmids had been prepared, they would be used to transfect with 293T cells in the protocol of calcium phosphate precipitation.8 hours after transfection, the current medium must be changed to the maximal medium and these 293T cells should be continued to culture for 48-72 hours untill the viral vectors were secreted from them. The supernatant, which contained abundant lentiviral vectors, was collected to concentrate to high concentrated solution. In the last step, the virus titer was measured by means of coubling dilution.The pure frozen EPCs were thawed and planted into the culture dish in the density of 1×105/cm2. As soon as the EPCs had become 60% confluence on the bottom of dish, the culture medium should be changed. At the same time, the concentrated lentiviral vectors were diluted to transfect with EPCs in the MOI for 50. These EPCs were co-cultured with lentiviral vectors for 24h and would be passaged as soon as they were 80%-85% confluence. These post-transfected EPCs could be amplified to a large number after several passages. At last step, we identified the function of these acquired EPCs in proliferation、migration、differentiation, angiogenesis and the resistance for ischemia、anoxia、inflammatory factors.Result:The final titer of lentiviral vectors was 5.0×108 TU/m in the concentrated viral solution. For these viral vectors, the average of efficiency for transfection was 87.9%±5.6%, when EPCs were transfected in the MOI for 50. The function of post-transfected EPCs showed that the ability of migration and angiogenesis were a little bit more enhanced, however, the proliferation、differentiation and the anti-apoptotic ability were significantly enhanced.Conclusion:When EPCs were transfected by our four plasmids system lentiviral vectors, the gene of hVEGF165-GFP carried by viral vectors could be transfered into these cells’ genome. Furthermore this exogenous hVEGF165-GFP gene could enhance the function of EPCs in proliferation、migration、differentiation、angiogenesis and anti-apoptotic ability. These carriers of lentiviral vectors could play a critical role in the acquirement of high anti-apoptotic and more powerfully targeted migratory EPCs. However, the improvement for the ability of post-transfected EPCs in differentiation made these kinds of projenitor cells transfer to mature cells faster than untransfected ones. This characteristic could reduce the self-renewal potentiality of EPCs in vitro.Part3:Transplantation of untransfected and post-transfected endothelial progenitor cells to prevent and treat MODSObjective:to investigate the effect of transplantion of the post-transfected EPCs that come from bone marrow and assess their clinical value on post-trauma multiple organ dysfunction syndrome treatment. Moreover, we aim to compare the effect of treatment in the transplantation with the untransfected and post-transfected EPCs.Methods:Experimental animal bone marrow was in advance taken in accordance with the aforementioned method for EPCs isolation, culturation and amplification. The MODS animals were randomly divided into five groups:A:transplantation with LV/ hVEGF165-GFP-EPCs to the MODS animals (12 rabbits); B:transplantation with LV-GFP-EPCs to the MODS animals (12 rabbits); C:transplantation with untransfected EPCs to the MODS animals (12 rabbits); D:intramuscular injections of hVEGF165 to the MODS animals (12 rabbits); E:none treatment for MODS animals as the control group (12 rabbits). The distribution of transplanted EPCs in main organs was observed in the use of fluorescence microscope and the improvement of these main organs was also assessed after transplantation.Result:The mortality of the experimental animals for untransfected EPCs transplantation group (50%; 6/12) was significantly higher than those of LV/hVEGF165-GFP-EPCs transplantation group (25%; 6/12), but lower than those of injection of hVEGF165 group (83.3%,10/12) and MODS group (75%; 9/12) (P＜0.05), however, there was no statistical difference to LV-GFP-EPCs group(58.3%; 7/12) (P＜0.05). In addition, the survival time of experimental animal in LV/hVEGF165-GFP-EPCs group (136.48±42.27hours) was significantly longer than those in LV-GFP-EPCs group (87.79±24.12 hours), untransfected EPCs group (90.34±23.92 hours), injecting hVEGF165 group(59.21±20.35 hours) and MODS group(63.45±23.55 hours). Moreover, the examine result of WBC, GRAN, SALT, SAST, Cr, BUN in LV-GFP-EPCs group had no difference to those in untransfected EPCs group, but was slightly worse than those in LV-VEGF-GFP-EPCs group, and significantly better than those in the control group.Conclusion:In our study, we proved that exogenous hVEGF165 gene could significantly improve the anti-apoptotic ability of the EPCs when they had been transferred into EPCs gemone. Furthermore, these post-transfecetd EPCs definitely could enhance the repair ability for microcirculation. In short, EPCs transplantation could improve the post-traumatic rehabilitation and reduce the mobidity and mortality of MODS animals.Summary:When the body suffers to severe inflammatory trauma, the reduction and dysfunction of EPCs may be the key point in the development of MODS. Since the transplanted EPCs could migrate to different organs or tissue and transfer to vascular ECs in vivo, these EPCs might play a critical role to repair the microcirculation and could enhance the ability of angiogenesis, both of which can ameliorate the organs’dysfunction. When EPCs were transfected by the lentiviral vectors that carried hVEGF165-GFP gene, they could become stronger in the anti-apoptotic ability for SIRS and possess higher target therapeutical effect on the injured organs and circulation. These transplanted EPCs could prevent MODS, improve the MODS animal’s prognosis and extend their survival time.更多还原