摘要
背景：早在1997年,Asahara等第一次报道了内皮祖细胞(EPCs)参与动物缺血组织中的血管新生；EPCs属于造血干细胞的一个亚群,可以在体内外分化为成熟的内皮细胞,并参与血管的新生,EPCs在血管新生、伤口愈合、缺血损伤、肿瘤生长等生理和病理过程中发挥关键作用。循环血EPCs归巢到血管新生部位并在原位分化为内皮细胞,这个过程称之为出生后的血管发生。骨髓起源的CD34+细胞包含内皮祖细胞(EPCs)、内皮细胞(EC)以及造血干细胞,CD34+细胞作为内皮祖细胞和内皮细胞的来源参与组织损伤的血管新生,实验证实移植CD34+细胞改善缺血性心脏病心肌功能并增加心肌血管新生,同时CD34+细胞可以归巢到卒中脑组织区域,可是关于脑创伤后CD34+细胞的变化及与创伤脑组织修复的关系却暂无报道。最近的报道提示：EPCs被移植到实验动物的下肢和心脏缺血模型后具有相应的治疗效果,这为EPCs的临床应用奠定了基础。
目的
1.大鼠外周血中内皮祖细胞的分离、培养、扩增和鉴定；2.观察脑创伤后大鼠外周血CD34+细胞动员与创伤区脑组织血管新生和修复的关系。3.探讨大鼠脑创伤后损伤组织中血管新生与神经新生的相关性。
方法
1.采集大鼠外周静脉血,采用密度梯度离心法分离外周血中的单个核细胞(MNCs),在包被人纤维连接蛋白(hFN)的培养皿内接种至EGM-2培养基,于37℃、5%CO2培养箱中进行培养；培养2天后经换液去除未贴壁细胞,以后每3天换液1次,倒置相差荧光显微镜下动态观察培养细胞的形态变化特征。借助免疫荧光技术检测培养细胞CD34、CD133、Ⅷ因子(vWF)等内皮祖细胞系列标志的表达；并通过硫氰酸荧光素标记的乙酰化低密度脂蛋白(DiI-ac-LDL)摄取试验和荆豆凝集素(FITC-UEA-1)结合实验,进行内皮祖细胞功能鉴定。体外培养至第7天,采用流式细胞仪检测CD34+CD133双荧光标记阳性细胞所占比率和CD34单荧光标记阳性细胞所占比率。2.本部分取雄性Wistar大鼠98只,随机分为创伤组和假手术组,每组49只,各组分为7个亚组(每组7只),大鼠制作液压脑创伤模型成功后,各将一个亚组分别于伤前、伤后24,48,72,120,and 168小时,取外周血测CD34+阳性细胞数。创伤组和假手术组分别于术前和术后1,3,7,14,21天处死一个亚组(7只),取脑行CD34免疫组化染色,研究血管新生,进一步探索内皮祖细胞和新生血管的关系。3.本部分取雄性Wistar大鼠42只,随机分为假手术组和创伤组各21只,创伤后3、7天14天假手术组和创伤组分别随机抽取7只大鼠,处死后取脑行脑组织切片免疫组化染色检测齿状回Brdu+细胞和Brdu/Neun+细胞数；取实验第二部分大鼠脑切片行免疫组化染色检测CD34和CD31阳性细胞数,探讨大鼠实验性脑创伤后血管新生和神经新生的相关性。
结果
1.内皮祖细胞经体外培养第2天细胞贴壁,第五天形成出现细胞克隆样集落,集落周围有内皮样细胞,培养细胞表达CD133、CD34、vWF等内皮祖细胞标志,表明具有内皮祖细胞特征。倒置相差荧光显微镜下观察显示胞质和胞膜呈绿色和红色双色荧光,说明内皮祖细胞能特异性吸附异DiI-ac-LDL和结合FITC-UEA-1,具有内皮细胞功能。体外培养至第7天,流式细胞仪检测CD34单荧光标记阳性细胞所占比率为85.4%,CD34+CD133双荧光标记阳性细胞所占比率为36.5%。2.与假手术组相比较,大鼠脑创伤后循环中CD34+细胞数量显著增加,大鼠脑创伤区CD34+细胞显著增加,伤后创伤区周围及伤侧海马DG各时间点CD34+数于伤后7天达到峰值,随后有所下降；创伤区血管新生显著增加,循环CD34+细胞和脑创伤后血管新生密切相关。3.与假手术组相比较,脑创伤组伤侧海马DG各时间点BrdU+细胞、Brdu/Neun+细胞较假手术组明显增加；脑创伤后创伤区及伤侧海马DG各时间点CD34+、CD31+细胞和MVD均高于假手术组。相关分析显示：血管新生和神经新生密切相关。
结论
1.可以从大鼠外周血中分离和培养血管内皮祖细胞,为内皮祖细胞研究提供基础。2.脑创伤后大鼠外周CD34+细胞动员,循环CD34+细胞和脑创伤后血管新生密切相关。3.脑创伤后创伤组大鼠血管新生和神经细胞新生均显著增加,血管新生和神经新生密切相关,提示二者共同参与创伤组织的修复。
Background:In 1997, Asahara et al reported for the first time about endothelial progenitor cells (EPCs) that differentiated from participated in vasculogenesis in the animal hindlimb ischemic model. Circulating blood contains a subtype of progenitor cells that have the capacity to differentiate into mature endothelial cells in vitro and in vivo. These cells have been termed endothelial progenitor cells (EPCs). The isolation of EPCs by adherence culture or magnetic microbeads has been described. CD34+-enriched mononuclear cells in peripheral blood include a hematopoietic stem cell population, and were shown to be incorporated into neovascularization. This finding, that circulating EPCs may home to sites of neovascularization and differentiate into endothelial cells in situ, is consistent with "vasculogenesis," a critical paradigm for embryonic neovascularization, and suggests that vasculogenesis and angiogenesis may constitute complementary mechanisms for postnatal neovascularization. There has been extensive research on how CD34+ cells or specifically EPCs contribute to the process of tissue repair in ischemic injury such as myocardial infarction and stroke. Previous reports demonstrating therapeutic potential of EPC transplantation in animal models of hindlimb and myocardial ischemia opened the way to the clinical application of cell therapy.
Objective
1. To study on the isolation, culture and identification of EPCs from rat peripheral blood, and provided basic research for neurologic research.2. The study was designed to test the hypothesis that CD34+ cells correlate with posttraumatic angiogenesis and tissue repair.3. To test the hypothesis that angiogenesis correlate with posttraumatic neurogenesis and tissue repair.
Methods
1.The mononuclear cells were isolated from rat peripheral blood by Ficoll density gradient centrifugation and were cultured in vitro in a special medium(EGM-2). The expression of CD133、CD34、vWF were assessed by immunofluorescent staining and flow cytometer. The biological functions of endothelial cells were examined by the adsorption of ulex-agglutinin (UEA) labeled by fluorescein isothiacyanate (FITC) and Dil-Ac-LDL internalization on 7th day.2. The rats were randomly assigned to control and TBI group (49 rats in each group), each of which further divided into 7 subgroups (7 rats in each subgroup). Fluid percussion injury was performed over the right parietal lobe in TBI groups. Blood samples (1 ml) were collected from retro-orbital venous plexus at baseline,24,48,72, 120, and 168 hrs after TBI from only one subgroup from TBI groups and control group. CD34+ cells in peripheral blood were evaluated by flow cytometry. Rats in control and TBI groups were sacrificed before and 1st,3rd,7th,14th, and 21st days after TBI (7 rats in each time point). brains were collected and processed for 5 mm coronal paraffin sections through the TBI zone and used for immunohistochemistry staining to access changing of the CD34+ cells in brain tissue, further to test the hypothesis that CD34+ cells correlate with posttraumatic angiogenesis and tissue repair.3.42 Adult male Wistar rats were divided into two groups:(1) control group (n=21); (2) TBI group (n=21), Brdu, Brdu/Neun were measured by immunohistochemistry on frozen sections and paraffin sections 3rd,7th,14th days after TBI. CD34 and CD31 were measured by immunohistochemistry on paraffin sections 0,1st,3rd,7th,14th, and 21st days after TBI in the second section. Further to test the hypothesis that angiogenesis correlate with posttraumatic neurogenesis and tissue repair.
Results
1. The cells attached after 2 days, exhibited the clone-like morphology after 5d cultivation and proliferated faster then. Immunofluorescent staining showed that the adherent cells were positive for CD133、CD34 and vWF. The cells could take up DiI-acLDL and bind to FITC-UEA-1, and showed double-positive fluorescence. The CD34 and CD133 double-positive ratio of cultured cells were 36.5%, and the CD34 positive ratio were 85.4% by flow cytometer analyzing.2. The number of circulating CD34+ cells and CD34 and CD133 double-positive cells (per 1×104 mononuclear cells) increased 24 hrs in rats being exposed to TBI and those underwent only surgery. The number of CD34+ cells in the injured tissue and ipsilateral hippocampus was greater than that in control group. In the TBI group, CD34+ cells increased rapidly, reaching plateau at about 1 week after TBI and then declined, whereas MVD showed a steady increase during the same period.3. The number of CD34+ cells and MVD in the injured tissue and ipsilateral hippocampus was greater than that in control group. The number of BrdU+ cells and BrdU/NeuN+ cells was greater than that in control group.
Conclusion
1. Relatively purified EPCs can be obtained by certain procedure of isolation and culture from rat peripheral blood, which provides the basic for clinical application of EPCs transplantations.2. CD34+ cells correlate with posttraumatic angiogenesis and tissue repair. Our data strongly indicate that angiogenesis in the TBI zone originate as the result of homing, migration, proliferation and differentiation of CD34+ cells. Circulating CD34+ cells may play an important role in regeneration of brain injury. Mobilized CD34+ cells eventually contribute to the formation of new vessel in the injured tissue, a critical event for neural recovery after traumatic injury.3. Angiogenesis correlate with posttraumatic neurogenesis and tissue repair. Angiogenesis and neurogenesis take part in the regeneration of TBI brain together.
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