内皮祖细胞对创伤性血脑屏障通透性及血管新生的研究

发布时间：2018-07-14 17:02

【摘要】：背景：颅脑创伤是一种获得性脑损伤,它可以导致血脑屏障破坏、神经元死亡以及神经功能障碍。血管新生作为颅脑创伤后修复的关键因素,它可以为神经重塑提供重要的血管基质,从而有利于伤后长期的功能恢复。内皮祖细胞(endothelial progenitor cells, EPCs)是一种单一谱系的干细胞,当受到外伤、炎症、缺血等刺激后,它可以从骨髓(或其它一些组织、器官)中动员至外周血,归巢至损伤的部位并分化为血管内皮细胞,通过整合入已存在的血管(血管新生)或直接产生新的血管(血管发生)来发挥其作用。EPCs的出现打破了血管发生仅在胚胎阶段出现的观念。前期的动物实验已证实移植EPCs可促进肢体缺血、心肌缺血、脑梗塞等疾病的功能恢复。本课题组首次报道了颅脑创伤患者循环血EPCs数目与临床预后呈正相关,应用促红细胞生成素、他汀、孕酮等刺激动员机体自身的EPCs水平升高从而促进了小鼠颅脑创伤后的功能恢复。目的：本后续研究旨在通过颅脑创伤后脑室移植脐血内皮集落形成细胞(endothelial colony-forming cells, ECFCs,目前被认为是“真正意义”的EPCs群体),证实其减少静脉移植造成细胞的全身分布,同时可以修复血脑屏障、促进血管新生,从而利于外伤后功能恢复,为临床治疗提供新的策略。方法：从人脐血中分离的单个核细胞经过培养和扩增成为ECFCs。成年裸鼠通过液压打击建立颅脑创伤模型,于伤后1天侧脑室内移植ECFCs,实验分为ECFCs组、生理盐水组和假手术组。采用细胞染料SP-DiIC18(3)和Y染色体荧光原位杂交技术对移植的细胞进行追踪。采用脑组织伊文氏蓝渗透法和干湿重法来测量血脑屏障的通透性和脑含水量。紧密连接蛋白ZO-1、claudin-5和细胞因子Ang1、Ang2的表达通过免疫印迹和实时荧光定量PCR来检测。微血管密度应用VWF免疫荧光染色的方法来计数。应用改良神经功能评分和Morris水迷宫来评价颅脑创伤后的功能结果。结果：人脐血中分离的单个核细胞培养5-8天可产生ECFCs,大约14天形成集落,ECFCs可摄取乙酰化低密度脂蛋白和结合荆豆凝集素,同时它表达干细胞标志CD34和内皮细胞标志KDR、VWF、VE-cadherin。通过细胞染料SP-DiIC18(3)和Y染色体荧光原位杂交技术于移植后3天在创伤区域可检测到移植的细胞。ECFCs组比生理盐水组减少了伊文氏蓝的渗漏和脑含水量,增加了ZO-1和claudin-5的表达,提高了Angl:Ang2比值,增加了微血管密度。同时,改良神经功能评分和Morris水迷宫也说明了ECFCs移植促进了鼠颅脑创伤后运动功能、空间获取和记忆功能的恢复。结论：本实验证实经脑室途径移植的ECFCs可归巢至脑创伤区域,其通过修复破坏的血脑屏障、增强血管新生来促进颅脑创伤后神经功能的恢复。ECFCs移植未来用于临床可能会有利于颅脑创伤患者的功能恢复。
[Abstract]:Background: traumatic brain injury is a kind of acquired brain injury, which can lead to blood-brain barrier damage, neuronal death and neurological dysfunction. Angiogenesis, as a key factor in the repair of traumatic brain injury, can provide an important vascular matrix for neural remodeling, which is beneficial to the long-term functional recovery after traumatic brain injury. Endothelial progenitor cells (endothelial progenitor cells, EPCs) are single lineage stem cells, which can mobilize peripheral blood from bone marrow (or other tissues, organs) when stimulated by trauma, inflammation, ischemia, etc. Homing to the site of the injury and differentiating into vascular endothelial cells, By integrating existing blood vessels (angiogenesis) or directly producing new vessels (angiogenesis) to play its role. The emergence of EPCs breaks the idea that angiogenesis occurs only in the embryonic stage. Previous animal experiments have proved that transplantation of EPCs can promote the functional recovery of limb ischemia, myocardial ischemia, cerebral infarction and other diseases. Our group reported for the first time that the number of circulating EPCs in patients with traumatic brain injury was positively correlated with the clinical prognosis. Progesterone stimulates the increase of EPCs and promotes the recovery of function after traumatic brain injury in mice. Objective: the purpose of this follow-up study was to confirm that endothelial colony-forming cells (ECFCs) in umbilical cord blood after craniocerebral trauma (TBI) can reduce the whole body distribution of cells induced by vein transplantation. At the same time, it can repair the blood-brain barrier, promote angiogenesis, which is conducive to the recovery of post-traumatic function, and provide a new strategy for clinical treatment. Methods: the mononuclear cells isolated from human umbilical cord blood were cultured and amplified into ECFCs. Adult nude mice were used to establish traumatic brain injury model by hydraulic attack. ECFCswere transplanted into lateral ventricle one day after injury. The rats were divided into three groups: ECFCs group, saline group and sham operation group. Cell dye SP-DiIC18 (3) and Y chromosome fluorescence in situ hybridization were used to track the transplanted cells. The permeability of blood-brain barrier and brain water content were measured by Yi Wen's blue osmotic method and dry wet weight method. The expression of tight junction protein ZO-1 claudin-5 and cytokine Ang-1 Ang2 was detected by Western blotting and real-time fluorescent quantitative PCR. Microvessel density was counted by VWF immunofluorescence staining. The modified neurological function score and Morris water maze were used to evaluate the functional results after traumatic brain injury. Results: cultured mononuclear cells isolated from human umbilical cord blood for 5-8 days could produce ECFCsand form colony ECFCs for 14 days to absorb acetylated low density lipoprotein and binding agglutinin. It also expressed stem cell marker CD34 and endothelial cell marker VWFFVE-cadherin. Three days after transplantation, SP-DiIC18 (3) and Y chromosome fluorescence in situ hybridization were used to detect that the Yi Wen blue leakage and brain water content were decreased and the expression of ZO-1 and claudin-5 were increased in the transplanted cells. The ratio of Angl:Ang2 and microvessel density were increased. At the same time, the improved neurological function score and Morris water maze also showed that ECFCs transplantation promoted the recovery of motor function, spatial acquisition and memory function after craniocerebral trauma in rats. Conclusion: this study demonstrated that ECFCs transplanted through the ventricular pathway can be homogenized to the traumatic area of the brain, which can repair the damaged blood-brain barrier. Enhanced angiogenesis to promote the recovery of neural function after traumatic brain injury .ECFCs transplantation in the future may be beneficial to the functional recovery of patients with traumatic brain injury.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R651.15

【共引文献】