内皮祖细胞来源外泌体对脑损伤小鼠血管修复作用的实验研究

发布时间：2018-04-13 00:27

本文选题：创伤性脑损伤 + 内皮祖细胞　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:颅脑创伤(Traumaticbraininjury,TBI)严重危害人类健康,具有较高的致残率和死亡率。颅脑创伤后神经血管的修复决定患者的预后,其中创伤灶周围血管的修复又是其关键影响因素之一。本课题组前期研究证实颅脑创伤后内皮祖细胞(endothelialprogenitor cells,EPCs)动员增加,且在外周血中呈现先抑后扬的变化趋势,也有研究证实内皮祖细胞能够归巢定位到创伤灶局部,促进创伤灶周围的血管新生,但是内皮祖细胞是否直接分化为血管内皮细胞并参与血管新生的机制目前仍不清楚。已有的报道证明了内皮祖细胞是通过旁分泌作用促进损伤区血管残端的内皮细胞增殖分化形成新生的微血管,而不是直接分化为血管内皮细胞参与修复作用。内皮祖细胞被证实可以分泌外泌体,并且内皮祖细胞外泌体通过其内包含的多种生物学物质发挥作用,但是内皮祖细胞来源的外泌体在促进脑创伤后的神经血管修复中的作用机制尚不明确。本实验通过观察内皮祖细胞外泌体在体内外实验观察内皮祖细胞外泌体对损伤内皮细胞的作用及其机制,为应用内皮祖细胞外泌体治疗创伤性脑损伤提供理论参考。方法:(1)应用梯度密度离心的方法体外获取脐带血单个核细胞(MNCs),培养纯化内皮祖细胞并鉴定其功能;(2)经超速离心的方法获取体外培养的内皮祖细胞分泌到培养基中的外泌体,应用透射电镜、westernblot的方法观察检测内皮祖细胞外泌体形态特征以及表面特异蛋白分子。(3)借助低氧培养箱体外制作的内皮细胞低氧损伤模型,应用Western blot的方法检测内皮祖细胞外泌体干预后其紧密连接蛋白的表达变化以及促进血管生成相关蛋白的表达情况。(4)应用脑皮质损伤仪(CCI)制备小鼠颅脑损伤模型,穿刺小鼠尾静脉移植内皮祖细胞外泌体,应用免疫荧光和westernblot的方法检测移植内皮祖细胞外泌体后的脑创伤小鼠创伤灶周围新生血管密度,紧密连接蛋白的变化,血脑屏障伊文思蓝的渗漏,应用PCR技术检测紧密连接蛋白、MMP9、VEGF表达情况,应用Western blot技术检测促血管生成蛋白PTEN表达水平变化。结果:成功的应用梯度密度离心的方法体外获取脐带血单个核细胞,并在体外扩增培养;经超速离心的方法获取内皮祖细胞来源的外泌体,通过透射电镜、westernblot的方法鉴定内皮祖细胞能够分泌外泌体;利用荧光染料PKH26对外泌体进行标记,证实内皮细胞能够吞噬内皮祖细胞外泌体;体外实验证实内皮祖细胞促进损伤内皮细胞紧密连接蛋白CLN5、ZO-1、OCLN表达增高,PTEN表达水平降低,p-Akt水平升高。体内实验提示脐带血内皮祖细胞外泌体能够减轻进脑创伤后血脑屏障伊文思蓝渗透率;PCR技术证实创伤灶紧密连接蛋白CLN5、ZO-1、OCLN表达增高,VEGF表达水平增高,MMP9表达水平升高;Western blot表明外泌体能够减少第3天和第7天创伤灶PTEN的表达水平。结论:内皮祖细胞能够分泌大量的外泌体,体外实验证实内皮祖细胞促进内皮细胞增殖和损伤后的的修复。促进脑创伤后的血管修复蛋白表达水平增高,直接移植内皮主细胞外泌体比移植干细胞更安全、有效,临床应用前景广阔,但是仍需要对外泌体的基础和临床研究深入探讨。
[Abstract]:Objective: traumatic brain injury (Traumaticbraininjury, TBI) serious harm to human health, with high morbidity and mortality. After craniocerebral trauma neurovascular repair decide the prognosis of patients, including repair of peripheral vascular trauma lesions is one of the key influencing factors. The subject of previous study confirmed that endothelial progenitor cells after traumatic brain injury (endothelialprogenitor cells, EPCs) mobilization increased, the trend xianyihouyang and in the peripheral blood, there is also evidence of endothelial progenitor cells capable of homing to the local traumatic lesions, promote traumatic lesions surrounding angiogenesis, but endothelial progenitor cells are directly differentiation into vascular endothelial cells and participate in angiogenesis at present is still not clear. Some reports proved that endothelial progenitor cells through paracrine action to promote the injury of the vascular stump of the endothelial cells to proliferate and differentiate to form new blood Tube, and not directly involved in the repair of differentiation into vascular endothelial cells. Endothelial progenitor cells have been shown to secrete exosomes, a variety of biological substances and endothelial progenitor cell exosomes contain the internal play a role, but the mechanism of nerve vascular repair exosomes derived endothelial progenitor cells in promoting after traumatic brain injury in it is not clear. In this experiment, through the observation of endothelial progenitor cells in exosomes in vivo endothelial progenitor cell exosomes to damage endothelial cells and its mechanism, to provide theoretical reference for the application of endothelial progenitor cell exosomes in treatment of traumatic brain injury. Methods: (1) from umbilical cord blood mononuclear cells in vitro the application of the method of density gradient centrifugation (MNCs), cultured and purified endothelial progenitor cells and identify its function; (2) by ultracentrifugation method obtained in vitro endothelial progenitor cell exosomes secreted into the culture medium, Application of transmission electron microscope, Westernblot method to observe the morphological characteristics of endothelial progenitor cells in exosomes and surface specific protein molecules. (3) by hypoxic endothelial cell injury model in vitro hypoxia box production method, application of Western blot detection of endothelial progenitor cell exosomes after intervention the expression changes of the tight junction protein expression and promote angiogenesis related proteins (4). The application of cerebral cortex injury instrument (CCI) preparation of craniocerebral injury in mice, puncture tail vein transplantation of endothelial progenitor cells exosomes, methods using immunofluorescence and Westernblot detection of endothelial progenitor cell transplantation exosomes after brain trauma wound around the focus of microvascular density changes the tight junction protein, blood brain barrier leakage of Evans blue, PCR was used to detect the tight junction protein, MMP9, expression of VEGF, application of Western blot technology to promote blood detection The expression of Guan Shengcheng protein PTEN. Results: the acquisition of umbilical cord blood mononuclear cells in vitro by gradient density centrifugation method successfully, and were cultured in vitro; exosome by ultracentrifugation method to obtain the source of endothelial progenitor cells by transmission electron microscopy, Westernblot method for identification of endothelial progenitor cells can secrete exosomes; for labeled with fluorescent dye PKH26 on the secretion of endothelial cells, endothelial progenitor cells can be confirmed phagocytosis of exosomes; in vitro endothelial progenitor cells promote injured endothelial cell tight junction protein CLN5, ZO-1, OCLN expression increased, PTEN expression decreased, increased the level of p-Akt. In vivo experiments suggested that umbilical cord blood endothelial progenitor cells can reduce into exosomes after traumatic brain injury of blood brain barrier permeability by Evans blue; PCR technology confirmed that traumatic lesions of tight junction protein CLN5, ZO-1, OCLN expression increased, VEGF expression level increased, MMP9 The expression level of Western increased; blot showed that exosomes can reduce the expression levels of third and seventh days PTEN traumatic lesions. Conclusion: endothelial progenitor cells can secrete exosomes of in vitro endothelial progenitor cells promote endothelial cell proliferation and repair after injury. To promote vascular repair protein expression after traumatic brain injury in the direct transplantation of endothelial cells and exosomes than transplanted stem cells more safe, effective, broad prospects of clinical application, but still need basic and clinical research on foreign body secretion in depth.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15

【参考文献】