【作者】 王大鹏；

【导师】 陈誉华；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2003， 硕士

【摘要】 背景及目的 血脑屏障主要由脑微血管内皮细胞构成，内皮细胞间形成紧密连接以严格限制大分子物质、致病因子及细胞进入脑内。许多神经系统疾病的发生都与血脑屏障相关。因此研究血脑屏障的主要组成部分人脑微血管内皮细胞的功能将有助于了解血脑屏障稳定性的调节机理，认识神经系统疾病的发病机制。 血管内皮细胞生长因子—VEGF是内皮细胞特异性有丝分裂原，在正常发育以及创伤，肿瘤等许多病理过程中诱导新生血管的形成。VEGF通过其受体VEGFR2(FLK-1)和VEGFR1(FLt-1)调节内皮细胞的诸多功能，例如增殖、分化、迁移等。VEGF既是一种旁分泌因子，又是一种自分泌因子。最新体内外的研究结果表明VEGF具有促进血管内皮细胞以及神经细胞存活的功能，这可能是通过与FLK-1的结合激活PI3-K／AKT和MEK／ERK途径来实现。这都说明VEGF能促进内皮细胞自身保持稳定，抵抗缺氧、缺血、营养匮乏引起的应激，而这些应激同时也必然会引起细胞内环境的紊乱，从而启动一些细胞内的保护性机制。 UPR是内质网到细胞核之间的一条信号传导通路，这条通路的最终结果是使位于ER上某些与蛋白折叠有关的分子伴侣(GRP78、GRP94、PDI等)基因转录水平上调，从而帮助蛋白质完成正确的折叠过程，缓解应激导致的ER中未折叠和错误折叠蛋白的负荷，从而维持细胞内环境的稳定。 既然UPR过程与VEGF-FLK-1通路都能使应激状态下的细胞维持自身的稳定，这似乎暗示它们之间存在某些关联。因此，本研究以血脑屏障的主要成分人脑微血管内皮细胞(HBMEC)为研究对象，在确定HBMEC内存在GRP78和GRP94表达的基础上检测了HBMEC内在UPR过程中VEGF及其受体FLK一1的表达情况，从而为探讨血脑屏障功能的研究积累资料。方法 1.人脑微血管内皮细胞培养至形成单层后，加人不同浓度(2，5，10，25林扩Inl)衣霉素(加m记amyein)诱导6小时 2.利用RT一PCR检侧GR功8、GRP94、VEGFm朋A的表达 3.利用蛋白质免疫印迹(Westem blot)方法检测GR即8、GR即4、VEGF及FLK一1蛋白的表达 4.利用免疫组织化学方法检测GR卫78、G刊四4、VEGF的表达结果 1.GRF78、GR即4、VEGF及FLK一1在人脑微血管内皮细胞内存在基础表达。 2.人脑微血管内皮细胞在tunicamycin诱导前后，其细胞内GR即8的表达存在明显的差异性，诱导后较诱导前GR种8的mR-NA和蛋白水平都明显增高，并且随着tunicajtnycin诱导浓度的增加，二者的表达量也随之增加。 3.人脑微血管内皮细胞在tunic田mycin诱导前后，其细胞内GR印4的表达存在明显的差异性，诱导后较诱导前GR即4的mR-NA和蛋白水平都明显增高，并且随着tunic和甲ycin诱导浓度的增加，二者的表达量也随之增加。 4.人脑微血管内皮细胞在加mic别mycin诱导前后，其细胞内VEGF的表达存在明显的差异性，诱导后较诱导前VEGF的mRNA和蛋白水平都明显增高，并且随着tunicajmycin诱导浓度的增加，二者的表达量也随之增加。 5.人脑微血管内皮细胞在tunicamycin诱导前后，其细胞内FLK一1的表达存在明显的差异性，诱导后较诱导前FLK一l的蛋白水平明显增高，并且随着tunicamycin诱导浓度的增加，其表达量也随之增加。结论 1.GRf口8、G和四4及v’EcF在人脑微血管内皮细胞中存在基础表达，推测在人脑微血管内皮细胞内存在VEGF的自分泌调节通路。 2.人脑微血管内皮细胞中VEGF及其受体FLK一1的表达水平与其UPR诱导水平成正相关。 3.推测VEGF及其受体FLK一1可能为UPR的靶基因，这可能是人脑微血管内皮细胞在应激状态下保持自身稳定性的一种机制。更多还原

【Abstract】 ObjectiveThe blood - brain barrier ( BBB) is formed by the complex tight junctions between the endothelial cells of the brain capillaries. This structure results in the capillary wall that behaves as a continuous lipid bilayer controlling the access of solutes and toxic substances to the central nervous system (CNS) so as to maintain brain homeostasis. So, study of human brain microvascular endothelial cell ( HBVEM ) will lead us to understand the regulative mechanism of BBB homeostasis , and pathogenesis of some CNS disorders.Vascular endothelial growth factor ( VEGF) is a highly specific mitogen for vascular endothelial cells and induces neovascularization in development, trauma and tumor. Through its receptors, VEGFR2 (FLK - 1) VEGFR1 (FLt -1), VEGF induces endothelial cell proliferation and differentiation, promotes cell migration, and inhibits ap-optosis. In addition of its paracrine function, VEGF is an autocrine factor. VEGF can promotes endothelial cell survival and resist apopto-sis, which is mediated through the VEGFR-2 receptor and the phos-phatidylinositol 3’- kinase (PI3 - K)/Akt and MEK/ERK signaling pathway. These indicate that VEGF can promote endothelial cell homeostasis under stress situation, such as hypoxia and ischemia. And that these stresses also result in disturbance of homeostasis unavoidably , and launch some stress response, including unfolding protein re-sponse.UPR is a signal passway from endoplasmic reticulum to nucleus and results in up-regulation of expression of molecular chaperon, such as GRP78, GRP94 and PDI. These molecular chaperon help protein fold accurately, and reduce the overloading of unfolded and wrongly - folded protein resulted from stress.Since UPR and VEGF - FLK - 1 passway can help cell maintain homeostasis under stress, we can assume some relationship between them. So, taking the main part of BBB - HBMEC as the object, we tested Expression of VEGF and FLK - 1 in HBMEC under the state of UPR and accumulate more data for exploring the function of BBB thereby.Methods1. HBMEC Cell culture and induction by tunicamycin of different concentration(2,5 ,10,25g/ml)2. detection of GRP78,GRP94,VEGF mRNA by RT - PCR3. detection of GRP78,GRP94,VEGF protein by western blot4. detection of GRP78,GRP94,VEGF protein by immunohisto-chemistryResults1. Basal expressions of GRP78, GRP94,VEGF and FLK - 1 are detected in HBMEC.2. Levels of both mRNA and protein of GRP78 of HBMEC in pre - induction and post-induction are of evident difference. Levels inpost - induction are far higher than in pre - induction, and with the increase o f the concentration o f tunicamycin, Levels of mRNA andprotein also increase.3. Levels of both mRNA and protein of GRP94 of HBMEC in pre- induction and post - induction are of evident difference. Levels in post - induction are far higher than in pre - induction, and with the increase o f the concentration o f tunicamycin, Levels of mRNA and protein also increase.4. Levels of both mRNA and protein of VEGF of HBMEC in pre- induction and post - induction are of evident difference. Levels in post - induction are far higher than in pre - induction, and with the increase o f the concentration o f tunicamycin, Levels of mRNA and protein also increase.5. Levels of protein of FLk-1 of HBMEC in pre - induction and post - induction are of difference. Levels in post - induction are higher than in pre - induction, and with the increase of the concentration o f tunicamycin, Levels of protein also increase.Conclusion1. In HBVEM, there are basal expression of GRP78,GRP94 VEGF and VEGF may function as a autocrine survival factor.2. Expression of VEGF and FLk -1 correlates to inductive level of UPR in HBVEM.3. VEGF and its receptor-FLK - 1 may be the target gene of UPR, suggesting a mechanism of homeostasis of HBMEC under stress.更多还原