基于络脉理论的脑微血管内皮细胞调控脑微环境的分子机制研究

【作者】 杜欢；

【导师】 李澎涛；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2011， 博士

【摘要】 脑血管病和脑退行性病变呈现高发病率、高致残率的特点,目前尚无一疗效突破性药物,尤其是仍未使脑损害的致残得以根本解决。该类脑疾病所呈现的临床表现和病机转归属于中医“络病”范畴,“毒损脑络”是其病机关键,临床从络脉的功能特点入手进行论治取得了显著的疗效。络脉渗灌气血、贯通营卫,是经脉气血实施调节与营养的场所。络脉病变导致气血渗灌失常,营养失调；营卫失和,卫气壅滞,化生火毒,进而损伤络脉,在脑之络脉病变则形成“毒损脑络”之病机。目前络病生物学基础的研究认为,血管内皮细胞在络病的多个病理环节中发挥着重要作用。现代医学研究也日益认识到血管内皮细胞对脑微环境的多种调节作用,除调节物质转运、血管细胞生长、迁移、基质降解、血栓形成和白细胞粘附外,还可活化多种细胞受体和信号传导途径。另外,内皮细胞产生的细胞因子通过旁分泌或自分泌的形式在其生成的微环境直接发挥作用,对维持内环境的平衡稳定具有重要的调节和控制能力。目的：本文在中医络脉理论的指导下,以脑微血管内皮细胞为切入点,研究脑微血管内皮细胞上的特定蛋白质对脑微环境的影响,探讨微血管内皮细胞对脑微环境实现调控作用的特征及其物质基础,以期为“毒损脑络”病机理论提供现代生物学依据,为中药治疗中枢神经系统疾病的作用途径与靶点提供有意义的思路。方法：本研究工作共分为三部分：第一部分：观察脑微血管内皮细胞上的特定蛋白质调节血液和脑组织中某些物质的转运,影响脑组织生物功能的特征。第二部分：研究脑微血管内皮细胞上的特定蛋白质参与物质转运,影响脑组织生物功能的细胞机制。第三部分：探讨脑微血管内皮细胞上的特异蛋白质对神经元功能的影响以及相关机制。具体实验方案为：第一部分：建立AD转基因动物模型,动物实验分为四组,分别是在脑血管过度表达APP组(TgKAPP)和在脑血管过度表达DN-RAGE组(TgDNPPET), TgKAPP和TgDNPPET交配得到TgKAPP/DNPPET组以及空白对照组(nonTg),观察各组脑组织、血浆中Aβ含量变化,Aβ沉积部位以及炎症相关蛋白MMP-2、VCAM-1和ET-1的表达水平。第二部分：通过培养小鼠脑微血管内皮细胞,研究RAGE介导Aβ损伤内皮细胞的相关机制。第三部分：通过大鼠大脑中动脉栓塞模型和内皮细胞培养,观察胎盘生长因子(P1GF)在缺血性损伤脑组织的表达以及神经保护机制。结果：1.与TgKAPP比较,TgKAPP/DNPPET组Aβ沉积明显减少(P=0.0074)。进一步对各组脑皮质3D6染色的脑血管数量统计后显示,与TgKAPP比较,TgKAPP/DNPPET组3D6染色的脑血管数量降低更加明显(P=0.002)。另外,DN-RAGE对脑组织、血浆中Aβ含量都有一定影响,尽管DN-RAGE对脑组织中Aβ42含量的影响没有统计学意义,但是DN-RAGE并没有升高Aβ42的含量,趋势还是降低的。2.与nonTg相比较,TgKAPP组MMP-2的活性和VCAM-1、ET-1的表达显著升高,而TgDNPPET组无明显差异；与TgKAPP相比较,TgKAPP/DNPPET组MMP-2的活性和VCA-M-1、ET-1的表达明显降低。3.与nonTg相比较,TgKAPP组Phospho ERK/Total ERK和Phospho JNK/Total JNK的比值都显著升高；与TgKAPP相比较,TgKAPP/DNPPET组Phospho ERK/Total ERK和Phospho JNK/Total JNK的比值都降低。4.Aβ作用内皮细胞后,MMP-2的表达明显升高。而ERK和JNK激酶的抑制剂PD98059和SP600125作用内皮细胞后,可以抑制Aβ引起MMP-2表达的升高。5.与Aβ作用内皮细胞相比较,anti-RAGE作用内皮细胞后表现出MMP-2的表达明显降低。6.脑缺血性损伤后,P1GF在脑组织微血管和组织间隙表达增加,并且缺血损伤72h是P1GF的表达高峰,同时对侧皮质区P1GF的表达呈反应性增高。7.与正常对照组比较,P1GF作用正常神经元后,VEGFR-2的含量有升高趋势；与正常对照组比较,缺血损伤组神经元VEGFR-2的表达增加(P<0.01)；与缺血损伤组神经元比较,P1GF作用缺血损伤神经元后,VEGFR-2的含量明显增加(P<0.01)结论：1.在AD转基因动物模型研究发现,DN-RAGE参与了AD Aβ沉积这一病理过程,尤其是影响了脑血管部位的Aβ沉积。DN-RAGE通过激活JNK、ERK信号通路,影响MMP-2的活性和VCAM-1、ET-1的表达,参与神经性炎症反应,发挥一定的保护作用。说明RAGE不仅可以作为Aβ的细胞表面结合位点,更重要的是它作为一种可以参与细胞信号转导的受体,参与Aβ介导的损伤机制。另外也再次证实了炎症反应在AD发病过程中的重要性。2.培养内皮细胞验证了RAGE通过与Aβ结合,激活JNK和ERK细胞信号转导,影响内皮细胞MMP-2的表达,参与神经性炎症反应的发生,最终导致细胞损伤以及AD血管病理学变化。3.通过大脑中动脉栓塞动物模型,证实在脑微血管内皮细胞表达P1GF,发生脑缺血性损伤后P1GF的表达上调,并旁分泌至脑组织之中发挥神经元保护作用,P1GF的神经保护机制可能与VEGFR-2介导的信号转导通路有关。4.脑微血管内皮细胞上的RAGE参与Aβ在血液-脑组织的转运,影响脑组织生物功能；内皮细胞可通过分泌P1GF,发挥神经元保护作用。可以认为脑微血管内皮细胞通过自身的分泌功能而维系脑微环境稳定和改善病理状态下的微环境以保护神经元是络脉渗灌气血发挥对脑神营养作用的生物学基础之一。更多还原

【Abstract】 Cerebrovascular diseases and brain neurodegenerative diseases are becoming the leading captured items in the domain of medicine, which have the characteristic of high incidence rate, high crippling rate, and high recurring rate. In recent years, through the repeatedly clinical observation and practice, it is thought that these diseases belong to the category of’collateral disease’in Chinese medicine, and ’toxin hurts brain collaterals’ is the key point in its pathogenesis. It is also realized that brain microvascular endothelial cells (BEC) are not only the substance exchange barrier of blood and tissue, but also have important incretion function. Our researches pay more attention to BEC rather than neurons, which is a meaningful probe to guide Traditional Chinese Medicine mordernation in brain diseases.The major goal of this study was to assess the molecular mechanism of brain microvascular endothelial cells affects cerebral micro-environment.The experiments are divided into three parts:First:We employed transgenic (Tg) mice model. Tg DNPPET mice were crossed with Tg KAPP animals to produce Tg KAPP/DNPPET animals, as well as single Tgs (Tg KAPP, Tg DNPPET) and nonTg littermates. We measured MMP-2 activity, VCAM-1 and ET-1 expression and involved signal transduction in four groups.Second:By culture mice brain microvascular endothelial cells, we observed Receptor for advanced glycation end products (RAGE) mediated MMP-2 expression in Aβinvolved BEC and signal transduction.Third:We examined the expression of Placental growth factor (PlGF) in cerebral ischemia, utilizing a permanent middle cerebral artery occlusion (MCAO) model in the rat. The effects of PlGF upon neuronal vascular endothelial growth factor receptor-1 (VEGFR-1) and vascular endothelial growth factor receptor-2 (VEGFR-2) expression were also examined.Research Results:1. By immunohistochemical analysis with 3D6 antibody in four genotypes of mice, there was a significant decrease in the extent of total Aβdeposition in the cortex in Tg KAPP/DNPPET mice compared with Tg KAPP mice. In contrast to the parenchymal, a more striking decrease in the amount of cerebral vascular A(3 was observed in the Tg KAPP/DNPPET mice.2. MMP-2 activity, VCAM-1 and ET-1 expression have been shown to be elevated in Tg KAPP mice compared with non Tg mice, however, we found markedly decreased MMP-2 activity and protein levels of VCAM-1 and ET-1 in Tg KAPP/DNPPET mice compared with Tg KAPP mice.3. Levels of phosphorylated JNK and ERK 1/2 were significantly increased in brain extracts of KAPP and KAPP/DNPPET mice, as compared to nonTg mice, while KAPP/DNPPET mice revealed significantly less phosphorylation of JNK and ERK 1/2, as compared with KAPP mice.4. Compared with control group, MMP-2 expressions were significantly increased in Aβ-induced BEC. Pretreatment with ERK, JNK MAP kinase inhibitor PD98059, SP600125 resulted in a highly significant reduction in Aβ-induced upregulation of MMP-2.5. Introduction of anti-RAGE to block RAGE exerted similar suppressive effects on Aβ-stimulated upregulation of MMP-2.6. PlGF immunoreactivity cells were increased in cells of the mesenchyma and in the vascular interstitium in the MCAO group compared with the sham group, and its peak time of expression is at 72h.7. In normal control neurons, VEGFR-2 cellular content was increased by the addition of PlGF. In comparison with the normal control neurons, the VEGFR-2 expression of OGD-treated neurons was significantly increased (p<0.01), confirming the results of the western blotting. VEGFR-2 cellular content in OGD neurons was also significantly increased by the addition of PlGF to OGD-treated neurons (p<0.01). Conclusions are made as follows:1. By transgenic mice model TgDN-RAGE, it was shown that DN-RAGE can affect deposition of Aβ, particularly on the cerebral vasculature. It also had influence on the levels of total brain Aβ, although there was not a significant different, notably, elevated the levels of plasma Aβ. It was suggested that RAGE was involved with facilitating diffuse Aβdeposition in the parenchyma, and also in the cerebral vasculature.2. DN-RAGE displayed decreased activity of MMP-2 and VCAM-1, ET-1 expression. Detailed mechanisms linked activation of JNK MAP kinase and ERK1/2. These data provided support for the concept that RAGE functions as a signaling receptor, rather than solely as a site tethering ligands to the cell surface and also suggested that DN-RAGE can weaken vascular inflammatory stress in Alzheimer disease.3. In cultured BEC, our data suggested that RAGE mediated MMP-2 expression in Aβ-induced endothelial cells, and ERK, JNK MAP kinase signal transduction involved. In all, our experiments supported the possibility that blockade of vessel RAGE might have protective effects, especially with respect to attenuate the pathogenesis of vascular inflammation in AD.4. It was shown that PlGF was expressed in rat brain and this expression was found mainly in brain microvascular endothelial cells. PlGF expression was significantly increased after cerebral ischemia injury and this increase has a neuroprotective effect in response to OGD injured neurons. We also found that VEGFR-2 signaling may play a role in PlGF-mediated neuroprotection. PlGF is, therefore, a promising target for therapeutic intervention in ischemic injury.更多还原