通络救脑注射液对脑微血管内皮细胞活性影响的特征

目的：观察通络救脑注射液对培养的正常及缺血脑微血管内皮细胞的活性影响。揭示其通络作用的效应靶点与特征。方法：原代培养大鼠脑皮质微血管内皮细胞,传至第三代。分为正常及拟缺血组,采用培养基氧糖刺夺（OGD）法建立拟缺血模型。通过四甲基偶氮唑盐（MTT）比色分析法测定不同浓度的通络救脑注射液对正常及OGD内皮细胞的活性影响。结果：通络救脑注射液作用于正常脑微血管内皮细胞,与未加药组比较,小剂量药物抑制细胞活性趋势,大剂量促进细胞活性趋势,剂量总趋势呈反抛物线形;通络救脑注射液作用于OGD组脑微血管内皮细胞,小剂量范围促进内皮细胞的增殖活性,呈显著和极显著差异,而大剂量组则抑制细胞活性,剂量总趋势呈抛物线形。结论：通络救脑注射液对正常及缺血脑微血管内皮细胞具有双向调节作用,药物剂量与细胞增殖活性呈非线性关系。天剂量与小剂量可能是不同的作用机制,反映了中药复方药效的多维性。     

通络救脑注射液对大鼠脑微血管内皮细胞活性 及其条件培养液影响的初步研究

目的：观察通络救脑注射液对正常及拟缺血大鼠脑微血管内皮细胞的活性影响,并初步探讨细胞条件培养液内蛋白分泌的时效特征、奠定可溶性蛋白深入分析的技术基础。方法：通络救脑注射液作用于正常夏拟缺血脑微血管内皮细胞之后,用MTS／PMS比色分析法测定细胞的活性,Bradford法测定细胞培养液总蛋白含量,比色分析法测定细胞培养液乳酸脱氢酶（LDH）漏出值,同步观察了5个时间点的细胞活性及条件培养液总蛋白量及LDH释放量。结果：通络救脑注射液能够提高拟缺血细胞的活性,且抑制LDH释放量;正常组分泌总蛋白量3h达到高峰,此时细胞活性最佳,LDH释放量亦少。拟缺血组分泌总蛋白量是6h达到高峰,此时LDH释放量最少,但细胞活性与3h比较有所下降。结论：通络救脑注射液对拟缺血细胞损伤具有保护作用：以3h至6h的细胞条件培养液做为收集目标是研究条件培养液的最佳时间段。     

脑微血管内皮细胞条件培养液对星形胶质细胞活性的影响

目的:观察脑微血管内皮细胞与星形胶质细胞的相互关系,探讨血脑屏障维持脑内环境稳定的生理学基础.方法:原代培养大鼠脑皮质微血管内皮细胞,传至三代,收集在指数生长期细胞生长48 h后的务件培养液;将条件培养液分别按20%、30%、40%、50%、60%、70%、80%、90%、100%不同浓度作用于星形胶质细胞,MTT法检测不同浓度内皮细胞条件培养液作用于星形胶质细胞24 h、48h后的活性变化.结果:48h时间点的各浓度内皮细胞条件液组与相应的正常对照组相比差异均有显著统计学意义(P<0.01),内皮细胞条件液对星形胶质细胞表现出显著的抑制效应,而24 h的70%、80%、90%、100%浓度组与相应正常对照组相比也有显著统计学意叉的差异(P<0.01),且有浓度依赖性.结论:正常脑微血管内皮细胞条件培养液抑制了正常星形胶质细胞的活性.     

脑微血管内皮细胞条件培养液对MIP-1β诱导大鼠小胶质细胞迁移影响的机制

目的:研究经通络方药作用后的脑微血管内皮细胞条件培养液对MIP-1β诱导的大鼠小胶质细胞迁移的影响,以及由MIP-1β在小胶质细胞上的受体-CCR5介导细胞信号转导通路的调控作用.方法:将通络方药作用后的大鼠脑微血管内皮细胞条件培养液加入到由5nM MIP-1β刺激6h后的原代大鼠小胶质细胞中,利用Transwell细胞迁移系统来观察小胶质细胞迁移,用Western blot法检测小胶质细胞CCR5,p-p38,P-JNK表达情况.结果:脑微血管内皮细胞条件培养液能够显著减少小胶质细胞迁移到Transwell下层的细胞数量(P<0.01),降低小胶质细胞上MIP-1β的受体CCR5表达,同时抑制了其下游信号蛋白p38和JNK的磷酸化.结论:通络方药作用后的脑微血管内皮细胞务件培养液可抑制由MIP-1β诱导的大鼠小胶质细胞迁移,其作用发挥可能是通过抑制MIP-1β的受体CCR5表达,降低了其下游通路上p38和JNK蛋白磷酸化程度实现.     

脑微血管内皮细胞氧糖剥夺条件培养液对PC12细胞活性的影响

目的:观察脑微血管内皮细胞氧糖剥夺后条件培养液对PC12细胞的影响.方法:原代培养大鼠脑皮质微血管内皮细胞,传至三代.制备其正常、氧糖剥夺、复糖复氧三种状态条件液,并观察内皮细胞在这三种状态下的形态改变;将这三种条件培养液作用于PC12细胞,分4组:正常对照组(Normal)、正常内皮细胞条件液组(N-CM)、氧糖剥夺内皮细胞条件液组(I-CM)、复糖复氧内皮细胞条件液组(R-CM).每组分别设6h、12 h、24 h、48 h、72 h、96 h、120 h、144 h 8个时间点,MTS/PMS法检测三种脑微血管内皮细胞条件液作用后的不同时间点PC12细胞活性的变化.结果:氧糖剥夺后脑微血管内皮细胞发生核固缩等明显病理改变,复糖复氧后这些改变有所恢复.N-CM组、R-CM组与相应时间点的Normal组PC12细胞活性相比差异均有统计学意义(P＜0.01).正常和复糖复氧内皮细胞条件液显著抑制了PC12细胞的增殖和活性.N-CM组、R-CM组与相应时间点的I-CM组PC12细胞活性相比差异均有统计学意义(P＜0.01).结论:正常脑微血管内皮细胞条件液抑制了PC12细胞的活性,氧糖剥夺后的脑微血管内皮细胞务件液抑制效应消失,复糖复氧后,这种抑制效应也同时恢复.     

缺氧对大鼠脑微血管内皮细胞增殖和凋亡的影响及其机制研究

目的:探讨缺氧对大鼠脑微血管内皮细胞增殖和凋亡的影响及其可能的分子机制。方法:从2周龄的SD大鼠中提取脑微血管内皮细胞。体外模拟脑缺氧微环境,将体外培养的脑微血管内皮细胞分别置于常氧(21%O_2)和低氧环境(1%O_2)下处理6、12和24小时,采用四甲基偶氮唑盐(MTT)法观测不同时间点细胞增殖能力的变化;用AnnexinV-FITC/PI双标流式细胞术观察不同时间点细胞凋亡情况;Real time-PCR和Western blot法检测细胞中(Hypoxia-inducible factor-1α) HIF-1αm RNA和蛋白的表达。进一步使用HIF-1αsi RNA靶向沉默HIF-1α基因,再检测低氧环境下HIF-1αm RNA和蛋白表达、细胞增殖以及凋亡水平的变化。结果:随着低氧处理时间的延长,大鼠脑微血管内皮细胞的增殖能力被显著抑制,同时凋亡水平显著增加,HIF-1αm RNA和蛋白表达水平显著升高。使用HIF-1αsi RNA特异性阻断HIF-1α表达后,低氧环境下HIF-1αm RNA和蛋白表达明显降低,同时细胞活性增加,细胞凋亡率显著下降。结论:缺氧微环境能够通过上调HIF-1α的表达抑制大鼠脑微血管内皮细胞增殖并促进其凋亡。     

星形胶质细胞在血脑屏障发育与稳态维持中的作用机制

血脑屏障(blood-brain barrier, BBB)由脑微血管内皮细胞及包绕内皮细胞的基膜、周细胞和星形胶质细胞的足突构成，它将血液与脑组织分隔开来，从而维持神经功能包括神经环路、突触连接和重塑等微环境的稳定。BBB稳态失衡与包括神经退行性疾病在内的许多中枢神经系统疾病有关，但目前BBB稳态维持与失衡的机制尚不清楚。星形胶质细胞作为BBB的组成成分，也是神经血管单元中联系神经元与脑微血管的枢纽，在BBB发育特别是BBB稳态维持中起重要作用。本文在简要介绍BBB的发育过程之后，综述了星形胶质细胞诱导BBB发育、成熟及其在BBB稳态维持中的作用和机制的研究进展，并指出了与BBB稳态失衡有关的A1型星形胶质细胞异质性的概念，以期为深入研究BBB稳态维持机制及加深理解BBB稳态失衡诱发神经退行性疾病提供新启示。     

IGF1在脑缺血大鼠脑组织中的表达与通络救脑注射液的脑保护作用

目的:观察脑缺血模型大鼠血清及脑组织中IGF1蛋白表达水平,以及通络救脑注射液对该因子表达水平的影响,分析通络救脑注射液的脑保护作用途径。方法:以线栓法制作大鼠大脑中动脉缺血模型(MCAO),将SD大鼠随机分为空白对照组、模型组及通络救脑注射药组,分别在造模后12小时、24小时、3天、7天4个时间点采取动物血清和脑组织,以免疫组化、酶联免疫(ELISA)以及RTPCR的方法,分别检测IGF1的表达部位及定量检测该因子的蛋白及mRNA表达水平,并测定血清NSE含量。结果:IGF1在正常脑组织有表达,在脑缺血后24小时表达增多,此后随脑缺血时间的延长不断减少;通络救脑组IGF1各时间点表达均高于模型组。模型组血清NSE水平显著升高,各时间点与正常组均有显著差异,通络救脑注射液组血清NSE水平在12小时、1天和7天时显著低于模型组。结论:大鼠脑缺血损伤后IGF1表达减少与神经元的坏死有相关性,通络救脑注射液能够提高其表达水平,对缺血损伤的脑组织发挥保护作用。     

脑缺血后的脑微血管变化

大脑微血管具有独特的组织结构,这种结构对脑组织起到了保护性屏障作用,局部脑缺血可以引起这种屏障功能破坏,导致血液成分渗出,以及与炎症反应密切相关的整合素表达明显增加,促使炎性细胞以及血小板等向缺血局部聚集和迁移,从而造成局部微血管阻塞。同时,血管内皮细胞基质金属蛋白酶表达明显增加,内皮细胞和星形胶质细胞表面的结构整合素以及对应的基质配体丢失,使微血管细胞间的紧密联系破坏。以上这些变化伴随着神经细胞的损伤,同时,与血管生成和神经发生相关的受体上调,缺血局部区域出现血管生成和神经发生现象,这些过程可能与缺血后期脑功能的恢复相关。本文主要就脑缺血以后脑微血管的变化进行了综述,并对其中的问题以及今后脑血管病研究的发展进行了探讨。     

大鼠大脑皮质星形胶质细胞条件培养液促进小脑皮质神经元的生长

本研究从大鼠大脑皮质分离、纯化星形胶质细胞,再经培养后收集星形胶质细胞的无血清条件培养液。用盖玻片培养法与快速自动比色微量分析法研究了星形胶质细胞条件培养液对小脑皮质神经元生存以及神经元活力的影响。发现星形胶质细胞条件培养液能够明显提高小脑皮质神经元的体外存活率,增强神经元的活力。表明星形胶质细胞具有神经营养性作用。     

An in vitro three-dimensional coculture model of cerebral microvascular angiogenesis and differentiation

Summary The microvasculature of the developing brain is plastic and responds differently to the many insults associated with preterm birth. We developed three-dimensional in vitro culture models for the study of the responses of the developing cerebral micro-vasculature. Beagle brain microvascular endothelial cells (BBMEC) were isolated by differential centrifugation from newborn beagle pups on postnatal Day 1 and placed in three-dimensional culture dispersed in a collagen gel. Alternatively, BBMEC were placed in a three-dimensional coculture with neonatal rat forebrain astrocytes. Cultures were analyzed for extracellular matrix components at 1 and 6 d, and total RNA was extracted for Northern analyses. Urokinase plasminogen activator activity was assayed in both mono- and cocultures of the two cell types. Studies of three-dimensional BBMEC/astrocyte cocultures demonstrated progressive tube formation with only low levels of endothelial proliferation. By 6 d in three-dimensional coculture, the BBMEC formed capillarylike tubes with a wrapping of glial processes, and basement membrane protein synthesis was noted. Urokinase plasminogen zymography suggested intercellular signaling by the two cell types. These data suggest that the three-dimensional beagle brain germinal matrix microvascular endothelial cell/neonatal rat astrocyte coculture provides a good model for the investigation of microvascular responses in the developing brain.     

Ischemic neurons activate astrocytes to disrupt endothelial barrier via increasing VEGF expression

Blood–brain barrier (BBB) disruption occurring within the first few hours of ischemic stroke onset is closely associated with hemorrhagic transformation following thrombolytic therapy. However, the mechanism of this acute BBB disruption remains unclear. In the neurovascular unit, neurons do not have direct contact with the endothelial barrier; however, they are highly sensitive and vulnerable to ischemic injury, and may act as the initiator for disrupting BBB when cerebral ischemia occurs. Herein, we employed oxygen–glucose deprivation (OGD) and an in vitro BBB system consisting of brain microvascular cells and astrocytes to test this hypothesis. Neurons (CATH.a cells) were exposed to OGD for 3‐h before co‐culturing with endothelial monolayer (bEnd 3 cells), or endothelial cells plus astrocytes (C8‐D1A cells). Incubation of OGD‐treated neurons with endothelial monolayer alone did not increase endothelial permeability. However, when astrocytes were present, the endothelial permeability was significantly increased, which was accompanied by loss of occludin and claudin‐5 proteins as well as increased vascular endothelial growth factor (VEGF) secretion into the conditioned medium. Importantly, all these changes were abolished when VEGF was knocked down in astrocytes by siRNA. Our findings suggest that ischemic neurons activate astrocytes to increase VEGF production, which in turn induces endothelial barrier disruption.

     

桃红四物汤对缺糖缺氧大鼠脑微血管内皮细胞VEGF、VEGFR-2、Akt基因及蛋白表达的作用机制研究

摘要 目的：探讨桃红四物汤对缺糖缺氧（OGD）大鼠脑微血管内皮细胞（rBMECs）血管内皮生长因子（VEGF）、血管内皮生长因子受体2（VEGFR-2）、蛋白激酶B（Akt）基因及蛋白表达的影响及其作用机制。方法：取rBMECs进行培养并随机分为正常组培养、模型组培养、桃红四物汤（0.4 mg/mL）组和桃红四物汤（0.8 mg/mL）组,除正常组外,其余三组采用氧糖剥夺实验构建OGD损伤模型。桃红四物汤组在造模前1 h按0.4 mg/mL、0.8 mg/mL浓度加入桃红四物汤,而正常组及模型组则加入等量体积生理盐水。实时定量聚合酶链反应（qRT-PCR）、蛋白免疫印迹试验检测VEGF、VEGFR-2、Akt基因及蛋白表达情况。结果：与正常组比较,模型组细胞凋亡率显著升高（P＜0.05）。与模型组比较,桃红四物汤（0.4 mg/mL）组和桃红四物汤（0.8 mg/mL）组均降低,且以桃红四物汤（0.8 mg/mL）组最为显著（P＜0.05）。与正常组比较,模型组rBMECs中Akt、VEGFR-2、VEGF相对mRNA和蛋白表达水平显著升高（P＜0.05）。与模型组比较,桃红四物汤（0.4 mg/mL）组和桃红四物汤（0.8 mg/mL）组显著降低,且以桃红四物汤（0.8 mg/mL）组最为显著（P＜0.05）。结论：桃红四物汤可能通过降低OGD条件下rBMECs中VEGF、VEGFR-2、Akt基因及蛋白表达水平,从而发挥抑制内皮细胞的凋亡,保持内皮细胞的完整性,这可能是桃红四物汤对脑保护的机制之一。     

Hypoxia/Aglycemia-Induced Endothelial Barrier Dysfunction and Tight Junction Protein Downregulation Can Be Ameliorated by Citicoline

This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs.     

Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte-derived soluble factor(s)

The purpose of the present study was to clarify the expression, transport properties and regulation of ATP-binding cassette G2 (ABCG2) transporter at the rat blood-brain barrier (BBB). The rat homologue of ABCG2 (rABCG2) was cloned from rat brain capillary fraction. In rABCG2-transfected HEK293 cells, rABCG2 was detected as a glycoprotein complex bridged by disulfide bonds, possibly a homodimer. The protein transported mitoxantrone and BODIPY-prazosin. In rat brain capillary fraction, rABCG2 protein was also detected as a glycosylated and disulfide-linked complex. Immunohistochemical analysis revealed that rABCG2 was localized mainly on the luminal side of rat brain capillaries, suggesting that rABCG2 is involved in brain-to-blood efflux transport. For the regulation study, conditionally immortalized rat brain capillary endothelial (TR-BBB13), astrocyte (TR-AST4) and pericyte (TR-PCT1) cell lines were used as an in vitro BBB model. Following treatment of TR-BBB13 cells with conditioned medium of TR-AST4 cells, the Ko143 (an ABCG2-specific inhibitor)-sensitive transport activity and rABCG2 mRNA level were significantly increased, whereas conditioned medium of TR-PCT1 cells had no effect. These results suggest that rat brain capillaries express functional rABCG2 protein and that the transport activity of the protein is up-regulated by astrocyte-derived soluble factor(s) concomitantly with the induction of rABCG2 mRNA.     

Transmigration of Neural Stem Cells across the Blood Brain Barrier Induced by Glioma Cells

Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE₂, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.     

Blood-brain barrier modeling with co-cultured neural progenitor cell-derived astrocytes and neurons

In vitro blood-brain barrier (BBB) models often consist of brain microvascular endothelial cells (BMECs) that are co-cultured with other cells of the neurovascular unit, such as astrocytes and neurons, to enhance BBB properties. Obtaining primary astrocytes and neurons for co-culture models can be laborious, while yield and heterogeneity of primary isolations can also be limiting. Neural progenitor cells (NPCs), because of their self-renewal capacity and ability to reproducibly differentiate into tunable mixtures of neurons and astrocytes, represent a facile, readily scalable alternative. To this end, differentiated rat NPCs were co-cultured with rat BMECs and shown to induce BBB properties such as elevated trans-endothelial electrical resistance, improved tight junction continuity, polarized p-glycoprotein efflux, and low passive permeability at levels indistinguishable from those induced by primary rat astrocyte co-culture. An NPC differentiation time of 12 days, with the presence of 10% fetal bovine serum, was found to be crucial for generating NPC-derived progeny capable of inducing the optimal response. This approach could also be extended to human NPC-derived astrocytes and neurons which similarly regulated BBB induction. The distribution of rat or human NPC-derived progeny under these conditions was found to be a roughly 3 : 1 mixture of astrocytes to neurons with varying degrees of cellular maturity. BMEC gene expression analysis was conducted using a BBB gene panel, and it was determined that 23 of 26 genes were similarly regulated by either differentiated rat NPC or rat astrocyte co-culture while three genes were differentially altered by the rat NPC-derived progeny. Taken together, these results demonstrate that NPCs are an attractive alternative to primary neural cells for use in BBB co-culture models.