胶质细胞诱导内皮细胞系血脑屏障特点的实验研究

目的探索胶质细胞在血脑屏障形成中的重要意义,建立一套可靠、简便的血脑屏障体外研究方法。方法采用内皮细胞系ECV304与星形胶质细胞体外接触共培养的方法,探索星形胶质细胞对内皮细胞系多种血脑屏障特点的诱导作用。结果星形胶质细胞可以诱导内皮细胞系形成紧密连接并产生较高的跨内皮阻抗(transendothelial electricalresistance, TER),同时,内皮细胞系的血脑屏障特征酶γ-谷氨酰转肽酶(γ-glutamyl transpeptidase, γ-GT)与碱性磷酸酶(alkaline phosphatase , ALP)活性明显增强。结论星形胶质细胞可以诱导ECV304细胞产生紧密连接等血脑屏障特点,ECV304细胞与星形胶质细胞的体外共培养可以作为研究血脑屏障结构与功能的一种可靠而简便的体外实验方法。     

一种改进的星形胶质细胞原代培养方法

目的改进大鼠大脑皮质星形胶质细胞的体外培养,旨在通过较简便的方法获得高纯度形态典型的星形胶质细胞,为研究星形胶质细胞的生物学作用提供实验模型。方法以新生大鼠为星形胶质细胞来源,利用不同规格的吸头相互叠套,逐步机械吹打,使细胞分散,制备单细胞悬液;获得的单细胞悬液用差速黏附处理去除成纤维细胞,原代培养14d后,恒温振荡法去除小胶质细胞等杂质细胞;胶质细胞原纤维酸性蛋白(GFAP)和S100β免疫荧光共染色,鉴定细胞纯度。结果这种改进的星形胶质细胞培养方法 ,分离培养出星形胶质细胞,阳性率达95%以上。结论采用机械吹打方法建立原代星形胶质细胞培养体系,是一种比较简便、值得推广的可用于体外细胞培养研究工作的有效方法。     

星形胶质细胞源性因子对神经干细胞分化的实验研究

目的探讨星形胶质细胞源性因子对神经干细胞分化的影响。方法分离和培养新生大鼠脑组织的神经干细胞;采用差速贴壁法和振荡法分离纯化星形胶质细胞,用免疫细胞化学染色法,胶质纤维酸性蛋白(GFAP)标记星形胶质细胞,进行细胞的纯度鉴定;将星形胶质细胞和神经干细胞在互不接触的情况下进行共培养,免疫荧光法观察神经干细胞分化后神经元特异性烯醇化酶(NSE)、GFAP和酪氨酸羟化酶(TH)的表达。结果纯化的星形胶质细胞GFAP抗体标记阳性,细胞纯度达98%;星形胶质细胞与神经干细胞共培养时,神经干细胞贴壁分化加快,NSE阳性细胞及TH阳性细胞明显多于对照组(P<0·05)。结论星形胶质细胞源性因子可快速诱导神经干细胞向神经元细胞、包括多巴胺神经元细胞分化,提示星形胶质细胞支持神经元发生。     

血脑屏障模型的建立与评价

离体血脑屏障模型是研究血脑屏障的重要工具，本文概述了血脑屏障模型的发展过程，重点介绍了脑微血管内皮细胞、星形胶质细胞的培养，血管内皮细胞和星形胶质细胞共同培养建立血脑屏障模型的方法，以及血脑屏障模型的评价手段。     

实验性血脑屏障细胞模型及其评价

目的 :建立模拟在体血脑屏障 (blood brainbarrier ,BBB)形态、功能的体外细胞模型。方法 :将纯化的 1型星形胶质细胞、脑微血管内皮细胞分别接种于涂有鼠胶原的Transwell多聚酯膜两侧 ,共培养。分别检测共培养后BBB特异性酶γ 谷氨酰转酞酶 (γ GT)含量 ;测定荧光素钠 (FLU)、12 5Ⅰ 牛血清白蛋白 (BSA)在该模型上的通透量。结果 :共培养后内皮细胞表现出典型细胞形态 ;γ GT含量随共培养酶活性增强 ;模型对大分子蛋白质和小分子物质都具有良好限制通透的作用。结论 :脑微血管内皮细胞与 1型星形胶质细胞共培养能够形成与在体BBB类似的结构 ,并具有在体屏障的限制物质通透、酶屏障的功能     

黄芩苷作用于脑微血管内皮细胞和星形胶质细胞对

背景：实验室前期研究表明黄芩苷可以提高体外培养的神经干细胞分化为神经元的比例。但考虑到体内实验中血脑屏障的存在,黄芩苷能否通过血脑屏障主要细胞成分而发挥诱导神经干细胞定向分化为神经元的能力还不清楚。 目的：分别将大鼠脑微血管内皮细胞、星形胶质细胞与神经干细胞共培养,观察在模拟体内复杂微环境条件下,黄芩苷能否定向诱导神经干细胞向神经元分化并促进分化神经元的成熟。 设计、时间及地点：细胞水平的体外对照观察,于2007-09/2008-03在天津中医药大学中医药研究院中药药理学重点实验室和南开大学医学院完成。 材料：取孕14 d SD大鼠,用以分离培养神经干细胞。 方法：利用Transwell装置,分别将脑微血管内皮细胞、星形胶质细胞和神经干细胞共培养。用含有10 μmol/L黄芩苷的培养基作用7 d,并设置空白对照组。以β-tubulinⅢ标记未成熟神经元,MAP-2标记成熟神经元,胶质纤维酸性蛋白标记星形胶质细胞。 主要观察指标：应用细胞免疫荧光化学染色检测神经干细胞分化后β-tubulinⅢ、MAP-2和胶质纤维酸性蛋白阳性细胞比例,以实时荧光定量反转录-聚合酶链反应技术检测黄芩苷对脑微血管内皮细胞和星形胶质细胞血管内皮细胞生长因子、神经生长因子和血小板衍生生长因子 mRNA表达的影响。 结果：与脑微血管内皮细胞共培养条件下,与空白对照组比较,黄芩苷可显著增加β-tubulinⅢ阳性细胞比例（P < 0.05）。与星形胶质细胞共培养条件下,与空白对照组比较,黄芩苷对β-tubulinⅢ、MAP-2和胶质纤维酸性蛋白阳性细胞比例均无明显影响（P > 0.05）。与脑微血管内皮细胞、星形胶质细胞共培养条件下,与空白对照组比较,黄芩苷可显著增加MAP-2阳性细胞比例（P < 0.01）。黄芩苷作用于脑微血管内皮细胞48 h,可以显著上调血小板衍生生长因子基因表达（P < 0.01）;作用72 h可显著上调星形胶质细胞血管内皮细胞生长因子、神经生长因子和血小板衍生生长因子基因表达（P < 0.01）。 结论：黄芩苷作用于脑微血管内皮细胞可诱导神经干细胞向神经元分化,黄芩苷同时作用于脑微血管内皮细胞和星形胶质细胞可诱导神经干细胞向神经元定向分化并促进其成熟,可能与黄芩苷调控脑微血管内皮细胞和星形胶质细胞生长因子分泌,改善微环境有关。     

胶质细胞对共培养的脑微血管内皮细胞增殖及功能的影响

目的 研究体外培养条件下，胶质细胞对脑微血管内皮细胞(BMEC)增殖及功能的影响。方法 模拟血-脑脊液屏障结构及内皮细胞、胶质细胞间相互影响的途径，建立内皮细胞与胶质细胞共培养模型，采用细胞计数、细胞活性检测、酶含量与细胞吞饮量测定对内皮细胞增殖和功能进行研究。结果 共培养和条件培养时，内皮细胞增殖能力减弱，细胞活性以及酶含量增高，细胞吞饮量则无明显变化。结论 胶质细胞可通过两种途径影响内皮细胞的生长。胶质细胞可诱导和维持微血管内皮细胞的脑表型，但并不能促进内皮细胞生长。     

SD大鼠星形胶质细胞的原代培养

目的 设计原代培养SD大鼠星形胶质细胞。方法 取新生第2-3dSD红皮鼠，无菌操作下取大脑皮质，剪碎、胰酶消化结合机械吹打使细胞分散，网筛过滤，差速粘附处理去除成纤维细胞，将未粘附的细胞悬液接种培养9～12d，置摇床．舍弃含脱落细胞的细胞悬液，以去除少突胶质细胞和小胶质细胞。细胞传代，GFAP免疫组化染色鉴定。结果 功分离培养了原代神经胶质细胞，并进行了星形胶质细胞的纯化，GFAP鉴定星形胶质细胞比例为95％以上。结论 建立星形胶质细胞体外培养方法，总结操作关键，以此为实验材料对于比较学研究是非常有帮助的。     

体内外不同环境对大鼠胚胎神经干细胞分化的影响

目的 探讨体内外不同环境条件对大鼠胚胎神经干细胞分化影响。方法 孕龄16天的大鼠胚胎神经干细胞体外扩增后移植至大鼠脑内尾状核出血部位；同时体外将其与新生大鼠Schwann细胞共培养，应用免疫组织化学和免疫荧光方法分另检测神经细胞、星形胶质细胞及少突胶质细胞特异性标志蛋白tubulin-β、MAP2，GFAP和GalC的表达。结果 移植到脑内的BrdU阳性细胞环绕脑出血区域分布，大部分细胞GFAP阳性，少部分细胞tubulin-β或ＧalC阳性；而与Ｓchwann细胞共培养后，神经干细胞大部分呈tubulin-β和ＭＡＰ２免疫荧光阳性，少部分呈ＧＦＡＰ或ＧalC免疫荧光阳性。结论 脑内尾状核出血环境诱导神经干细胞主要分化成神经胶质细胞；而体外与Ｓchwann细胞共培养主要诱导其分化成神经细胞。     

脑白质低灌注损伤后星形胶质细胞缝隙连接蛋白43的表达变化

目的研究慢性脑白质缺血后星形胶质细胞和缝隙连接蛋白Connexin43(Cx43)的变化。方法原代培养星形胶质细胞,建立体外慢性缺氧模型;双侧颈总动脉狭窄法,建立慢性低灌注脑白质损伤小鼠模型;免疫荧光共染观察星形胶质细胞活化与Cx43表达。Western蛋白定量分析髓鞘相关指标髓鞘相关糖蛋白MAG,星形胶质细胞标记物GFAP和Cx43的表达。结果与对照组相比,细胞慢性缺氧7d后,星形胶质细胞明显增生活化,伴随Cx43表达水平明显上调。Western blot发现,在慢性脑白质缺血过程中,MAG的表达逐渐降低,GFAP持续增高,Cx43表达明显上调。免疫荧光共标记可见,星形胶质细胞中Cx43表达上调,主要分布于胼胝体中央区。结论慢性脑白质缺血损伤过程伴随星形胶质细胞Cx43表达增加,Cx43可能成为临床治疗血管性认知障碍的新靶点。     

Development of a three-dimensional, all-human in vitro model of the blood-brain barrier using mono-, co-, and tri-cultivation Transwell models

In vitro models of the blood-brain barrier (B-BB) generally utilise murine or porcine brain endothelium and rat astrocytes which are commonly grown in foetal calf serum supplemented conditions which modulate cell growth rates. Consequently, results gained from these experimental models can be difficult to extrapolate to the human in vivo situation since they are not of human origin. The proposed in vitro Transwell model of the B-BB is a multi-culture human cell system. It requires reconstruction of the human derived B-BB components in vitro (cerebral microvascular endothelial cells, astrocytes, and brain vascular pericytes) in a three-dimensional (3D) configuration based on Transwell filters. Different cell permutations (mono-, co-, and tri-cultivation) were investigated to find the most effective model in terms of tight junction resistance of the human cerebral microvascular endothelial cells. The B-BB model permutations comprised of human astrocytes (CC-2565 and SC-1810), human brain vascular pericytes (HBVP), and human cerebral microvascular endothelial cells (hCMEC/D3), under human serum supplementation. The models were assessed by trans-endothelial electrical resistance (TEER) measurements using an epithelial voltohmmeter, to validate the tight junction formation between hCMEC/D3 cells. Mono-, co-, and tri-cultivation Transwell models constructed with human brain-derived cells under human serum supplementation demonstrated that co-cultivation of astrocytes with endothelial cells produced the most successful model, as determined by TEER. Pericytes on the other hand improved tight junction formation when co-cultured with endothelial cells but did not improve the model to such an extent when grown in tri-cultivation with astrocytes.     

Assessments of tight junction proteins occludin, claudin 5 and scaffold proteins ZO1 and ZO2 in endothelial cells of the rat blood-brain barrier: cellular responses to neurotoxicants malathion and lead acetate

The blood-brain barrier (BBB) is essential for central nervous system (CNS) normal function. It is formed by endothelial cells with special characteristics, which confer the BBB with low permeability and high transendothelial electrical resistance (TEER). We previously demonstrated that malathion and lead, two neurotoxicants widely present in the environment, decrease TEER and increase permeability in in vitro models of the BBB. In this study we assessed tight junction disruption at the protein and gene expression levels using a rat brain microvascular endothelial cell line (RBE4) exposed to lead acetate at 10(-5)M and 10(-6)M, malathion at 10(-5)M, malaoxon at 10(-6)M, and their combinations. Cells were incubated with treatments for 2h, 4h, 8h, 16h, and 24h periods. Immunoblotting assessments demonstrated that protein levels of tight junction proteins occludin and claudin 5, and scaffold proteins ZO1 and ZO2 were decreased after treatments. Gene expression determinations did not correlate with the decreases in protein, indicating that the effects on these proteins were post-translational.     

eNOS解偶联对大脑中动脉栓塞后大鼠神经血管单元的作用及机制

目的探讨大鼠大脑中动脉栓塞模型中,内皮型一氧化氮合酶(e NOS)解偶联对神经血管单元的作用及机制。方法将80只雄性SD大鼠随机分为假手术组(n=40)和大脑中动脉栓塞(MCAO)组(n=40)。参照Longa法制作MCAO模型。栓塞后6 h,通过蛋白印记法检测缺血区e NOS不同偶联状态的表达情况及3-硝基酪氨酸(3-NT)的水平;通过尼氏染色比较缺血区神经元的形态及存活情况;通过免疫组织化学染色比较缺血区星形胶质细胞、微血管内皮细胞以及血脑屏障紧密连接对应的标记物GFAP、VWF、Occludin的表达水平;通过MRI的T2加权序列和动脉自旋标记(ASL)反应栓塞后脑组织病灶范围及血流情况。结果 MCAO后栓塞部位在T2序列上呈高信号,在ASL序列上血流信号减低。e NOS二聚体/单体的比例明显低于假手术组(P0.05);3-NT的含量明显高于假手术组(P0.05)。尼氏染色显示MCAO后神经元大量空泡形成,核固缩变性,数量显著减少;免疫组化结果显示,VWF、Occludin的表达较假手术组明显减少,而GFAP则明显增多(P0.05)。结论 MCAO会导致e NOS解偶联。e NOS解偶联后NO合成受限,而具有毒性作用的3-NT产生增多,炎症反应加重,从而对神经血管单元结构及功能产生破坏作用。     

Interferon-beta stabilizes barrier characteristics of brain endothelial cells in vitro

Multiple sclerosis (MS) is accompanied by a breakdown of the blood-brain barrier (BBB) leading to edema formation and aggravation of the disease. Interferon-beta (IFN-beta) has been approved for the treatment of MS and besides its immunomodulatory effects has been demonstrated to lead to a stabilization of BBB integrity in vivo. To investigate whether human recombinant IFN-beta exerts direct effects on the BBB, we used an in vitro BBB model in which brain endothelial cells in coculture with astrocytes form a tight permeability barrier for 3H-inulin and 14C-sucrose. Removal of the astrocytes from the coculture or alternatively addition of histamine resulted in an increased paracellular permeability for small tracers across the brain endothelial cell monolayer. Strikingly, in the presence of IFN-beta, permeability increase under both conditions was inhibited. Permeability changes were accompanied by minor changes in the staining for tight junction-associated proteins in brain endothelial cell monolayers. Taken together, our data demonstrate a direct stabilizing effect of IFN-beta on BBB cerebral endothelial cells in vitro that might significantly contribute to the beneficial effects of IFN-beta treatment in MS in vivo.     

Tyrosine phosphatase inhibition induces loss of blood-brain barrier integrity by matrix metalloproteinase-dependent and -independent pathways

Tight junctions between endothelial cells of brain capillaries form the structural basis of the blood-brain barrier (BBB), which controls the exchange of molecules between blood and CNS. Regulation of cellular barrier permeability is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins. We have analysed the impact of tyrosine phosphatase inhibition on tight junction proteins and endothelial barrier integrity in a primary cell culture model based on porcine brain capillary endothelial cells (PBCEC) that closely mimics the BBB in vitro. The tyrosine phosphatase inhibitor phenylarsine oxide (PAO) induced increased matrix metalloproteinase (MMP) activity, which was paralleled by severe disruption of cell-cell contacts and proteolysis of the tight junction protein occludin. ZO-1 and claudin-5 were not affected. Under these conditions, the transendothelial electrical resistance (TEER) was markedly reduced. PAO-induced occludin proteolysis could be prevented by different MMP inhibitors. Pervanadate (PV) reduced the TEER similar to PAO, but did not increase MMP activity. Cell-cell contacts of PV-treated cells appeared unaffected, and occludin proteolysis did not occur. Our results suggest that tyrosine phosphatase inhibition can influence barrier properties independent of, but also correlated to MMPs. Evidence is given for a role of MMPs in endothelial tight junction regulation at the BBB in particular and probably at tight junctions (TJs) in general.     

Compartmentalized coculture of rat brain endothelial cells and astrocytes: a syngenic model to study the blood-brain barrier

The specific structure of the blood-brain barrier (BBB) is based on the partnership of brain endothelial cells and astrocytes. In the last decade, cocultures of these two cell types have been developed as in vitro models. However, these studies did not allow close contacts between both cell types. We report here a syngenic coculture model using rat endothelial cells on one side of a polyethylene terephtalate filter and rat astrocytes on the other. Endothelial cells retain their typical morphology and are factor VIII and OX 26 positive. We optimized the diameter of the membrane pores to establish very close contacts between the cells through the membrane pores without mixing the two cell types. Transmission electron microscopy showed evidence of tight junction formation between the endothelial cells and few pinocytic vesicles. The cocultures reached high electrical resistances up to 1000 Omegacm(2) showing their ability to limit the passage of ions. A 15-fold increase in gamma-glutamyl transpeptidase activity was measured in the endothelial cells in coculture compared to endothelial cell monoculture. Our syngenic coculture represents a useful in vitro model of the rat BBB that may prove to be valuable for studying the passage of substances across the barrier as well as other aspects of the BBB function.     

HIV-1 Tat protein alters tight junction protein expression and distribution in cultured brain endothelial cells

Disruption of the blood-brain barrier (BBB) is widely believed to be the main route of human immunodeficiency virus (HIV) entry into the central nervous system (CNS). Although mechanisms of this process are not fully understood, alterations of tight junction protein expression can contribute, at least in part, to this phenomenon. Tight junctions are critical structural and functional elements of cerebral microvascular endothelial cells and the BBB. The aim of the present study was to examine the effects of HIV-1 Tat protein on expression of tight junction proteins. Primary cultures of brain microvascular endothelial cells (BMEC) were employed in these experiments. A 24-hr exposure of BMEC to Tat(1-72) resulted in a decrease of claudin-1, claudin-5, and zonula occludens (ZO)-2 expression, whereas total levels of occludin and ZO-1 remained unchanged. In addition, a short (3-hr) exposure of BMEC to Tat(1-72) induced cellular redistribution of claudin-5 immunoreactivity. Tat(1-72)-induced alterations of claudin-5 expression also were confirmed in vivo where Tat(1-72) was injected into the right hippocampus of mice. These findings indicate that HIV-1 Tat protein can markedly affect expression and distribution of specific tight junction proteins in brain endothelium. Alterations of only distinct tight junction proteins suggest a finely tuned effect of Tat(1-72) on the BBB. Because tight junction proteins are critical for the barrier function of the BBB, such alterations can lead to disturbances of the BBB integrity and contribute to HIV trafficking into the brain.     

A comparison of the induction of immortalized endothelial cell impermeability by astrocytes

The suitability of various commercially available endothelial cell lines in studies of astrocytic/endothelial cell interactions was assessed. The endothelial-like cell line ECV304 was compared with T24/83, Eahy929, and b.End5 and rat cerebral endothelial cells in their ability, when co-cultured with rat (C6) glioma cells, to form a transendothelial electrical resistance (TEER), an indicator of tight junction formation which is an important property of the blood-brain barrier. As reported previously, the basal TEER of ECV304 cell monolayers was significantly enhanced upon co-culture, an effect reproduced by human 1321N1 astrocytes and primary rat astrocytes. T24/83 cells formed a patchy, gapped monolayer, which produced a poor basal TEER with little in the way of an increase upon co-culture. Similarly, all the other cell monolayers analysed demonstrated poor TEERs that were only moderately increased upon co-culture. These data confirm that while no endothelial cell line with ideal features is available, ECV304 cells remain an appropriate choice especially for studies of astrocyte/endothelial cell interactions.     

Persistent SIV infection of a blood-brain barrier model

In order to better model HIV infection of the brain, a dynamic, in vitro model of the blood-brain barrier (the DIV-BBB) was characterized. The model was composed of simian brain microvascular endothelial cells (MVEC) cocultured with human fetal astrocytes under conditions of media flow. Simian immunodeficiency virus (SIV) was introduced into the DIV-BBB model in order to determine whether SIV infection has an effect on the blood-brain barrier (BBB). The cells of the DIV-BBB model were maintained for 127 days, during which a low permeability to sucrose developed. SIV infection of the BBB model was readily accomplished with cell-free virus. Results from ELISA for viral p27 protein, s-MAGI assays, and coculture techniques indicate that SIV productively and persistently infected the BBB model. These studies indicate that SIV can persist in MVEC without overtly compromising BBB function, and suggest that the DIV-BBB will be a highly valuable and suitable model for studies of HIV neuropathogenesis.