两种体外血脑屏障细胞模型的比较

目的 :比较两种不同体外血脑屏障 (BBB)模型的功能差异。方法 :采用大鼠脑微血管内皮细胞 (BMEC)、人脐静脉内皮细胞(HUVEC)分别与大鼠脑TIA共同培养于Transwell多聚酯膜的两侧 ,建立两种体外血脑屏障模型。测定两种模型中荧光素钠、牛血清白蛋白的通透量以及屏障特异性酶γ GT的含量。结果 :BEMC构建的BBB模型对低分子量荧光素钠和大分子量蛋白的通透性都小于HUVEC组。在细胞生物化学方面 ,BMEC组γ GT含量为 ( 11 3± 0 66)mgprotein ,而HUVEC组则未测出。结论 :同种属的BMEC与TIA共培养构建的BBB模型是模拟体内BBB的理想选择。     

体外血脑屏障模型的建立及功能测定

目的利用大鼠原代微血管内皮细胞及星形胶质细胞建立体外血脑屏障模型,并通过跨内皮细胞电阻(Trans-epithelium electrical resistant,TEER)方法对血脑屏障模型进行功能测定。方法原代分离纯化SD大鼠脑微血管内皮细胞和星形胶质细胞,用免疫荧光检测内皮细胞标志物VWF,紧密连接蛋白ZO-1,星形胶质细胞标志物GFAP;用微血管内皮细胞和星形胶质细胞在Transwell小室上建立体外血脑屏障模型,观察TEER值的动态变化。结果原代的微血管内皮细胞培养至融合后具有典型的梭形"铺路石"样外观,VWF鉴定细胞纯度达到95%以上,ZO-1免疫荧光鉴定证实细胞间形成紧密连接;原代培养的星形胶质细胞呈现具有多个突起的典型形态,GFAP鉴定细胞纯度达到95%以上;在第10 d,单独微血管内皮细胞血脑屏障模型的TEER值为(42±1.41)Ωcm2,内皮细胞和星形胶质细胞共培养血脑屏障模型的TEER值为(65±1.42)Ωcm2。结论建立了体外血脑屏障模型,通过TEER值测定证明共培养使模型更加完整,更加接近在体血脑屏障模型的特性。     

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。     

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。     

缺氧应激下血管内皮细胞调控小胶质细胞表型及其机制

目的 研究缺氧应激下内皮细胞形态表型改变对小胶质细胞活化、趋化和吞噬活性的调控及机制,解释血-脑屏障(BBB)开放的双时相现象.方法 采用鼠小胶质细胞系(BV2)和鼠血管瘤内皮细胞(EOMA)细胞株进行实验,制作内皮细胞缺氧损伤模型;将BV2细胞分别与正常及缺氧EOMA细胞共育培养,观察前者形态表型变化,检测其趋化和吞...     

干扰素λ对氧糖剥夺/复氧诱导的血-脑屏障模型通透性的影响

目的 探究干扰素-lambda(interferon-lambda, IFN-λ)对氧糖剥夺/复氧(oxygen glucose deprivation/reoxygenation, OGD/R)诱导的血脑屏障(blood brain barrier, BBB)模型通透性的影响及机制。方法 通过Transwell共培养模型,将b.End3细胞在Transwell的上腔室培养,星形胶质细胞在下腔室培养,构建BBB模型。将BBB模型分为对照组、OGD/R处理组、IFN-λ干预组,每组设3个复孔。OGD/R处理组给予OGD/R处理,IFN-λ干预组在给予OGD/R处理的基础上给予IFN-λ干预。采用qRT-PCR检测IFN-λ在OGD/R模型中是否发挥作用;通过qRT-PCR实验确定构成Transwell共培养模型的星形胶质细胞和脑微血管内皮细胞(b.End3)能否表达IFN-λ的受体;根据Transwell渗透性实验FITC-dextran-10000渗透情况,评估IFN-λ处理后对OGD/R模型BBB通透性的影响;通过qRT-PCR、Western blot和荧光共聚焦实验检测b.End...     

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。     

星形胶质细胞、周皮细胞与血脑屏障

血脑屏障（blood-brain barrier,BBB）是一个调节中枢神经系统内环境的细胞屏障,维持脑内离子、激素和递质等动态平衡。由脑毛细血管内皮细胞及细胞间紧密连接、星形胶质细胞终足、基膜和周皮细胞共同组成的一个细胞复合体。近年来,许多研究都是针对内皮细胞及其紧密连接,而对星形胶质细胞和周皮细胞研究相对较少,并且二者在BBB中的作用尚不清楚,本文就此方面的研究进展进行综述。[第一段]     

星形胶质细胞诱导成年骨髓基质细胞分化成神经元样细胞的实验研究

目的探讨星形胶质细胞能否在体外诱导成年大鼠骨髓基质细胞向神经元方向分化。方法采用新生鼠海马组织来源的星形胶质细胞与转染有绿色荧光蛋白基因的骨髓基质细胞(MSCs)进行共培养,并分成三组:共培养组、共培养 脑源性神经营养因子(BDNF)组、单纯碱性成纤维生长因子(bFGF) BDNF诱导分化组,在相差显微镜下每日观察、记录MSCs的诱导分化状况,并应用免疫荧光染色技术对分化后的MSCs进行鉴定,同时应用流式细胞术测定MSCs分化前后及星形胶质细胞的DNA含量。结果共培养第4天,部分MSCs已初步具备神经元形态:折光性强的锥形或圆形胞体及长的多极突起,免疫荧光染色呈微管相关蛋白(MAP-2ab)、神经元特异性烯醇化酶(NSE)阳性;DNA的含量测定结果显示诱导分化后第7天未发现有四倍体、六倍体细胞。结论(1)星形胶质细胞可以在体外诱导成年大鼠MSCs向神经元方向分化,这种分化并不是由细胞融合引起的。(2)星形胶质细胞具有调控神经元的分化、促进神经元成熟的功能。     

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。     

The perineurial and blood-nerve barriers in experimental diabetes

Summary The permeabilities of the blood-nerve barrier and the perineurial barrier were investigated in alloxan and streptozotocin diabetic rats and in the mutant diabetic mouse [C57BL/Ks(db/db)]. In the mouse model both fluorescence and electron microscopic techniques were employed using Evans blue albumin (EBA) and horseradish peroxidase (HRP), respectively, as exogeneous tracers. In the rat models only HRP was used. Tracers were applied locally around the sciatic nerve in order to investigate the perineurial barrier, and systemically to detect changes in the blood nerve barrier.None of the models was found to show increased permeability across either of the barriers.Supported by Grant MA 5857 from the Medical Research Council of Canada     

Serum-CSF protein gradients,the blood-CSF barrier and the local immune response

For the majority of proteins there is a steady state equilbrium between the serum and the CSF compartment which depends upon the hydrodynamic radii of the passively transferred molecules. For clinical purposes the serum-CSF concentration ratios of albumin (Aalb) and alpha2-macroglobulin (Qalpha2 M) have proven to be a reliable barrier parameter, which is more sensitive than the total protein level in certain diseases, e.g. disk protrusions, degenerative processes and metabolic disorders. The immunoglobulins G and A cope with the passive transfer mechanism in both normal conditions and all degrees of pure barrier impairments but deviate in cases with local immunoglobulin production. The method described produces a quantitative differentiation between the locally synthesized and the serum-derived immunoglobulin fractions. A humoral immune response within the central nervous system was found in certain stages of acute infectious diseases and with chronic inflammatory processes such as subacute sclerosing panencephalitis, neurolues and multiple sclerosis.     

Fluorescence-microscopic localization of in vivo injected ethidium bromide in the nervous system of the mouse

Summary Ethidium bromide is a compound which can suppress DNA, RNA, and protein synthesis in mammalian cells. It is a very useful tool in experimental neuropathology for studies on myelin lesions taking place in the spinal cord after injury to oligodendroglial cells following intracisternal or intraspinal administration.By using a technique described in this short original communication we can now directly trace the distribution of the compound in various cells of the central and the peripheral nervous systems after its administration to a living experimental animal. Therefore, in the future direct correlations can be made between the cellular distribution of the compound and its cytotoxic effects.Supported by grants from the Swedish Medical Research Council, projekt B12-12X-03020, Åhlénstiftelsen, Söderbergs stiftelser, Svenska Läkarsällskapet, and Trygg-Hansa     

黄体酮对脑梗死大鼠血脑屏障保护作用的机制研究

Objective To investigate the inhibitory effects of progesterone on the inflammatory response and its influence on the structure of the blood brain barrier (BBB) of rats after cerebral infarction. Methods Progesterone was injected intrapefitoneally following permanent middle cerebral artery occlusion (MCAO) in rats. Western blotting was used to detect the expressions of tumor necrosis factor-alpha (TNF-α) and claudin-5, the major component of tight-junction proteins associated with the BBB. The water content in the injured brain tissue was also examined. Results The expression level of TNF-α increased but claudin-5 expression decreased in the brain tissue of all the rats following MCAO. Progesterone treatment significantly reduced the TNF-α expression in comparison with the vehicle-treated rats, resulting also in ameliorated claudin-5 protein loss and reduced water content in the injured brain tissue. Conclusion Progesterone inhibits the inflammatory response and mitigates the brain damage after cerebral infarction in rats, suggesting the role of this steroid as a neuroprotective agent.     

In situ quantitative estimates of the age-related and diabetes-related changes in cerebral endothelial barrier antigen

To determine whether aging or diabetes, conditions known to alter the blood-brain barrier (BBB) is associated with changes in endothelial barrier antigen (EBA), a rat BBB-specific protein, in situ quantitation of forty vibrotome sections of cerebral tissue of 5 young (4 months old), 5 aged (26 months old) and 5 streptozotocin-induced diabetic rats after 4 weeks of diabetes were studied using immunohistologic techniques. The anti-EBA-stained microvessels were normalized against the total microvessels identified with either anti-EBA binding or anti-Glut-1 binding. The results indicate that the EBA-stained microvessels of the hippocampus, but not of other cerebral cortical areas or white matter, are reduced in aged rats(88.02±2.19%vs.80.65±2.44%, P < 0.01). There were no significant changes in the diabetic rats. These results support the notion that changes in the BBB correlate with the aging of the central nervous system.     

Antigen presentation by rat brain and retinal endothelial cells

Brain and retinal endothelial cells (EC) form the blood-brain and vascular blood-retinal barriers, respectively, and are believed to play a role in mediating T cell responses in the central nervous system. In this study, Lewis rat retinal and brain EC grown in vitro were capable of expressing MHC class II I-A but not I-E molecules following treatment with interferon-γ. In the presence of their antigen, CD4⁺ antigen-specific T cells were able to mediate lysis of retinal EC monolayers to a similar extent as brain EC. T cell proliferation was poorly supported by confluent retinal or brain EC monolayers, but subconfluent EC monolayers supported proliferation in a MHC class II (I-A)-restricted manner (P < 0.001). Exposure of T cells to confluent retinal EC monolayers resulted in them becoming less responsive to subsequent antigen presentation by thymocytes. Conversely, pre-exposure with subconfluent EC had no such effect. These results suggest that a non-proliferating EC monolayer is able to downregulate T cell responsiveness which may have important implications during lymphocyte traffic across the blood-tissue barriers of the central nervous system.     

Differential localization of alkaline phosphatase in barrier tissues of the frog and rat nervous systems: a cytochemical and biochemical study

We investigated the localization of alkaline phosphatase (AP) in the peripheral and central nervous systems of the frog (Rana pipiens) and rat. In the frog sciatic nerve, AP reaction product was seen as a precipitate within caveolae and vesicular profiles of perineurial cells, and frequently filled the extracellular space. In the rat peripheral nerve, AP reaction product appeared as small tufts on the cell surfaces and within vesicular profiles of endoneurial blood vessels. AP reaction product was not detected in the rat perineurium or in endoneurial blood vessels of the frog. In the frog central nervous system, AP reaction product was detected in the arachnoid membrane adjacent to the subarachnoid space, but not in the cerebral or pial vessels, whereas in the rat it was detected in the outer arachnoid membrane and in the cerebral and pial blood vessels. Biochemical analysis indicated a sevenfold higher AP activity in the frog perineurium over the endoneurium, whereas in the rat, threefold more activity was measured in the endoneurium over the perineurium. Levamisole, an AP inhibitor, decreased the enzyme activity by 95% in rat tissues, and by 70% in frog tissues and in plasma from both animals. Similar decrements were observed cytochemically. This study suggests that: (1) the distribution of AP varies between species, but that it is always present in at least one component of the blood-brain and blood-nerve barriers, (2) because barrier tissues of the nervous system have enzymatic activity, they may biochemically modify the adjacent environment, (3) vesicular profiles and caveolae in the blood vessels and perineurium may function as microenvironments for enzymatic activity, and (4) in the rat and frog, different isozymes of AP may be present.     

Characterization of a rat retinal endothelial cell culture and the expression of P-glycoprotein in brain and retinal endothelium in vitro

Retinal vascular endothelia form one aspect of the blood-retinal barrier and, like the blood-brain barrier, control the passage of molecules and cells into the parenchyma. To facilitate comparative in vitro studies, rat retinal endothelial cells have been cultured and characterised. Using immunocytochemical techniques, retinal endothelium was positive for von Willebrand's factor, tight junction-associated polypeptide (ZO-1) and the transferrin receptor. The cells also expressed high-affinity uptake of acetylated low-density lipoprotein. Using the monoclonal antibodies JSB-1 and C219, the product of the multidrug resistance gene, P-glycoprotein, was found to be expressed on primary cultures of both brain and retinal endothelium.     

Frequency of blood-retina and blood-brain barrier changes in multiple sclerosis

The frequency of blood-retina barrier (BRB) and blood-brain barrier (BBB) alterations was studied in 20 cases of Multiple Sclerosis (MS) (12 relapsing and 8 chronic-progressive). BBB impairment was found in 7 out of 20 patients (35%), 3 of whom had the chronic-progressive form of the disease. Alterations to BRB were found in 9 out 20 cases (45%): 2 out 12 (17%) of the relapsing cases and 7 out 8 of the chronic-progressive cases (87.5%). BBB impairment was found in 3 of the 9 cases (33%) with BRB alterations. Our findings indicate that BRB and BBB alterations do not occur simultaneously. We propose that the higher frequency of BRB alterations in chronic-progressive MS may be a sign of persistent antigenic stimulation.

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Sommario è stata studiata l'incidenza di alterazioni della barriera emato-retinica e emato-encefalica in 20 casi di Sclerosi a placche (12 recidivanti e 8 cronico-progressivi). è stato dimostrato un danno della barriera ematoencefalica nel 35% dei casi di cui 3 appartenevano alla forma cronico-progressiva. Il 45% dei casi, di cui il 17% recidivanti e l'87.5% cronico-progressivi, presentavano alterazioni della barriera emato-retinica. Il 33% dei casi con alterazioni della barriera emato-retinica avevano alterazioni della barriera emato-encefalica. I nostri risultati dimostrano che le alterazioni della barriera emato-retinica non sono simultanei a danni della barriera ematoencefalica. è ipotizzato che la maggiore incidenza di alterazioni della barriera emato-retinica nella S.P. cronico-progressiva può essere un segno di persistente stimolazione antigenica.