大鼠脑星形胶质细胞的体外培养及鉴定

目的 探讨新生大鼠大脑皮质星形胶质细胞的分离、培养以及鉴定方法.方法 取出生1～2 d的Wistar大鼠大脑皮质,用机械法和胰酶消化法分散细胞,制成细胞悬液,差速黏附处理后,将未贴附的细胞接种培养.待细胞铺满瓶底后,置摇床摇晃,舍弃含脱落细胞的培养液,细胞传代.GFAP免疫细胞化学染色鉴定.结果 成功分离培养了原代星形胶质细胞,传代培养的星形细胞形态典型,纯度可达95%以上.结论 成功建立了大鼠星形胶质细胞体外培养方法.     

非接触式共培养体外血脑屏障模型的跨膜电阻及通透性

目的建立大鼠脑毛细血管内皮细胞(BCEC)与星形胶质细胞共培养血脑屏障模型并评价其功能。方法采用SD大鼠原代分离培养获得BCEC和星形胶质细胞。经细胞形态学观察、免疫组化检测相关抗原后建立非接触式共培养血脑屏障模型,测定共培养模型所形成的跨细胞电阻及荧光素钠的通透性。采用LC-MS检测6个化合物透过血脑屏障模型的通透性,并与文献报道的体内数据进行比较。结果培养的BCEC多数为短梭形外观,免疫组化检测可见细胞高表达因子Ⅷ;星形胶质细胞呈现具有细胞突起的典型形态,免疫组化检测可见细胞高表达胶质纤维酸性蛋白。共培养的体外血脑屏障模型跨BCEC单层的电阻值为(373±41)Ω·cm2,荧光素钠跨BCEC单层的通透性为(0.34±0.14)×10-3cm·min-1,符合体外血脑屏障模型要求。对所选6个化合物体内外透过BBB模型渗透系数的比较,表明具有一定的相关性(R2=0.7679,P<0.05)。结论建立的体外BBB模型在跨内皮电阻和通透性方面具备了在体BBB的基本特性,可以用于模拟体内环境,进行药物早期筛选方面的研究。     

一种高纯度和高活力的大鼠脑微血管内皮细胞的提取及原代培养方法

目的建立一种纯度和活力较高、简单实用的大鼠脑微血管内皮细胞分离及原代培养方法,为建立体外血脑屏障提供材料。方法采集1-2周SD大鼠大脑皮质,应用Ⅱ型胶原酶和分散酶/胶原酶连续消化法,筛网过滤法及20%BSA和44%Percoll两次梯度离心法获得脑微血管段后,接种于培养瓶中进行原代培养,采用倒置显微镜对所培养的细胞进行形态学观察,以Ⅷ因子相关抗原免疫染色法对其进行鉴定。结果体外培养2 h后,微血管内皮细胞爬出血管段进行贴壁生长,3⁴ d呈典型的铺路卵石样结构,Ⅷ因子相关抗原免疫组化检测内皮细胞表达呈阳性,可见细胞胞质呈棕色,阳性细胞占99%以上。结论该方法能成功地分离并培养出高纯度的大鼠脑微血管内皮细胞,对体外血脑屏障的建立以及脑微血管内皮细胞的生物学特性和功能的深入研究具有重要意义。     

脑微血管内皮细胞的原代培养方法概述

脑微血管内皮细胞(brain microvascular endothelialcells,BMECs)是构成血脑屏障的主要成分,与脑水肿、脑缺血以及脑肿瘤等脑血管疾病的发生、发展密切相关。BMECs原代培养模型已被广泛应用于血脑屏障、脑血管疾病的病理、生理及分子生物学研究。该文对近年来国内外有关BMECs原代培养的方法进行综述,为脑血管疾病的研究提供基础。     

血脑屏障及其体外模型研究进展

血脑屏障是介于血液循环系统和脑部中枢神经系统之间的一种动态界面,主要由脑微血管内皮细胞、星形胶质细胞、周细胞及一系列转运体和紧密连接蛋白构成,它们形成了一种致密的屏障,维持脑部内环境的稳态平衡,但同时也对作用于中枢神经系统的药物的开发造成了困难。血脑屏障的体外模型有成本低、易操作、条件可控等优点,可以一定程度上预测体内实验的结果,减少在体实验成本,更符合伦理学,可用于脑部病理生理的研究和药物的筛选。综述了血脑屏障的组成和体外血脑屏障模型常用的细胞及其特点,旨在为血脑屏障体外模型的构建提供参考。     

大鼠脑皮质星形胶质细胞的原代培养、纯化及采用HCA技术进行GFAP鉴定

目的探索大鼠脑皮质星形胶质细胞(astrocyte,AS)的分离、原代培养及纯化方法,采用高内涵细胞成像分析技术(high content analysis,HCA)对其进行胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)鉴定。方法取2、3 d的新生SD大鼠,体视显微镜下获取大脑皮质,机械分离成1 mm3组织碎块,采用质量分数为0.25%的胰蛋白酶和0.2%的胶原酶(体积比为1∶1)消化后进行体外培养,分别观察接种1 h、3 d、5 d后原代AS基本形态结构,酶消化3 min后,接种30 min,5 d后传代AS基本形态结构;采用多次、多阶段的差速贴壁和震荡相结合的方法纯化细胞;采用HCA技术对AS进行GFAP免疫细胞化学鉴定。结果采用本法培养的大鼠脑皮质星形胶质细胞,数量多、活性佳、纯度高,胞突丰富且细长,并相互交织成网,呈现典型而良好的生长状态;经本法纯化后的星形胶质细胞,细胞纯度明显提高;经HCA技术进行GFAP鉴定,AS纯度质量分数达98%以上,且图片质量佳。结论建立了一种大鼠脑皮质星形胶质细胞原代培养及纯化方法,采用高内涵细胞成像分析技术进行GFAP鉴定的图片质量明显优于传统方法,该技术值得推广和应用。     

三七总皂苷对抗H_2O_2所致大鼠脑微血管内皮细胞损伤的物质基础研究

目的研究三七总皂苷对H2O2致大鼠脑微血管内皮细胞损伤产生保护作用的物质基础。方法分离并培养大鼠脑微血管内皮细胞(RBMEC),以MTT和LDH的释放为指标,观察三七总皂苷(TPNS)、三七皂苷R1、人参皂苷Rg1、Rd、Re和Rb1对RBMEC损伤的保护作用。结果500μmol·L-1的H2O2对原代培养的RBMEC产生明显的损伤。20～200mg·L-1的TPNS,16·0μmol·L-1的R1,37·5～75·0μmol·L-1的Rg1,8·0～16·0μmol·L-1的Rd及5·4～54·0μmol·L-1的Rb1对H2O2致RBMEC的损伤具有明显的保护作用(P<0·05或P<0·01),Re则无此作用。结论TPNS对H2O2所致原代培养的RBMEC损伤具有明显的保护作用,产生这一作用的主要物质基础可能为Rb1、Rg1和Rd。     

PAS-Na对锰致大鼠基底核原代星形胶质细胞损伤的拮抗模型探讨

目的探讨对氨基水杨酸钠(PAS-Na)对锰(Mn)致大鼠基底核原代星形胶质细胞损伤的拮抗作用,为深入Mn中毒机制及其防治研究提供科学技术平台。方法取SPF级新生24 h内SD大鼠基底核进行消化、传代培养,用荧光免疫法鉴定星形胶质细胞。给予不同浓度Mn (125、250、500和1 000μmol/L)和PAS-Na (5、50、500和5 000μmol/L)对星形胶质细胞处理24 h,用MTT法测定细胞存活率,按细胞存活率75%为Mn细胞毒性的筛选标准,细胞存活率100%为PAS-Na无细胞毒性标准,选择染Mn和PAS-Na干预剂量。结果染不同浓度Mn 24 h后,星形胶质细胞出现不同程度的肿胀或皱缩,并随染锰浓度增加而加重。正常培养液(对照)、125、250、500和1 000μmol/L Mn组细胞存活率依序为100. 0%、91. 5%、83. 3%、78. 2%和55. 6%。与对照组比较,250、500和1 000μmol/L Mn组细胞存活率均降低(P<0. 05)。随着Mn浓度增高,细胞存活率呈剂量-反应性下降(r=0. 979)。给予5、50、500和5 000...     

Development of an in vitro blood-brain barrier model based on immortalized porcine brain microvascular endothelial cells

Immortalized porcine brain microvessel endothelial cells (PBMEC/C1-2) were used to develop a model for measurement of blood-brain barrier permeation of central nervous system active drugs. Previous studies showed that a system using C6 astrocyte glioma conditioned medium leads to cell layers with transendothelial electrical resistance values up to 300 Omega cm(2) and a permeability coefficient P(e) of 3.24 +/- 0.14 x 10(-4) cm/min for U-[(14)C]sucrose, which is in good agreement to published values and thus indicates the formation of tight junctions in vitro. However, commercially available inserts for the Transwell system were not permeable for highly lipophilic compounds, such as diazepam. Systematic studies with different insert showed, that inserts with a pore width of 1 microm proved to be optimal for permeation studies of lipophilic compounds. Permeability studies with a set of three benzodiazepines further supported this finding.     

Establishment and functional characterization of an in vitro model of the blood-brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes.

OBJECTIVE: The aim was to establish a flexible, abundantly available, reproducible and functionally characterized in vitro model of the blood-brain barrier (BBB). METHODS: In a first step, bovine brain capillaries and newborn rat astrocytes were isolated. Subsequently, a co-culture of primary brain capillary endothelial cells (BCEC) on semi-permeable filter inserts, with astrocytes on the bottom of the filter was established. The cell material was characterized on the basis of specific cell-type properties and (functional expression of) specific BBB properties. RESULTS: BCEC displayed: (1) characteristic endothelial cell morphology; (2) expression of endothelial cell markers (i.e., CD51, CD62P, CD71 and cadherin 5); (3) marginal F-actin localization; (4) tight junction formation between the cells; (5) expression of gamma-glutamyl-transpeptidase (gamma-GTP); (6) expression of P-glycoprotein (Pgp); (7) functional transendothelial transferrin transport and uptake; (8) restriction of paracellular transport; and (9) high transendothelial electrical resistance (TEER). Astrocytes displayed characteristic astrocyte morphology and expressed glial fibrillary acidic protein (GFAP). Co-culture with astrocytes increased TEER and decreased paracellular transport. In addition, expression of the glucocorticoid receptor (GR) was demonstrated in the endothelial cells of the BBB, while no expression of the mineralocorticoid receptor (MR) was found. CONCLUSIONS: A high quality and mass-production in vitro BBB model was established in which experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and pharmacodynamics) and pathophysiological (e.g., disease influence on BBB permeability) objectives can be reproducibly performed.     

Evaluation of blood-brain barrier transporters by co-culture of brain capillary endothelial cells with astrocytes

To investigate the transport function of the blood-brain barrier (BBB), we employed an in vitro model of the BBB, consisting of a co-culture of porcine brain capillary endothelial cells (BCECs) with rat astrocytes. Porcine BCECs were cultured on a filter insert with rat astrocytes on the underlying plastic well. Rat astrocytes induced characteristic BBB properties of porcine BCECs, such as gamma-glutamyl-transpeptidase activity and intercellular adhesion of porcine BCECs. Next, the transport properties of P-glycoprotein (P-gp) substrate and several anionic compounds across the co-cultured porcine BCECs were characterized. Expression of P-gp was detected by immunocytochemistry, and efflux-directed transport of the P-gp substrate [(3)H]daunomycin was observed. Luminal-to-abluminal transport of the monocarboxylic acid transporter 1 (MCT1) substrate [(14)C]benzoic acid was saturable, and the K(m) value (3.05 mM) was similar to that for brain uptake observed in vivo. Abluminal-to-luminal transport of [(14)C]benzoic acid was also saturable, indicating that the monocarboxylic acid transporter of the BBB contributes to the efflux from the brain as well as to blood-to-brain influx. Abluminal-to-luminal transport of organic anions, [(3)H]dehydroepiandrosterone sulfate, [(3)H]estrone sulfate and [(3)H]estradiol 17beta-D-glucuronide was significantly higher than the corresponding luminal-to-abluminal transport. These results demonstrate the presence of multiple efflux transport pathways in this in vitro model.     

大鼠心肌微血管内皮细胞的体外培养

目的 探讨大鼠心肌微血管内皮细胞离体培养方法.方法 选取1～2周龄的SD大鼠,无菌条件下快速取出心脏,彻底去除大血管、心房、右心室和心内外膜.用植块法培养心肌微血管内皮细胞.倒置显微镜下形态学观察结合免疫细胞化学方法检测第Ⅷ因子相关抗原和CD34对培养细胞进行鉴定.结果 细胞呈单层贴壁生长,融合后呈"铺路石样";第Ⅷ因子相关抗原和CD34免疫细胞化学鉴定阳性.结论 成功培养出纯度较高的大鼠心肌微血管内皮细胞,方法简便,可用于心血管疾病的研究.     

建立体外共培养血脑屏障模型评价纳米粒的胞吞转运和毒性

目的建立大鼠脑毛细血管内皮细胞(BCECs)和星形胶质细胞共培养模型，评价纳米粒的跨血脑屏障(BBB)转运和对内皮细胞紧密连接的毒性。方法采用复乳/溶媒蒸发法制备载荧光探针6-香豆素的聚乙二醇-聚乳酸纳米粒。首先，分别从新生鼠大脑分离培养BCECs和星形胶质细胞并进行免疫组化鉴定。然后，将BCECs接种于细胞培养池微孔膜的上面，将星形胶质细胞接种于膜下面建立共培养模型。分别测定¹⁴C-蔗糖和纳米粒的渗透系数。结果载6-香豆素纳米粒的平均重均粒径为(102.4±6.8) nm，zeta电位为(-16.81±1.05) mV。BCECs的VIII因子表达呈阳性；星形胶质细胞的胶质原纤维酸性蛋白表达呈阳性。模型的跨内皮细胞电阻值为(313±23) Ω·cm²。扫描电镜和透射电镜观察发现，共培养模型中的BCECs形成紧密连接。纳米粒的质量浓度低于200 μg·mL^-1时，不影响¹⁴C-蔗糖渗透系数的改变，表明其不会影响BBB内皮细胞的紧密连接。10 μg·mL^-1载6-香豆素纳米粒的渗透系数为0.29×10^-3 cm·min^-1。结论该大鼠BBB模型与体内情况接近，适合用于评价纳米粒的脑内转运和毒性。     

The role of pericytes in blood-brain barrier function and stroke

Central nervous system pericytes have critical and complex inductive, structural, and regulatory roles interacting with other cell types of the neurovascular unit, especially endothelial cells and astrocytes. Pericyte-endothelial interactions are particularly prominent for blood-brain barrier (BBB) maintenance, with profound effects on basement membrane and endothelial tight junction structure and function. Under experimental conditions of hypoxia-ischemia mimicking stroke, pericytes migrate from their usual microvascular location and influence, directly or indirectly, BBB permeability. The contractile properties of pericytes provide the capacity to regulate capillary blood flow, but this may have detrimental effects on ischemic injury. Stem cell characteristics of pericytes imply an important regenerative role following stroke. Pericytes thus appear to orchestrate multiple critical functions in stroke, involving blood flow, permeability, and repair of the neurovascular unit.     

The cytotoxicity of methylmercury in human microvascular endothelial cells and pericytes in culture

Methylmercury is an environmental neurotoxin that induces severe neurological damage in the brain of humans and animals. The main pathological characteristic of methylmercury neurotoxicity is the location of the damage; lesions are localized around the deep sulci and fissures in the cerebral cortex, such as the calcarine fissure, and the granule cell layer of the cerebellum. Since the localization of the damage is suggested to be a result of secondary damage occurring due to edematous change in the white cortex, the toxicity of methylmercury to cells that compose the microvessels-endothelial cells and pericytes-may be important for understanding the neurotoxicity of methylmercury. We investigated the toxicity of methylmercury to human brain microvascular endothelial cells and pericytes using a cell culture system. It was revealed that the toxicity of methylmercury to microvascular cells depends on the cell type and density. It is suggested that vascular tissue is one of the targets of methylmercury toxicity and that this may contribute to the progression of edematous change in the brain. Methylmercury may also be involved in the progression of cardiovascular diseases.     

Endotoxin-induced arterial endothelial barrier dysfunction assessed by an in vitro model.

Using an in vitro model in which albumin transfer across monolayers of bovine aortic endothelial cells (BAEC) was measured, we have shown that lipopolysaccharide (LPS) induces a concentration-dependent increase in endothelial permeability. This increase was biphasic, having an early peak at 2 h and rising again by 24 h. Both peaks were abolished by polymixin B (PMB) but were unaffected by N omega-monomethyl-L-arginine, N omega-nitro-L-arginine methyl ester or dexamethasone. Furthermore, LPS did not stimulate nitric oxide production by BAEC following 24 h exposure. Thus, the LPS-induced increase in permeability may account for the vascular leakage of septic shock, but the L-arginine-nitric oxide system does not appear to be involved.