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。     

一种新型微孔底膜插入式培养皿的制作方法

目的探讨一种新型微孔底膜插入式培养皿的制作方法, 为进行体外细胞共培养提供技术支持.方法利用塑料离心管, 微孔滤膜为材料制备微孔底膜插入式培养皿; 在该插入式培养皿中接种大鼠脑微血管内皮细胞 (brain capillary endothelial cells, BCECs)(在星形胶质细胞条件培养液影响下), 以跨内皮细胞阻抗(trans-endothelial electronic resistance, TEER)为指标对该插入式培养皿进行评价.结果该插入式培养皿在培养条件下(未接种细胞)15 天内未出现破损; 共培养条件下TEER增幅明显:由(66.1±13.3) Ωcm2升至(182.2±6.7) Ωcm2.结论该微孔底膜插入式培养皿制作简便, 脑微血管内皮细胞在上生长良好, 并具有可调节性和可重复使用的特点, 是一种可用于体外细胞共培养相关研究工作有效的工具.     

幼儿语言中枢微血管的研究

脑血管形态的定量研究，对探讨脑微循环动力学的规律，阐明脑血管疾病的发病机制有重要意义。关于脑血管宏观分布及脑微血管密度的定量研究，已有不少报道。但有关脑语言中枢微血管的研究，尚未见报到。本文对幼儿语言中枢微血管进行定量研究、观察，从而为脑缺血时侧支循环建立机制的研究提供形态学基础。     

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。     

灯盏花素对大鼠脑微血管内皮细胞OGD损伤的保护作用及机制研究

<正>灯盏花素(breviscapine,Bre)是从短亭飞蓬中提取的黄酮类化合物,具有保护缺血性脑血管疾病、调节血管内皮功能、减轻炎性反应等作用[1]。本实验通过分离培养大鼠脑微血管内皮细胞(BMECs),建立氧糖剥夺(OGD)BMECs模型,研究Bre对BMECs损伤是否具有保护作用,并探讨其可能的机制。相关研究尚未见报道。1材料与方法     

血小板激活因子刺激血小板在脑微血管内皮细胞上粘附及药物的阻断作用(英文)

目的:研究血小板激活因子(PAF)刺激脑微血管内皮细胞导致血小板在内皮细胞上粘附及WEB,DMPP和粉防己碱的抑制作用. 方法:用[~3H]腺嘌呤标记血小板探讨PAF导致血小板在脑微血管内皮细胞上粘附和药物的抑制作用. 结果:PAF 10—100 nmol L~(-1)显著增加血小板与脑微血管内皮细胞的粘附率,WEB,DMPP和粉防己碱抑制由PAF刺激而导致的血小板在脑微血管内皮细胞上的粘附. 结论:DMPP和粉防己碱能够抑制PAF对脑血管的损害作用.     

血管紧张素Ⅱ诱导培养的牛脑微血管内皮细胞凋亡及其机制

目的　研究AngⅡ对牛脑微血管内皮细胞(BCMEC)有无诱导凋亡的作用。方法　利用流式细胞仪和透射电子显微镜检测体外培养的BCMEC凋亡。结果　流式细胞术显示AngⅡ (0 .0 1～ 10 μmol·L- 1)呈浓度依赖性诱导BCMEC凋亡。 10 μmol·L- 1AngⅡ诱导的BCMEC凋亡不能被 10 0 μmol·L- 1AT1受体拮抗剂氯沙坦所抑制 ,而能被 15 0kU·L- 1超氧化物歧化酶所部分抑制。BCMEC经 10 μmol·L- 1AngⅡ刺激 18h后 ,呈现胞浆浓缩、染色质凝集、核碎裂和凋亡小体形成等凋亡的超微结构变化。结论AngⅡ能诱导BCMEC凋亡 ,其机制可能与其刺激BCMEC产生的氧自由基有关。AngⅡ诱导脑血管内皮细胞凋亡可能是其参与脑血管疾病的机制之一     

牛视网膜微血管内皮细胞的培养及鉴定

目的 探讨牛视网膜微血管内皮细胞(BREC)的体外选择性培养和鉴定方法.方法 采用含0.075%胶原酶和0.03%牛血清白蛋白(BSA)的磷酸缓冲液(PBS)培养基选择性培养BREC.细胞鉴定采用经Ⅷ因子相关抗原免疫组化染色细胞鉴定内皮细胞.结果 通过选择性培养获得的BREC纯度达98%以上,并能连续稳定传代.BREC早期聚集在微血管碎片周围,呈片状鹅卵石样单层生长,存在接触性抑制.经Ⅷ因子相关抗原免疫组化染色可见胞质内棕色的免疫沉积物反应呈阳性.结论 本研究所采用的细胞培养方法可获得稳定生长与传代的较高纯度的视网膜微血管内皮细胞,简单、经济易操作.可为糖尿病视网膜病变等微血管病变研究提供可靠的(体外)试验方法.     

化合物EXP-2528对Ang II诱导大鼠脑微血管内皮细胞表达E-selectin和VCAM-1的影响

本研究利用体外培养大鼠脑微血管内皮细胞(brain microvascular endothelial cells，BMEC)模型，探讨了血管紧张素(Ang)II对大鼠脑微血管内皮细胞表达E-selectin和VCAM-1的影响及其机制，并评价了新型AT₁受体拮抗剂化合物EXP-2528对其的影响。采用RT-PCR和Western blotting分别检测大鼠脑微血管内皮细胞E-选择素(E-selectin)和血管细粘黏附分子-1(vascular cell adhesion molecule-1，VCAM-1)的mRNA及蛋白的表达。结果显示1×10^-7 mol·L^-1 Ang II分别孵育4 h和18 h可显著促进BMEC E-selectin及VCAM-1的mRNA及蛋白的表达，应用氯沙坦和新型选择性AT₁受体拮抗剂化合物EXP-2528选择性阻断AT₁受体对此有明显抑制作用。同时阻断AT₁受体和AT₂受体也可明显抑制Ang II诱导的E-selectin及VCAM-1表达的增加，但是与单独阻断AT₁受体相比无明显差异。而选择性阻断AT₂受体对Ang II诱导的E-selectin及VCAM-1的表达无明显影响。以上实验结果提示，Ang II可通过激动AT₁受体上调BMEC E-selectin和VCAM-1的表达，参与脑血管疾病的发生发展。     

烟碱对新生牛脑微血管内皮损伤的作用机制

烟碱是使人们吸烟成瘾的主要物质，已有研究表明，烟碱对心脑血管等组织和器官有直接损伤作用。本研究采用新生牛脑组织，分离培养脑微血管内皮细胞，观察烟碱作用于内皮细胞后细胞黏附分子的表达，探讨了烟碱对脑血管损伤的分子机制，主要结果如下：     

姜黄素抑制TNF-α诱导的BMEC与白细胞的粘附

目的研究姜黄素(curcumin)对脑微血管内皮细胞(brain microvascular endothelium cell,BMEC)和白细胞粘附的影响。方法采用髓过氧化酶法测定经肿瘤坏死因子(tumor necrosis factor-α,TNF-α)刺激的BMEC对白细胞的粘附;以RT-PCR和Western blot方法检测在TNF-α作用下BMEC表面ICAM-1表达。结果经TNF-α刺激的BMEC明显增加其与白细胞的粘附反应;curcumin(12.5~100mg.L-1)可剂量依赖性地抑制TNF-α所诱导的BMEC与白细胞的粘附;在TNF-α刺激前30min加入curcumin可抑制BMEC的ICAM-1表达。结论curcumin对TNF-α所致的BMEC损伤具有保护作用,该作用可能是curcumin下调ICAM-1表达,抑制BMEC与白细胞的粘附,从而保护内皮细胞免受损伤。     

Protective effects of orally administered honokiol on cerebral ischemia reperfusion in rats and on stroke in SHRsp

Honokiol is a protective agent for cerebral ischemia injury when administered intravenously. In the present study, we aimed to investigate the oral effect of honokiol microemulsion on cerebral ischemia-reperfusion (I-R) injury in rats and stroke in SHRsp. Both tMCAO and SHRsp models in rats were used to evaluate the efficacy of the microemulsion. Rat aortic segment contraction test, primary rat aortic endothelial cells and primary brain microvascular endothelial cells (BMECs) injured by OGD-R were used to explore its potential action mechanism. Oral honokiol microemulsion significantly reduced infarct volume, neurological score and brain water content in tMCAO model, and it evidently reduced neurological score and increased the survival rate of SHRsp. Moreover, honokiol significantly inhibited aortic contraction induced by KCl and phenylephrine, and L-NAME suppressed these inhibitory effects. On the other side, honokiol increased NO and p-eNOS levels in rat endothelial cells. In addition, it also protects BMECs against OGD-R injury and increased eNOS expression in BMECs. In conclusion, oral honokiol administration has protective effects in tMCAO and in SHRsp rats, and its action mechanism is likely to be associated with its vasodilative effect produced by eNOS activation and with its protective effect on BMECs.     

Lysophosphatidylcholine induces apoptosis and inflammatory damage in brain microvascular endothelial cells via GPR4-mediated NLRP3 inflammasome activation

Lysophosphatidylcholine (LPC), as the main active component of oxidized low-density lipoproteins (ox-LDLs), has significant effects in cerebrovascular disease. However, the complex mechanism by which LPC functions in brain microvascular endothelial cells (BMECs) is not clearly understood. In this study, BMECs were transfected with G protein-coupled receptor 4 (GPR4) siRNA or an NLRP3-overexpression plasmid, and GPR4 expression was identified by RT-qPCR and western blotting; IL-1β, IL-18, and IL-33 levels were evaluated by ELISA. Apoptosis was monitored by flow cytometry and Hoechst staining, while Caspase 3, ASC, NLRP3, and GPR4 protein expression were examined by western blotting. Our results showed that LPC significantly increased the levels of inflammatory cytokines (IL-1β, IL-18, and IL-33) and markedly induced apoptosis and NLRP3 inflammasome activation in BMECs. Moreover, LPC notably upregulated GPR4 in BMECs, and knockdown of GPR4 significantly attenuated the effects of LPC in BMECs. Above all, we also proved that LPC induced apoptosis and inflammatory injury in BMECs by causing GPR4 to activate NLRP3 inflammasomes. Therefore, GPR4-mediated activation of NLRP3 inflammasomes might be the underlying mechanism by which LPC promotes the progression of cerebrovascular disease. In summary we found that LPC is an important pathogenic factor in cerebrovascular disease, and can induce GPR4 to active NLRP3 inflammasomes.     

Exogenous expression of interferon-beta in cultured brain microvessel endothelial cells

Brain microvessel endothelial cells (BMECs) make up the blood-brain barrier (BBB) and regulate the passage of therapeutic proteins as well as drugs from the cerebrovasucular circulation to the brain. In the present study, we transferred mouse or human interferon-beta (IFN-beta) gene via cationic liposomes into primary cultures of bovine BMECs developed as an in vitro model of the BBB. The gene-transferred BMECs secreted transiently a substantial amount of IFN activity more efficiently during the growth phase than at confluence. This was suggested to be due to a difference in the potential for plasmid incorporation between growing and confluent BMECs in a series of cell association experiments with (32)P-labelled plasmid DNA. Furthermore, when BMEC monolayers in Transwell plates were transfected with the IFN-beta-expression vectors from the upper side, IFN-beta was predominantly detected in the upper compartments, suggesting polarized secretion of the transgene products in BMEC monolayers. These findings provide important basic information about therapeutic secretory protein gene delivery to BMECs.     

Alisol A 24-acetate protects oxygen–glucose deprivation-induced brain microvascular endothelial cells against apoptosis through miR-92a-3p inhibition by targeting the B-cell lymphoma-2 gene

ContextAlisol A 24-acetate has been used to treat vascular diseases. However, the underlying mechanisms still remain unclear.ObjectiveThe present study evaluated the antiapoptotic effect of alisol A 24-acetate on brain microvascular endothelial cells (BMECs) and explored the underlying mechanisms.Materials and methodsBMECs were injured through oxygen -glucose deprivation (OGD) after alisol A 24-acetate treatment. Cell viability and half-maximal inhibitory concentration (IC₅₀) were measured using CCK-8, whereas inflammatory factors and oxidative stress indicators were measured using enzyme linked immunosorbent assay. Cell invasion and wound healing assays were detected. Cell apoptosis was assessed using flow cytometry. B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X (Bax) expression were analyzed using Western blotting. Dual-luciferase assay was applied to detect target genes of miR-92a-3p.ResultAlisol A 24-acetate had an IC₅₀ of 98.53 mg/L and inhibited cell viability at concentrations over 50mg/L. OGD induced apoptosis and promoted miR-92a-3p overexpression in BMECs. However, alisol A 24-acetate treatment suppressed inflammation, improved migration and invasion abilities, increased Bcl-2 expression, inhibited Bax expression, and repressed apoptosis and miR92a-3p overexpression in OGD-induced BMECs. MiR-92a-3p overexpression promoted cell apoptosis and suppressed Bcl-2 expression, whereas its inhibitor reversed the tendency. Alisol A 24-acetate treatment relieved the effects of miR-92a-3p overexpression. Dual-luciferase assay confirmed that miR-92a-3p negatively regulated the Bcl-2 expression.ConclusionsThese findings suggest that alisol A 24-acetate exerts antiapoptotic effects on OGD-induced BMECs through miR-92a-3p inhibition by targeting the Bcl-2 gene, indicating its potential for BMECs protection and as a novel therapeutic agent for the treatment of cerebrovascular disease.     

Investigation of the mechanism of enhancement of central nervous system delivery of 2'-beta-fluoro-2',3'-dideoxyinosine via a blood-brain barrier adenosine deaminase-activated prodrug.

Enhanced central nervous system (CNS) delivery of certain poorly penetrating 2',3'-dideoxynucleosides has been achieved by designing prodrugs that are substrates for enzymes, such as adenosine deaminase (ADA), that are present at high activities in brain tissue. In this study, the potential role of adenosine deaminase localized within the endothelial cells of the blood-brain barrier (BBB) in providing enhanced intracellular and CNS delivery of an ADA-activated prodrug is assessed in vitro using cell culture models of the BBB. The kinetics of uptake and bioconversion of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA), a model ADA-activated prodrug of 2'-beta-fluoro-2',3'-dideoxyinosine, were determined in primary cultured bovine brain microvascular endothelial cells. Model-based simulations of CNS availability derived from in vitro estimates of parameters for simple passive diffusion and ADA-catalyzed deamination suggest that ADA that is localized within the BBB plays an important role in the conversion of F-ddA to 2'-beta-fluoro-2',3'-dideoxyinosine during its passage across the BBB. Consistent with in vivo observations, these simulations demonstrate that elevated levels of certain enzymes, such as ADA, in the brain microvascular endothelial cells of the BBB may be exploited in the design of brain-targeted prodrugs or drug-carrier conjugates, which brain tissue selectively converts.     

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.     

Salvianolic acid A prevented cerebrovascular endothelial injury caused by acute ischemic stroke through inhibiting the Src signaling pathway

Stroke is an acute cerebrovascular disease caused by ruptured or blocked blood vessels.For the prevention of ischemic stroke,the coagulation state of blood and cerebrovascular protection should be considered.Our previous study has shown that salvianolic acid A(SAA),which is a water-soluble component from the root of Salvia Miltiorrhiza Bge,prevents thrombosis with a mild inhibitory effect on platelet aggregation.In this study we investigated the preventive effects of SAA on cerebrovascular endothelial injury caused by ischemia in vivo and oxygen-glucose deprivation(OGD)in vitro,and explored the underlying mechanisms.An autologous thrombus stroke model was established in SD rats by electrocoagulation.SAA(10 mg/kg)was orally administered twice a day for 5 days before the operation.The rats were sacrificed at 24 h after the operation.We showed that pretreatment with SAA significantly improved the neurological deficits,intracerebral hemorrhage,BBB disruption,and vascular endothelial dysfunction as compared with model group.In human brain microvascular endothelial cells(HBMECs),pretreatment with SAA(10μM)significantly inhibited OGD-induced cell viability reduction and degradation of tight junction proteins(ZO-1,occludin,claudin-5).Furthermore,we found that SAA inhibited the upregulation of Src signaling pathway in vivo and vitro and reversed the increased expression of matrix metalloproteinases(MMPs)after ischemic stroke.In conclusion,our results suggest that SAA protects cerebrovascular endothelial cells against ischemia and OGD injury via suppressing Src signaling pathway.These findings show that pretreatment with SAA is a potential therapeutic strategy for the prevention of ischemic stroke.     

The blood-brain barrier sodium-dependent multivitamin transporter: a molecular functional in vitro-in situ correlation.

The molecular mechanism of biotin brain uptake was investigated using an in vitro bovine blood-brain barrier (BBB) cell model and an in situ mouse brain perfusion technique. A functional uptake/transport correlation of the in vitro and in situ characteristics of biotin uptake was investigated. Morphological and immunochemical characteristics (e.g., factor VIII expression) of the primary culture of brain microvessel endothelial cells (BMECs) were confirmed. Gene expression of the multidrug resistance (Mdr1) and sodium-dependent multivitamin (SMVT) transporters was also determined in BMECs. Biotin transport was saturable and Na(+)-dependent at the luminal side of the BBB. The estimated half-saturation concentrations (K(m)) of biotin uptake in vitro and in situ were 49.1 and 35.5 microM, respectively, supporting the presence of a carrier-mediated biotin transport system. Inhibition studies using various biotin derivatives and structural analogs demonstrated the structural requirements for biotin-SMVT interaction. Desthiobiotin and pantothenic acid significantly inhibited the uptake of biotin, whereas 2-iminobiotin and diaminobiotin were very weak inhibitors. Based on our results, there was a good correlation between the in vitro and in situ BBB models, suggesting that when a single membrane transporter is involved in substrate uptake, flexibility in choosing the experimental model can be afforded. The current results are also consistent with the suggestion that the properties of the BBB are likely to be organ-specific rather than species-specific. Further mechanistic and comparative studies are needed to validate these results. In conclusion, the in vitro transporter-based mechanism studies produced valuable molecular functional transport results that correlated well with in situ results.