阪崎肠杆菌单克隆抗体的制备与特性鉴定

目的:制备抗阪崎肠杆菌的单克隆抗体并对其生物学特性进行鉴定。方法:采用灭活的阪崎肠杆菌菌体为抗原,免疫BALB/c小鼠,取血清效价高的小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,间接ELISA法筛选阳性杂交瘤细胞,mAb亚类检测试剂盒鉴定单克隆抗体的亚型,通过Western blot和间接ELISA法鉴定该单克隆抗体的特性、效价及mAb相对亲和力。结果:获得2株能稳定分泌抗阪崎肠杆菌mAb的杂交瘤细胞株,分别命名为1H7、2B12,抗体Ig亚类分别为IgG1和IgG2b;交叉反应显示单抗具有良好的特异性;ELISA分析表明制备的单抗效价在1×107～2×107,相对亲和常数达109L/mol,染色体鉴定分别为104和106条,符合杂交瘤细胞的特性。结论:抗阪崎肠杆菌单克隆抗体的成功制备为其快速检测方法的建立奠定了基础。     

巨噬细胞炎症因子1α对人脑微血管内皮细胞通透性的影响

目的研究巨噬细胞炎症因子1α(MIP-1α)是否具有开通血脑屏障的作用。方法以重组人MIP-1α直接作用于人脑微血管内皮细胞(HBMEC),免疫荧光方法检测紧密连接蛋白ZO-1的分布变化、跨内皮细胞电阻、HRP穿过HBMEC单层的改变、HBMEC细胞CC趋化因子受体5(CCR5)的表达,以及MIP-1α中和抗体和分泌MIP-1α的模式细胞(6T-CEM)与HBMEC单层共同温育时ZO-1的分布变化。结果MIP-1α作用下,HBMEC单层紧密连接结构被破坏,通透性增加,引起HBMEC细胞CCR5受体的表达,MIP-1α中和抗体阻断6T-CEM细胞对HBMEC单层ZO-1分布的改变。结论MIP-1α可能通过CCR5改变HBMEC单层通透性促进T淋巴细胞穿过血脑屏障。     

剪切力对血管内皮细胞骨架的影响及其机理

哺乳动物血管系统中最里面的一层是内皮细胞层。这层细胞在整个生命过程中将始终受到血管中血液的流体力学作用。流体对内皮细胞单位面积作用的力称为应力。主要包括平行作用于细胞表面的切应力和垂直作用于内皮细胞表面的压应力，切应力即我们常说的剪切力。细胞骨架是内皮细胞中重要的组成成分，其三种结构基础：微丝、微管、中间丝各成系统，分别与细胞质膜上的蛋白质脂质分子相互连接而成为细胞运动、细胞形态和跨膜信息传递的结构基础［‘］。对剪切力主要起响应的结构是微丝，是由双股Ｆ－ａｃｔｉｎ螺旋式组成的。剪切力对血管内皮细…     

小细胞肺癌细胞诱发人脑微血管内皮细胞紧密连接的开放

目的小细胞肺癌易发生脑转移,但其机制尚不完全清楚,本文探讨小细胞肺癌细胞对人脑微血管内皮细胞单层细胞间紧密连接开放的作用。方法应用免疫荧光技术观察人脑微血管内皮细胞间紧密连接结构改变;应用Western blot技术分析小细胞肺癌细胞与微血管内皮细胞单层共培养时紧密连接蛋白occludin的可溶性片段和不可溶性片段在内皮细胞紧密连接处的分布情况;应用跨内皮迁移实验及辣根过氧化物酶渗漏实验分析小细胞肺癌细胞与内皮细胞单层共培养后其跨内皮细胞单层的迁移能力和内皮单层渗透性的改变;利用ROCK特异性抑制剂Y27632,分析Rho/ROCK信号通路是否参与小细胞肺癌细胞跨内皮单层迁移。结果小细胞肺癌细胞能够通过人脑微血管内皮细胞单层迁移,在0h到12h之间随培养时间的延长小细胞肺癌细胞的迁移率逐渐增加;共培养8h时人脑微血管内皮细胞紧密连接处紧密连接结构蛋白(ZO-1和occludin)表达减少,通透性明显增加;ROCK特异性抑制剂Y27632能够阻断小细胞肺癌细胞引起的内皮细胞间紧密连接处紧密连接结构蛋白ZO-1和occludin表达减少、内皮单层通透性的增加及小细胞肺癌细胞跨内皮单层迁移。结论小细胞肺癌细胞与人脑微血管内皮细胞单层共培养诱发人脑微血管内皮细胞间紧密连接的开放,利于小细胞肺癌细胞的跨内皮单层迁移。     

高浓度尿素诱导人脑微血管内皮细胞系产生炎性因子

目的研究高浓度尿素诱导人脑微血管内皮细胞系(HBMECs)产生炎性因子及其机制。方法以相同渗透压的甘露醇为对照,高浓度尿素(25 mmol/L)干预HBMECs 3、6、12和24 h后,免疫荧光法观察细胞内肿瘤坏死因子α(TNF-α)和诱导型一氧化氮合酶(i NOS)的表达。蛋白免疫印迹法(Western blot)检测TNF-α、i NOS、环氧合酶-2(cycloxygenase-2,COX-2)、核因子κB(NF-κB)/P65和p-P65的表达水平。一氧化氮(NO)试剂盒检测细胞NO含量。结果高浓度尿素增强细胞内TNF-α和i NOS表达。细胞TNF-α、COX-2和p-P65蛋白水平在3和6 h明显高于对照组(P0.01);i NOS蛋白水平持续增高(P0.01)。NO含量在3 h明显增多(P0.05)。结论高浓度尿素诱导人脑微血管内皮细胞产生炎性因子。     

猪链球菌胞壁蛋白SSU05_0272对人脑微血管内皮细胞β-catenin的调控

目的:研究猪链球菌细胞壁蛋白SSU05_0272对人脑微血管内皮细胞β-catenin的调控.方法:采用免疫荧光、实时定量PCR、Western blot等方法从基因转录和表达水平研究SSU05 _0272对人脑微血管内皮细胞β-catenin的调控.结果:实时定量PCR结果显示SSU05_0272蛋白能下调β-catenin在人脑微血管内皮细胞中的表达;免疫荧光和免疫印迹结果也显示,SSU05_0272蛋白能下调β-catenin在人脑微血管内皮细胞中的含量.结论:SSU05_0272调控人脑微血管内皮细胞β-catenin的表达.     

抗人脑微血管内皮细胞单域抗体P5的制备及对人和大鼠血脑屏障的影响

目的制备抗人脑微血管内皮细胞单域抗体P5,研究P5对大鼠血脑屏障结构的影响。方法采用PCR技术将已获得的P5基因片段克隆至载体pET-30a-c并测序确证,重组载体经转化诱导后通过Western blot鉴定。采用免疫荧光和免疫组织化学方法观察P5在人及大鼠脑微血管内皮细胞中的结合水平,透射电镜观察大鼠血脑屏障超微结构的变化。结果Western blot结果显示表达产物的分子量为128kD,随着单域抗体P5(1μg/ml)作用时间的延长,P5与人及大鼠的脑微血管内皮细胞结合水平增加。透射电镜结果显示随着给予单域抗体P5浓度的增大(1μg/kg,10μg/kg),脑微血管内皮细胞中吞饮小泡的数量呈现一定程度的增加。结论成功获得抗人脑微血管内皮细胞单域抗体P5,P5能增加血脑屏障的胞吞转运。     

CCR5介导6T-CEM细胞穿过人脑微血管内皮细胞单层能力分析

目的 为进一步研究T细胞在阿尔茨海默病(AD)患者脑内发挥的作用,探讨CCR5在6T-CEM穿过人脑微血管内皮细胞(HBMECs)过程中所发挥的生物学功能.方法 应用免疫荧光和Western blot等技术,集中探讨了HBMECs膜受体CCR5在6T-CEM细胞穿过HBMECs过程中作用.结果 在6T-CEM细胞与HBMECs单层单独孵育过程中,引起HBMECs膜受体CCR5表达变化;HBMECs膜受体CCR5的高表达使6T-CEM细胞穿过HBMECs单层能力增强.结论 HBMECs膜受体CCR5参与了6T-CEM细胞穿过HBMECs单层过程.     

电磁脉冲对血管内皮细胞骨架微丝聚合肽肌动蛋白表达的影响

目的研究不同脉冲次数电磁脉冲（electromagnetic pulse,EMP）作用对人脐血管内皮细胞（ECV-304）骨架聚合态肌动蛋白（F-actin）表达的影响。方法EMP采用0、100、200、400次脉冲数各辐照ECV-304细胞,用异硫氰酸荧光素-鬼笔环肽染色F-actin和PI染色胞核的双标染色法,观察受辐照血管内皮细胞内微丝形态学的变化,记录并测定细胞F-actin的平均荧光强度。结果对照组细胞中的大部分荧光样物质呈弥漫状态,胞膜荧光较弱,胞浆内可见少量肌动蛋白纤维丝,方向不规则。与对照组相比,各暴露组细胞均可见其胞浆中微丝F-actin明显粗大、变长,其间的荧光样物质大多为较长的粗大应力丝,沿细胞纵轴排列较多,细胞内数量和荧光强度明显增加（P〈0.01）,细胞膜结构完整且荧光增强。随着EMP脉冲次数的增加而微丝F-actin表达显著增多,以400次脉冲组最为明显（P〈0.01）。结论EMP可引起血管内皮细胞的骨架F-actin表达增高且存在着一定的量效关系。     

细胞松弛素B对内皮细胞损伤修复的影响

目的观察细胞松弛素B对内皮细胞损伤修复过程中微丝骨架系统的形态结构变化,研究阻断微丝功能对内皮细胞损伤修复的影响。方法以培养单层内皮细胞损伤模型,采用免疫荧光染色和3H-TdR掺入法,研究微丝功能对创面愈合及细胞增殖的影响。结果内皮细胞在损伤修复过程中伴随微丝特殊而有序的变化。用细胞松弛素B破坏微丝,可不同程度抑制创面的愈合及细胞增殖,并呈一定的时间-剂量依赖关系。结论微丝功能在促进内皮细胞修复过程中起重要作用,可通过直接或间接效应影响DNA合成,从而影响修复过程。     

Outer membrane protein A expression in Enterobacter sakazakii is required to induce microtubule condensation in human brain microvascular endothelial cells for invasion

Enterobacter sakazakii (ES) causes neonatal meningitis and necrotizing enterocolitis with case-fatality rates among infected infants ranging from 40 to 80%. Very little is known about the mechanisms by which these organisms cause disease. Here, we demonstrate that ES invades human brain microvascular endothelial cells (HBMEC) with higher frequency when compared with epithelial cells and endothelial cells from different origins. The entry of ES into HBMEC requires the expression of outer membrane protein A (OmpA), as the OmpA-deletion mutant was sevenfold less invasive than the wild type ES and the bacterium does not multiply inside HBMEC. Anti-OmpA antibodies generated against the OmpA of Escherichia coli K1, which also recognize the OmpA of ES, did not prevent the invasion of ES in HBMEC. ES invasion depends on microtubule condensation in HBMEC and is independent of actin filament reorganization. Both PI3-kinase and PKC-alpha were activated during ES entry into HBMEC between 15min and 30min of infection. Concomitantly, overexpression of dominant negative forms of PI3-kinase and PKC-alpha significantly inhibited the invasion of ES into HBMEC. In summary, ES invasion of HBMEC is dependent on the expression of OmpA similar to that of E. coli K1; however, the epitopes involved in the interaction with HBMEC appears to be different.     

活血定眩胶囊含药血清减轻小鼠脑微血管内皮细胞bEnd.3缺氧损伤

目的:研究活血定眩胶囊含药血清对抗缺氧所致小鼠脑微血管内皮细胞bEnd.3损伤的作用。方法:将30只大鼠随机分为空白对照组15只和活血定眩胶囊组15只,采集2组大鼠血清。将bEnd.3细胞分为正常组、缺氧模型组和活血定眩胶囊血清组,给药后,bEnd.3细胞缺氧6 h。显微镜下观察细胞形态,流式细胞术检测细胞凋亡率及细胞周期,按试剂盒方法检测细胞上清液超氧化物歧化酶(SOD)活性和丙二醛(MDA)的含量。结果:活血定眩胶囊含药血清可显著对抗缺氧造成的损伤,明显改善bEnd.3细胞的形态,使缺氧所致细胞凋亡数明显减少,有效抑制缺氧诱导的bEnd.3细胞发生G_1/S期阻滞,抑制MDA生成,增强SOD活性。结论:活血定眩胶囊对缺氧所致bEnd.3细胞的损伤具有明显的对抗作用,其机制与增强细胞抗氧化能力、抑制细胞凋亡有关。     

Time lapse phase contrast video microscopy of directed migration of human microvascular endothelial cells on matrigel

Migration of microvascular endothelial cells is an early and critical step in angiogenesis. Formation of branching and polygonal cellular aggregates by endothelial cells on matrigel has often been considered to be an in vitro model for angiogenesis, although formation of lumens has not always been confirmed. The dynamics of migration of living cells of a human dermal microvascular endothelial cell line (HMEC-1) on a reconstituted basement membrane matrix have been captured in real time using time lapse video microscopy. The cells exhibit periods of quiescence and directed rapid migration by formation of extensions towards a specific target cell. Cells repeatedly extend flexible protrusions from the cell body both within the plane of the matrix and out of the plane of the matrix into the incubation medium. Connections between protrusions and target cells are made frequently, but not all cells which start to form protrusions achieve connections with other cells. Some of these migrating cells which do not connect arrest before reaching the target, or arrest and retract to their origin. After formation of multicellular polygonal structures, the structures contract to form amorphous clusters of fused cells without visible effects on the underlying matrix. The study demonstrates that time lapse video microscopy is a simple but very useful approach to monitor the dynamics of movements which vary in speed and frequency during migration of living cells.     

Activation of sonic hedgehog signaling attenuates oxidized low-density lipoprotein-stimulated brain microvascular endothelial cells dysfunction in vitro

The study was performed to investigate the role of sonic hedgehog (SHH) in the oxidized low-density lipoprotein (oxLDL)-induced blood-brain barrier (BBB) disruption. The primary mouse brain microvascular endothelial cells (MBMECs) were exposed to oxLDL. The results indicated that treatment of MBMECs with oxLDL decreased the cell viability, and oxidative stress was involved in oxLDL-induce MBMECs dysfunction with increasing intracellular ROS and MDA formation as well as decreasing NO release and eNOS mRNA expression. In addition, SHH signaling components, such as SHH, Smo and Gli1, mRNA and protein levels were significantly decreased after incubation with increasing concentrations of oxLDL. Treatment with oxLDL alone or SHH loss-of-function significantly increased the permeability of MBMECs, and overexpression of SHH attenuated oxLDL-induced elevation of permeability in MBMECs. Furthermore, SHH gain-of-function could reverse oxLDL-induced apoptosis through inhibition caspase3 and caspase8 levels in MBMECs. Taken together, these results demonstrated that the suppression of SHH in MBMECs might contribute to the oxLDL-induced disruption of endothelial barrier. However, the overexpression of SHH could reverse oxLDL-induced endothelial cells dysfunction in vitro.     

Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis

Human umbilical vein endothelial cells (HUVECs) have played a major role as a model system for the study of the regulation of endothelial cell function and the role of the endothelium in the response of the blood vessel wall to stretch, shear forces, and the development of atherosclerotic plaques and angiogenesis. Here, we use HUVECs and human microvascular endothelial cells to study the role of the HMG-CoA reductase inhibitor, simvastatin, and the small GTP-binding protein Rho in the regulation of angiogenesis. Simvastatin inhibited angiogenesis in response to FGF-2 in the corneal pocket assay of the mouse and in vascular endothelial growth factor (VEGF)-stimulated angiogenesis in the chick chorioallontoic membrane. Furthermore, simvastatin inhibited VEGF-stimulated tube formation by human dermal microvascular endothelial cells and the formation of honeycomb-like structures by HUVECs. The effect was dose-dependent and was not secondary to apoptosis. Geranylgeranyl-pyrophosphate (GGPP), a product of the cholesterol metabolic pathway that serves as a substrate for the posttranslational lipidation of RhoA, was required for membrane localization, but not farnesylpyrophosphate (FPP), the substrate for the lipidation of Ras. Furthermore, GGTI, a specific inhibitor of GGPP, mimicked the effect of simvastatin of tube formation and the formation of honeycombs whereas FTI, a specific inhibitor of the farnesylation of Ras, had no effect. Adenoviral expression of a DN-RhoA mutant mimicked the effect of simvastatin on tube formation and the formation of honeycombs, whereas a dominant activating mutant of RhoA reversed the effect of simvastatin on tube formation. Finally, simvastatin interfered with the membrane localization of RhoA with a dose-dependence similar to that for the inhibition of tube formation. Simvastatin also inhibited the VEGF-stimulated phosphorylation of the VEGF receptor KDR, and the tyrosine kinase FAK, which plays a role in cell migration. These data demonstrate that simvastatin interfered with angiogenesis via the inhibition of RhoA. Data supporting a role for angiogenesis in the development and growth of atherosclerotic plaques suggest that this antiangiogenic effect of Statins might prevent the progression of atherosclerosis via the inhibition of plaque angiogenesis.     

舒洛地特对低氧状态下人真皮微血管内皮细胞凋亡的影响

目的:探讨舒洛地特(sulodexide,SDX)对低氧状态下人真皮微血管内皮细胞(human dermal microvascular endothelial cells,HDMECs)凋亡的影响及其分子机制。方法:对HDMECs进行分组培养:常氧对照组在常氧状态下培养;低氧对照组于1%O_2、5%CO_2、37℃条件孵箱培养24 h;处理组用不同浓度(0.25、0.5和1 LSU/m L)舒洛地特作用于HDMECs低氧培养24 h。CCK-8法检测细胞存活率;流式细胞术检测细胞凋亡率;caspase-3活性检测试剂盒测定细胞内caspase-3活性;Western blot和real-time PCR检测促凋亡因子P53、Bax和caspase-3及凋亡抑制因子Bcl-2的蛋白及m RNA表达。结果:低氧状态下舒洛地特可提高HDMECs生长活力,降低其凋亡率,抑制促凋亡因子P53、Bax和caspase-3的表达以及caspase-3的活性,提高抑凋亡因子Bcl-2的表达。结论:舒洛地特可以抑制低氧状态下人真皮微血管内皮细胞的凋亡,其分子机制与抑制线粒体凋亡通路有关。     

Reconstituting neurovascular unit with primary neural stem cells and brain microvascular endothelial cells in three‐dimensional matrix

Neurovascular dysfunction is a primary or secondary cause in the pathogenesis of several cerebrovascular and neurodegenerative disorders, including stroke. Therefore, the overall protection of the neurovascular unit (NVU) is a promising therapeutic strategy for various neurovascular diseases. However, the complexity of the NVU limits the study of the pathological mechanisms of neurovascular dysfunction. Reconstituting the in vitro NVU is important for the pathological study and drug screening of neurovascular diseases. In this study, we generated a spontaneously assembled three‐dimensional NVU (3D NVU) by employing the primary neural stem cells and brain microvascular endothelial cells in a Matrigel extracellular matrix platform. This novel model exhibits the fundamental structures and features of the NVU, including neurons, astrocytes, oligodendrocytes, vascular‐like structures, and blood–brain barrier‐like characteristics. Additionally, under oxygen‐glucose deprivation, the 3D NVU exhibits the neurovascular‐ or oxidative stress‐related pathological characteristics of cerebral ischemia and the injuries can be mitigated, respectively, by supplementing with the vascular endothelial growth factor or edaravone, which demonstrated that the availability of 3D NVU in ischemic stroke modeling. Finally, the 3D NVU promoted the angiogenesis and neurogenesis in the brain of cerebral ischemia rats. We expect that the proposed in vitro 3D NVU model will be widely used to investigate the relationships between angiogenesis and neurogenesis and to study the pathology and pharmacology of neurovascular diseases.     

Increased P-glycoprotein function and level after long-term exposure of four antiepileptic drugs to rat brain microvascular endothelial cells in vitro

To investigate whether long-term exposure to four typical antiepileptic drugs (AEDs): phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ) and valproic acid (VPA) can increase P-glycoprotein (P-gp) level and function in primary cultured rat brain microvascular endothelial cells (rBMECs) in vitro, the rBMECs were incubated in culture medium containing indicated drugs (PB, PHT, CBZ, VPA and rifampin) for 60 days in a gradient concentration manner. Age-matched cells were incubated in normal culture medium. After a 60-day exposure to the indicated drugs, P-gp function and level in cells were measured using rhodamine 123 (Rho123) accumulations and Western blot analysis, respectively. Lower Rho123 accumulation in drug-treated cells was found than that in age-matched cells. Cyclosporin A (CsA) and verapamil (Ver) increased Rho123 accumulation both in drug-treated cells and age-matched cells. The magnitude of increased Rho123 accumulation in drug-treated cells was larger than that in age-matched cells. Higher P-gp levels were found to be consistent with decrease of Rho123 accumulation in drug-treated cells. The results verified the hypothesis that long-term exposure to the four antiepileptic drugs can induce P-gp function and level in rBMECs.     

小鼠脑微血管内皮细胞的原代培养与纯化

目的:探讨纯度较高的小鼠脑微血管内皮细胞的分离与原代培养。方法:取8～14周龄BALB/c或C57/BL6小鼠,无菌分离脑组织,采用改进的两种方法即2次酶消化和1次密度梯度离心法及1次酶消化和1次密度梯度离心法分离小鼠脑微血管段,接种于涂布有Ⅳ型胶原的培养皿上进行原代培养,相差显微镜下观察细胞形态,并以免疫荧光法鉴定Ⅷ因子相关抗原。结果:改进的两种方法均可见内皮细胞在接种后12～16 h从微血管段周围爬出,5～7 d时细胞呈"漩涡状"生长,并可见细胞汇合,90%以上培养的细胞Ⅷ因子相关抗原表达阳性。结论:改进后的两种方法均能进行较高纯度的小鼠腑微血管内皮细胞的分离和原代培养。