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1.
目的研究白喉毒素的无毒突变体CRM197对豚鼠血脑屏障通透性的影响和作用机制。方法给予豚鼠不同浓度的CRM197后,采用伊文氏兰法检测血脑屏障通透性的变化;荧光显微镜观察给予示踪剂伊文氏兰和荧光素钠后血脑屏障完整性的改变;透射电镜观察血脑屏障中血管内皮细胞间紧密连接等结构的变化。结果随着给予CRM197浓度的增加,血脑屏障对伊文氏兰的通透性逐渐增大,其中100,300,500μg/kgCRM197剂量组血脑屏障的通透性显著高于对照组(P<0.01)。给予300μg/kgCRM197后30min,未见有伊文氏兰-白蛋白渗漏到脑血管外周。透射电镜观察,不同浓度的CRM197作用后30min血脑屏障的完整性无明显改变,随着CRM197浓度的增大,脑微血管内皮细胞中吞饮小泡的数量有一定程度的增加,未见紧密连接明显开放。结论CRM197以剂量依赖的方式增加血脑屏障的通透性,其机制主要与胞吞转运增加有关,血脑屏障的完整性不受影响。  相似文献   

2.
目的:在Transwell 细胞跨膜培养系统中培养T84 细胞单层模拟肠黏膜屏障,并对Th17/ Treg 细胞相关因子处理前后T84 细胞单层的跨上皮电阻(TER)和辣根过氧化物酶(HRP)通过率进行检测,以探讨其对肠黏膜屏障通透性的影响。方法:将不同浓度的IL-17、IL-22 和IL-10 因子分别加入到Transwell 跨膜系统培养的T84 细胞单层中,之后测量不同时间(0、6、12、24 和48 h)T84 细胞单层的TER 和不同时间(6、24 和48 h)HRP 通过率的变化。结果:当不同浓度IL-17 和IL-22 处理时间大于6 h 时,继续增加细胞因子的浓度和继续延长作用时间,T84 细胞单层TER 会逐渐下降并且HRP 通过率逐渐增加。当IL-17 和IL-22 浓度大于100 ng/ ml,继续增加其浓度,T84 细胞单层的TER 和HRP 通过率均未见明显变化。当不同浓度IL-10 作用于T84 细胞后,随着时间和浓度的变化,T84 细胞单层TER 和HRP 通过率均未发生明显变化。结论:在一定的浓度范围内,IL-17 和IL-22 均可以造成T84 细胞单层通透性的增加,并且随着浓度的增加和作用时间的延长,T84 细胞单层通透性的增加也越来越明显。但是IL-10 并不影响T84 细胞单层的通透性。  相似文献   

3.
目的: 探讨E1A激活基因阻遏子(CREG)诱导的人血管内皮细胞(ECs)单层通透性改变中的作用及机制。方法: 用CREG过表达及CREG表达下调的ECs为模型,Transwell chamber弥散模型观察ECs单层通透性的改变; 荧光倒置显微镜观察细胞骨架肌动蛋白F-actin及黏附连接蛋白VE-cadherin在ECs中的分布和形态学改变;酶联免疫吸附实验(ELISA)检测ECs血管内皮生长因子(VEGF)分泌。结果: CREG过表达的ECs (EO组) 较EN组单层通透性明显增高 (P<0.05);CREG表达下调的ECs(ES组)较EN组单层通透性有所下降(P<0.05)。与EN组相比较,EO组细胞中F-actin排列紊乱,形成大量应力纤维; ES组F-actin则主要呈细丝状分布于细胞周边,中央分布较少。同时,EO组VE-cadherin在细胞周边的正常拉链状结构减少或缺失,细胞间隙增宽;而ES组VE-cadherin在细胞周边呈正常拉链状分布,细胞之间连接紧密。ELISA检测显示EO组细胞上清中VEGF分泌较EN组明显增加(P<0.05);ES组VEGF分泌较EN组减少(P<0.05)。应用VEGF中和抗体阻断后,CREG过表达引起的EO通透性增加的现象明显受到抑制。结论: CREG过表达可能通过VEGF介导的信号途径引起F-actin重构及VE-cadherin减少,使血管内皮细胞单层通透性增加。  相似文献   

4.
目的探讨非对称性二甲基精氨酸(ADMA)对单层内皮细胞通透性的影响,以及氧化应激、p38丝裂原活化蛋白激酶(MAPK)通路在此过程中的作用。方法利用Transwell小室建立单层内皮细胞屏障结构,设立实验组和对照组,实验组经浓度25、50、100、200μmol/L ADMA作用24 h和100μmol/L ADMA分别刺激细胞4、8、16、24 h,或分别经烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶抑制剂、p38 MAPK抑制剂预处理细胞后再加入ADMA刺激。随后用异硫氰酸荧光素(FITC)标记的右旋糖酐漏出法测定单层内皮细胞的通透系数Pa值,并用免疫荧光染色显示细胞骨架及细胞间连接的形态学改变。结果 ADMA呈剂量及时间依赖性的增加单层内皮细胞的通透性,同时促进内皮细胞中应力纤维的形成并破坏细胞间连接。NADPH氧化酶抑制剂和p38 MAPK抑制剂均可对抗ADMA的上述作用。结论 ADMA通过引起氧化应激,激活p38 MAPK通路,改变细胞骨架及细胞间连接的结构,使单层内皮细胞通透性增高。  相似文献   

5.
 目的 探讨马兜铃酸(AA)是否通过ERK1/2信号传导途径诱导人脐静脉血管内皮细胞(HUVECs)凋亡。方法 通过MTT法检测细胞增殖能力;通过Hoechst 33258荧光染色观察细胞凋亡的形态学改变;通过Annexin V-FITC/PI染色采用流式细胞仪检测细胞凋亡率;通过Western blotting法测定细胞内p-ERK1/2的水平。结果 马兜铃酸呈浓度和时间依赖方式抑制了内皮细胞增殖。马兜铃酸可引起内皮细胞出现凋亡形态学改变,且呈浓度依赖方式增加了内皮细胞凋亡率。同时,马兜铃酸可降低内皮细胞内p-ERK1/2的水平。应用ERK1/2抑制剂PD98095预处理后,细胞内p-ERK1/2的水平与AA (10 mg/L)组相比显著增加,马兜铃酸诱导的内皮细胞凋亡率亦被明显抑制。结论 马兜铃酸可诱导血管内皮细胞凋亡,其机制可能是通过抑制ERK1/2信号传导途径。  相似文献   

6.
张京伟  李明梅  丁琼  王莹  胡鹏超  汤梦婕  魏蕾 《微循环学杂志》2012,22(3):14-16,75,5,8,9
目的:观察乳腺癌常规化疗药物表阿霉素(EPI)对血管内皮细胞通透性的影响,探讨其在静脉注射时造成血管渗漏的机制。方法:用0、0.1、1.0、10、100μg/mlEPI处理人脐静脉内皮细胞株HUVEC-CRL-1730,光学显微镜观察细胞形态改变,Transwell小室检测单层内皮细胞的通透性,MTT法检测细胞增殖效率,RT-PCR和Western blotting检测血管扩张刺激磷蛋白(VASP)mRNA和蛋白表达水平。结果:10μg/mlEPI处理内皮细胞24h后,与对照组相比,EPI能显著抑制内皮细胞增殖(P<0.05),细胞收缩、变形,单层内皮细胞通透性增加(P<0.05);同时,VASP mRNA和蛋白表达水平均降低(P<0.05)。结论:EPI静脉注射造成的血管渗漏可能与EPI抑制细胞增殖以及通过降低VASP蛋白表达导致的内皮细胞通透性增加有关。  相似文献   

7.
目的:观察纤维蛋白对离体培养的大鼠脑血管内皮细胞白细胞介素6(IL-6)转录及蛋白水平表达的影响。方法:大鼠脑血管内皮细胞分离后培养,加入不同浓度的纤维蛋白,通过real-time PCR检测脑血管内皮细胞中IL-6转录水平,应用酶联免疫方法(ELISA)定量检测培养基和细胞裂解液中的IL-6水平。结果:加入不同浓度(0.03 g/L、0.1 g/L、0.3 g/L和1.0 g/L)的纤维蛋白24 h后,1.0 g/L纤维蛋白组的培养基中IL-6水平显著增高(P<0.01);然后加入1.0 g/L纤维蛋白2、4、8、24 h,培养基中的IL-6水平均显著升高(P<0.05);而加入不同浓度的胶原蛋白不引起IL-6水平的明显变化;real-time PCR结果显示,脑血管内皮细胞IL-6 mRNA的上调呈现出剂量和时间依赖性增加。结论:纤维蛋白是血管内皮细胞活化剂,可以上调大鼠脑血管内皮细胞中IL-6的表达,在蛋白和mRNA均呈现出剂量和时间依赖性增加。  相似文献   

8.
目的 探究血必净注射液(XBJ)对脂多糖诱导的脓毒症大鼠肺微血管内皮细胞损伤的作用及其对细胞紧密连接通透性的影响。方法 选择大鼠肺微血管内皮细胞,利用5μg/mL的脂多糖(LPS)处理大鼠肺微血管内皮细胞24 h,构建大鼠脓毒症肺损伤细胞模型,XBJ治疗组采用生药浓度2.5、5、10、25、50 mg/mL的XBJ共同进行干预24 h。CCK-8检测各组细胞活性,筛选最佳XBJ浓度;Hoechst染色检测各组细胞凋亡率;Western blot检测各组细胞凋亡相关蛋白Bax、Bcl-2和cleaved caspase3的表达;Transwell小室检测单层大鼠肺微血管内皮细胞通透性;Western blot检测紧密连接相关蛋白ZO-1、ZO-2和occludin的表达。结果 CCK8实验结果显示,XBJ的最佳浓度为10mg/mL。LPS处理后,大鼠细胞凋亡率明显升高,凋亡相关蛋白Bax/Bcl-2的比值和cleaved caspase3的表达水平增加;而XBJ治疗后,细胞凋亡率下降。LPS处理导致FITC-dextran含量显著增加,ZO-1、ZO-2和Occludin的蛋白表达水平降...  相似文献   

9.
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。  相似文献   

10.
目的:探讨NADPH氧化酶(Nox)抑制剂对晚期氧化蛋白产物(AOPP)刺激下血管内皮的保护作用。方法:体外培养人脐静脉内皮细胞进行实验,人血清白蛋白(HSA)作为阴性对照,用不同浓度(50、100和200mg/L)AOPP-HSA共同孵育8 h后,利用5-氯甲基二乙酸荧光素标记人急性单核细胞白血病细胞株THP-1的细胞渗出数量反映内皮细胞的通透性,研究不同浓度AOPP-HSA对单层细胞通透性的影响。此外,另将细胞分为HSA组、AOPP-HSA组和AOPP-HSA+二联苯碘(DPI)组,进而探讨AOPP-HSA对Nox活化水平的影响以及DPI对内皮细胞骨架重构和细胞通透性改变的作用。结果:AOPP-HSA可使血管内皮细胞通透性明显增加(P0.05)。AOPP-HSA可导致Nox磷酸化水平上升,并呈剂量依赖性。Nox抑制剂DPI预处理组可抑制AOPP-HSA刺激下Nox磷酸化水平的上升,从而抑制血管内皮细胞通透性增加及细胞骨架重构。结论:AOPP-HSA可通过激活Nox导致血管内皮细胞通透性受损,Nox抑制剂DPI可以降低其通透性及细胞骨架重构,起到一定的保护作用。  相似文献   

11.
目的 研究白细胞介素-8(IL-8)对血管内皮细胞紧密连接的影响.方法 采用免疫荧光染色检测经不同浓度和时间IL-8处理后EA.hy926细胞的3种紧密连接蛋白occludin、claudin-5和ZO-1的形态和分布;逆转录PCR(RT-PCR)检测3种蛋白mRNA的表达水平.结果 IL-8可改变内皮细胞紧密连接蛋白...  相似文献   

12.
Adherens junctions of the endothelium play a key role in the maintenance of endothelial permeability and are composed of the vascular endothelial (VE)-cadherin/catenin adhesion complex. We report that following tumour cell (MDA MB231 cells) adherence to the HUVECs, there was a rapid (within 5 min) redistribution of VE-cadherin, resulting in its transient loss from regions of endothelial cell-cell contact. The molecule gradually reorganised within the endothelial cell contacts after this time. It was further shown that the overall expression of VE-cadherin did not change, however, the amount of alpha- and beta-catenins coprecipitated with VE-cadherin markedly decreased after 5 min of tumour cell adhesion to the HUVECs. Immunoprobing of these samples with anti-phosphotyrosine antibodies demonstrated that the tyrosine phosphorylation of VE-cadherin was significantly increased following 5 min of tumour cell adhesion. Together, these results suggest that the adhesion of tumour cells to HUVEC promotes the redistribution of VE-cadherin from interendothelial adherens junctions, an effect that may be attributed to the increase in tyrosine phosphorylation of members of the VE-cadherin/catenin adhesion complex. This, in turn, may increase vascular endothelial permeability and facilitate the transendothelial migration of tumour cells during extravasation.  相似文献   

13.
IL-8诱导血管内皮细胞迁移的实验研究   总被引:6,自引:1,他引:5  
为了探讨不同浓度IL-8诱导的血管内皮细胞迁移从而为进-步研究IL-8引起血管内皮细胞迁移的分子机理选择最佳的IL-8浓度。采用内皮细胞transwell小室迁移率分析法和刮除-迁移分析法,考察IL-8对血管内皮细胞迁移的影响。结果表明,各种浓度的IL-8均可促进血管内皮细胞迁移,其中以IL-8浓度为100ng/ml时内皮细胞迁移率最高。  相似文献   

14.
目的研究3种不同磁性纳米颗粒对体外培养的血管内皮细胞中活性氧(reactive oxygen species,ROS)水平和细胞间连接的影响,探讨二者之间的关联性。方法将原代人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)随机分为对照组和不同磁性纳米颗粒暴露组。利用动态光散射(dynamic light scattering,DLS)对纳米颗粒的粒径和电势进行表征;采用细胞计数试剂盒(cell counting Kit-8,CCK-8)法测定细胞活性;通过二氯二氢荧光素-乙酰乙酸酯(2',7'-dichlorofluorescin diacetate,DCFH-DA)荧光探针标记和流式细胞术检测细胞中ROS水平;利用普鲁士蓝染色和透射电镜方法观察内皮细胞对磁性纳米颗粒的摄取。对细胞表面血管内皮钙黏蛋白(vascular endothelial cadherin,VE-cadherin)进行免疫荧光标记,在激光共聚焦显微镜下观察细胞间连接,并通过Western blot检测VE-cadherin表达水平。结果磁性纳米颗粒能诱导内皮细胞内ROS水平上升,降低VE-cadherin表达水平,细胞间缝隙增大。抗氧化剂N-乙酰半胱氨酸处理可使ROS水平下降并减少细胞缝隙。由于组分、表面修饰、尺寸等因素不同,磁性纳米颗粒对内皮细胞活性、ROS水平及VE-cadherin产生不同程度的影响。结论不同磁性纳米颗粒对内皮细胞活性氧和细胞间连接的影响不同;在实验所采用的低剂量暴露下可影响内皮细胞连接的完整性。  相似文献   

15.
同型半胱氨酸对血管内皮细胞PAI-1活性及其mRNA水平的影响   总被引:4,自引:0,他引:4  
探讨血浆同型半胱氨酸(Hcy)致血管病变的机制。培养人脐静脉内皮细胞株,用发色底物法测定细胞上清的纤溶酶原激活物抑制物-1(PAI-1)活性,细胞原位杂交及辉度扫描检测PAI-1 mRNA水平。结果表明H 驻作用血管内皮细胞后,PAI-1活性随Hcy浓度增加和Hcy作用时间延长而呈递增趋势,PAI-1 mRNA灰度面积积分值随Hyc浓度的增加而增加。提示Hcy抑制内皮细胞纤溶能力是Hcy致血栓形成和血管病变的一个机制。  相似文献   

16.
IL-1β is a pleotropic cytokine that may mediate increased procoagulant activity and permeability in endothelial tissue during inflammatory conditions. The procoagulant effects of IL-1β are mediated through induction of tissue factor (TF) but its alterations on vascular permeability are not well characterized. We found that IL-1β induced a rapid and dose-dependent increase in TF activity in human umbilical vein endothelial cells (ECs) under routine culture conditions. However, IL-1β caused a rapid and marked increase in permeability across confluent EC monolayers using a two-compartment in vitro model only in the presence of factor VIII-deficient plasma that was completely abrogated by neutralizing anti-TF antibody pre-treatment. In vitro permeability was associated with loss of EC surface expression of VE-cadherin and contraction of F-actin cytoskeletal elements that resulted in EC intercellular gap formation. These data demonstrate that IL-1β induces marked changes in permeability across activated endothelium via a TF dependent mechanism and suggest that modulation of TF activity may represent a strategy to treat various acute and chronic inflammatory conditions mediated by this cytokine.  相似文献   

17.
In order to examine the possible implication of capsular polysaccharide (CP) types 5 and 8 (CP5 and CP8) from Staphylococcus aureus in the pathological mechanism associated with staphylococcal infections, we tested the immunomodulatory effects of CP5 and CP8 on human epithelial KB cells, endothelial cells, and monocytes. Using biotinylated CP5 and CP8, we provide evidence that both CPs bind to KB cells, endothelial cells, and monocytes in a dose- and calcium-dependent manner through specific interactions. These results were confirmed by competition experiments using soluble cell extracts. Furthermore, we show that CPs bind to identical cell membrane receptors on all three types of human cells and that human normal serum contains a factor(s) which inhibits the binding of both CPs to human KB cells, endothelial cells, and monocytes. The ability of CP5 and CP8 to stimulate the production of cytokines by the human cells was then examined. CP5 and CP8 trigger KB cells to produce interleukin-8 (IL-8); endothelial cells to produce IL-8 and IL-6; and monocytes to produce IL-8, IL-6, IL-1 beta, and tumor necrosis factor alpha. The release of cytokines by all three types of cells is time dependent and dose dependent, and the tumor necrosis factor alpha production by monocytes is not affected by the addition of polymyxin B. We further confirm that human normal serum inhibits the immunomodulatory effects of both polysaccharides on each kind of cell. These results confirm that S. aureus CPs act as bacterial adhesins having immunomodulatory effects for human cells.  相似文献   

18.
Electrical resistance across human umbilical vein endothelial cells (HUVECs) was measured using an electrical cell sensor system. The transendothelial electrical resistance (TEER) value was used to estimate the permeability through endothelial cells in vitro. Decrease in the TEER value was associated with increase in the passage of albumin through endothelial cells in the albumin permeability assay. The effects of cytokines and dengue virus infection on the permeability of HUVECs were examined by measuring the TEER value. Tumor necrosis factor alpha (TNF-alpha) at 1 and 0.1 microg/ml decreased the TEER value, but TNF-alpha at lower dose did not. Interferon-gamma (IFN-gamma) at 1 microg/ml also decreased the TEER value. In contrast, interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) or interferon-beta (IFN-beta) did not decrease the TEER value. The decrease in the TEER value was associated with the morphological changes of HUVECs. Dengue virus infection at a multiplicities of infection (m.o.i.) of 5 pfu/cell decreased the TEER value. Infection at an m.o.i. of 0.5 pfu/cell did not decrease the TEER value; however, addition of 0.01 microg/ml of TNF-alpha to these infected endothelial cells decreased the TEER value. The results suggest that TNF-alpha and dengue virus infection decrease synergistically the TEER value of endothelial cells. The TEER method is easy, reliable and can be applicable to further analysis of the increase in the permeability of endothelial cells in vitro induced by inflammatory cytokines and dengue virus infection.  相似文献   

19.
The pathogenesis of cerebrovascular lesions in hypertensive rats   总被引:4,自引:0,他引:4  
In this study we investigated the pathogenesis of hypertensive cerebrovascular lesions by light microscopy, immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. The brains of rats with experimentally induced hypertension exhibited severe edema and intracerebral hemorrhage. Light microscopy of the arteries showed severe medial lesions and the deposition of fibrinoid substance in the intima. Immunohistochemistry showed that intercellular adhesion molecule (ICAM)-1, platelet-endothelial cell adhesion molecule (PECAM)-1, interleukin (IL)-1alpha, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha endothelial cell expression was upregulated. Scanning electron microscopy of these arteries revealed the adhesion of neutrophils, monocytes, and a few platelets to endothelial cells, and their invasion of endothelial cell junctions and opened junctions. Transmission electron microscopy showed neutrophil and monocyte adhesion to the endothelial cells and neutrophil and monocyte invasion of endothelial cell junctions, intimal deposition of fibrinoid substance, and severe medial cell injury. Intravenously injected horseradish peroxidase insulated from endothelial cell junctions and, via pinocytotic vesicles, into the subendothelial space. These findings suggest that hypertension activates endothelial cells to increase the expression of adhesion molecules and cytokines, and induces neutrophil and monocyte adhesion and migration, resulting in endothelial cell injury and increased permeability of endothelial cells, which results in hypertensive arterial disease.  相似文献   

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