人脐静脉内皮细胞单层通透性改变的效应因子

背景：外源性刺激引起血管屏障功能损伤的分子机制是血管病理生理学尚未阐明的热点问题之一。 目的：探讨炎症递质脂多糖诱导的人脐静脉内皮细胞单层通透性改变的效应分子,寻找有效治疗靶点。 方法：应用脂多糖刺激并观察人脐静脉内皮细胞骨架蛋白F-actin和细胞单层通透性的改变。应用荧光免疫组化和Western blot方法检测脂多糖刺激前后细胞中RhoA和SRF等信号分子的改变。并通过阻断实验证实RhoA-SRF信号通路的作用。 结果与结论：100 µg/L脂多糖刺激6 h可引起人脐静脉内皮细胞中F-actin快速重构并形成大量应力纤维,细胞单层通透性明显增强。细胞中活化RhoA的表达明显增加,SRF发生明显的入核转位现象。应用特异性分子抑制剂Y27632抑制RhoA的活化后,细胞中F-actin重构现象消失,细胞单层通透性增加也受到明显抑制,SRF蛋白发生明显的出现转位。而应用Latrunculin B抑制脂多糖刺激的人脐静脉内皮细胞中F-actin应力纤维形成,对抗通透性增加,但RhoA活化未受到干扰,SRF入核现象则受到抑制。提示RhoA-SRF通路的活化介导了脂多糖诱导的人脐静脉内皮细胞中F-actin重构和内皮单层通透性增加,特异性抑制F-actin也可以阻断脂多糖引起的血管内皮单层通透性增加,同时反馈抑制SRF的入核活化现象。     

川芎嗪对内毒素脂多糖诱导的体外血脑屏障模型通透性增高的保护作用及其机制

目的:探讨川芎嗪对内毒素脂多糖(LPS)诱导的体外血脑屏障模型通透性增高的保护作用及其调控机制。方法:利用脑微血管内皮细胞与星型胶质细胞共培养建立体外大鼠血脑屏障模型,随机分为正常对照组、川芎嗪对照组、LPS干预组和川芎嗪治疗组。采用γ计数仪检测~(125)I-BSA通透量观察体外血脑屏障模型通透性的改变,Western印迹法检测紧密连接蛋白(zonula occludens-1,ZO-1)表达量的变化。结果:LPS使体外血脑屏障模型对~(125)I-BSA的通透量明显增加,脑微血管内皮细胞ZO-1蛋白表达下降,川芎嗪治疗组能明显拮抗LPS的上述作用。结论:川芎嗪对LPS诱导的体外血脑屏障通透性增高具有保护作用,其机制与它能影响血脑屏障紧密连接蛋白ZO-1表达有关。     

血管内皮细胞单层通透性研究的现状

血管壁通透性增高,血浆渗出是炎症等多种疾病过程中最重要的病理变化之一,其发生机理目前尚不清楚。常用的研究方法有整体动物、离体灌流器官和体外培养的内皮细胞单层(endothelial monolayer)三个不同层次的模     

MRP8/MRP14通过损伤细胞骨架蛋白和连接蛋白介导内皮细胞通透性增高

<正>目的:探讨多耐药相关蛋白8(MRP8)、多耐药相关蛋白14(MRP14)及MRP8-14复合物对单层内皮细胞通透性的影响及其机制。方法:分别采用不同浓度的MRP8、MRP14及MRP8-14复合物刺激单层脐静脉内皮细胞     

单层内皮细胞通透性电导测定方法的建立

根据通过溶液的电导受通过面积影响的原理，设计了单层内皮细胞通透性电导测定方法，并与蛋白滤过方法进行了比较。研究结果表明，滤膜孔隙间的电导值与滤膜孔隙的总面积有良好相关性，应用电导测定方法能反映单层内皮通透性。电导测定方法与白蛋白滤过方法的结合应用，能更好地反映内皮单层通透性变化的程度与范围。     

爆炸冲击波对单层肺微血管内皮细胞通透性的影响

采用不同压力梯度爆炸冲击波对培养单层肺微血管内皮细胞滤膜作用的模型,观察了不同压力梯度爆炸冲击波对培养的肺微血管内皮细胞单层通透性的影响。结果表明,电导法测定时,100kPa左右的爆炸冲击波压力作用即可见到单层内皮通透性的显著增加;轻度爆炸冲击波的损伤主要以内皮细胞间隙的增加为主要类型,而中强度以上的爆炸冲击波损伤,则主要以内皮细胞的脱落性损伤为主。     

微囊介导VE-Cad胞吞参与了LPS作用后血管通透性增高的形成

目的:观察脂多糖(lipopolysaccharide,LPS)作用后微囊介导的血管内皮钙黏蛋白(vascular endothelial cadherin,VE-Cad)胞吞,及其在血管通透性增高中的作用。方法:采用人血管内皮细胞株CRL-2922,以及免疫组化、免疫印迹、免疫共沉淀等技术方法,观察LPS处理后不同时点细胞质膜微囊重要结构蛋白小窝蛋白1(caveolin-1,Cav1)的蛋白表达和磷酸化,Cav1与VE-Cad的共沉淀和共定位,以及微囊抑制剂对LPS处理后Cav1与VE-Cad的共沉淀、VE-Cad质膜表达和单层细胞通透性的影响。结果:(1)LPS处理后Cav1蛋白表达无显著变化,但其Tyr14位点的磷酸化水平逐渐增高(P0.05),Cav1与VE-Cad的免疫共沉淀逐渐增多(P0.05),免疫组化激光共聚焦显微镜观察在4 h时可见明显的共定位;(2)细胞质膜微囊抑制剂非律平(5 mg/L)可以显著减少LPS处理4 h Cav1与VE-Cad的免疫共沉淀(P0.05),增强VE-Cad的质膜表达(P0.05),改善单层细胞通透性(P0.05)。结论:细胞质膜微囊介导VE-Cad胞吞参与了LPS作用后血管通透性增高的形成。     

E1A激活基因阻遏子促进人血管内皮细胞VEGF分泌和单层通透性增加

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。     

P38 MAPK激活在高血糖致大鼠微血管通透性增高过程中的作用

目的研究P38 MAPK信号传导通路在糖尿病大鼠微血管内皮细胞通透性增高中的作用机制。方法SD雄性大鼠,腹腔注射链脲佐菌素(STZ 55 mg/kg)制备糖尿病模型。分正常组、糖尿病组、溶剂对照组、MKK6b(A)组和MKK6b(E)组。溶剂组:给予相同容量的柠檬酸缓冲液;MKK6b(A)组:在糖尿病模型基础上,实验前24 h尾静脉注射MKK6b(A)2.5 mL/kg;MKK6b(E)组:在实验前24 h给正常大鼠尾静脉注射MKK6b(E)2.5 mL/kg。用荧光强度检测微血管通透性;用荧光染色法观察微血管内皮细胞骨架蛋白的形态。结果糖尿病大鼠的微血管通透性明显高于正常组;注射MKK6b(E)可以增高大鼠微血管的通透性,而注射MKK6b(A)后微静脉的高通透性则被抑制。结论P38 MAPK信号转导系统参与介导了糖尿病微血管通透性增高的反应过程。     

Modulation of endothelial monolayer permeability induced by plasma obtained from lipopolysaccharide-stimulated whole blood

The aim of this study was to elucidate the time course of the permeability response of endothelial monolayers after exposure to plasma obtained from lipopolysaccharide (LPS)-treated human whole blood; to investigate the role of apoptosis in monolayer permeability, and to inhibit the permeability increase, particularly after addition of the plasma stimulus. Human umbilical vein endothelial cells (HUVEC) were cultured on semiporous membranes and the permeability for albumin was measured after exposure, according to different schedules, to LPS-conditioned plasma. Apoptotic HUVEC were measured by both flow cytometry and ELISA. A variety of agents, including antibodies against cytokines, inhibitors of NF-kappaB, and a caspase inhibitor, were added to HUVEC, either prior to or after the stimulus. A maximum increase of the permeability was achieved after 4-6 h of exposure to LPS-conditioned plasma. This response was not accompanied by an increase in the number of apoptotic HUVEC. Administration of antibodies against both Tumour Necrosis Factor-alpha (TNF-alpha) and Interleukin-1beta (IL-1beta) to HUVEC within 1 h after stimulation significantly reduced the permeability increase. Similarly, pyrollidine di-thiocarbamate (PDTC), but not N-acetylcysteine, could prevent the permeability response, and was still effective when added within 2 h after LPS-conditioned plasma. The TNF-alpha/IL-1beta signal present in LPS-conditioned plasma appears to increase endothelial permeability through intracellular pathways that very likely involve the activation of NF-kappaB. Although poststimulatory inhibition of the permeability response proves to be possible with agents such as PDTC, the window of opportunity appears very small if placed in a clinical perspective.     

RhoA在内皮-单核细胞激活多肽-Ⅱ增强血肿瘤屏障通透性中的作用

目的探索信号分子RhoA在内皮-单核细胞激活多肽-Ⅱ(endothelial monocyte activating polypeptide-Ⅱ,EMAP-H)增强血肿瘤屏障(blood-tumor barrier,BTB)通透性中的作用。方法采集出生3~5d的Wistar胎鼠的大脑皮质,应用酶消化法及葡聚糖离心法获得脑微血管段后,接种于培养皿中进行脑微血管内皮细胞(brain microvascular endothelial cells,BMECs)原代培养;将BMECs与C6脑胶质瘤细胞共培养,构建体外BTB模型;共培养后的BMECs随机分成3组(每组6例):对照组、EMAP-Ⅱ组和C3 exoenzyme+EMAP-Ⅱ组。测定跨内皮阻抗值和辣根过氧化物酶流量评估各组BTB通透性变化情况;Western blot法检测BMECs上紧密连接相关蛋白ZO-1的表达水平;免疫荧光法检测ZO-1和细胞骨架蛋白F-actin在BMECs上的分布与表达。结果与对照组相比较,EMAP-Ⅱ组BTB通透性显著增高,ZO-1和F-actin在BMECs上的表达水平显著降低,应力纤维形成明显增多;EMAP-Ⅱ的上述作用受到RhoA抑制剂C3 exoenzyme预处理的显著抑制。结论信号分子RhoA在EMAP-Ⅱ增强BTB通透性中发挥着重要的作用。     

Interleukin-4 increases the permeability of human endothelial cells in culture

BACKGROUND: IL-4 plays a key role in the induction of allergic inflammation, but its role as an effector molecule is less well-established. Although some observations suggest that IL-4 may mediate increased vascular permeability, which is a characteristic feature of allergic inflammation, evidence for a direct effect on endothelial cell permeability is lacking. OBJECTIVE: To determine the effect of human IL-4 on the albumin permeability of cultured human endothelial cells. METHODS: Human umbilical vein endothelial cells were cultured on permeable membranes and the albumin permeability of endothelial monolayers was measured with and without exposure to recombinant human IL-4. Endothelial cells were exposed to various concentrations of IL-4 (0.001-100 U/mL), for various durations (6-24 h), either in the presence or absence of anti-IL-4 antibody. Recovery of endothelial barrier function following exposure to IL-4 was also examined. RESULTS: IL-4 induced a dose-dependent, reversible increase in endothelial permeability to albumin. Low concentrations of IL-4 (1 U/mL) induced a significant increase in endothelial permeability (P=0.004). IL-4-mediated endothelial leak occurred rapidly, within 6 h of exposure. CONCLUSIONS: IL-4 has the capacity to induce vascular leak by a direct effect on cultured endothelial cells, suggesting a potential effector role for IL-4 in the pathogenesis of vascular leak in allergic diseases.     

p38丝裂原活化蛋白激酶通路在非对称性二甲基精氨酸诱导内皮细胞通透性增高中的作用

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。     

Baicalin protects against thrombin induced cell injury in SH-SY5Y cells

Baicalin, an extract from the dried root of Scutellaria baicalensis Georgi, was shown to be neuroprotective. However, the precise mechanisms are incompletely known. In this study, we determined the effect of baicalin on thrombin induced cell injury in SH-SY5Y cells, and explored the possible mechanisms. SH-SY5Y cells was treated with thrombin alone or pre-treated with baicalin (5, 10, 20 μM) for 2 h followed by thrombin treatment. Cells without thrombin and baicalin treatment were used as controls. Cell viability was detected by MTT assay. Cell apoptosis was analyzed by flow cytometry. Real-time PCR was performed to determine the mRNA expression of protease-activated receptor-1 (PAR-1). Western blotting was conducted to determine the protein expression of PAR-1, Caspase-3 and NF-κB. Baicalin reduced cell death following thrombin treatment in a dose-dependent manner, with concomitant inhibition of NF-κB activation and suppression of PAR-1 expression. In addition, baicalin reduced Caspase-3 expression. The above findings indicated that baicalin prevents against cell injury after thrombin stimulation possibly through inhibition of PAR-1 expression and NF-κB activation.     

Albumin permeability and electrical resistance as means of assessing endothelial monolayer integrity in vitro

Summary Methods were developed to measure albumin permeability and electrical resistance of bovine aortic endothelial cell (BAEC) monolayers cultured on porous polycarbonate filters. Permeability to 1% bovine serum albumin (Pe) was quantified by measuring the flux of fluorescent-labeled albumin with an apparatus in which there were no transmural oncotic or hydrostatic pressure gradients. The effect of passage of BAEC monolayers in culture on permeability was studied using 60 BAEC monolayers of Passage 6 to 10. There was no significant difference in Pe between passages, and the mean Pe of all monolayers was 4.5 ± 0.5 (SEM) × 10^–6 cm/s. Using these same BAEC monolayers, a fluorescent technique was developed to examine en face permeability patterns. Most BAEC monolayers demonstrated diffuse permeability across the monolayer, whereas others had focal regions of enhanced permeability despite similar Pe values. In those monolayers with punctate permeability, there were 5.4 ± 0.6 (SEM) focal regions of enhanced permeability per 1000 cells. To study the effect of culture time on monolayer integrity, electrical conductivities of nine BAEC monolayers were measured daily using a Millipore electrical resistance system. Electrical resistance increased from 4.5 ohm·cm² at Day 2 to a peak level of 11.4 ohm·cm² at Day 7 and then decreased daily to 4.0 ohm·cm² by Day 12. The in vitro BAEC monolayer has many of the transport characteristics of intact vessels, making these techniques useful in physiologic studies of the endothelial transport barrier. These methods provide relatively simple means of assessing the integrity of endothelial cell monolayers grown on porous substrates.     

Nitric oxide induced by tumor cells activates tumor cell adhesion to endothelial cells and permeability of the endothelium in vitro

Human fibrosarcoma HT1080 cell surface phenotype analysis revealed the expression of "cluster of differentiation 15" (CD15) antigen and to a lesser extent, of "very late antigen-4" (VLA-4). Expression of "endothelial-leukocyte adhesion molecule-1" (ELAM-1) was negligible on resting human umbilical vascular endothelial cells (HUVECs), but its expression could be induced by HT1080 conditioned medium. HT1080 cell adhesion to HUVECs was partially dependent on CD15/ELAM-1 adhesion molecules. HT1080 cell adhesion to HUVECs induced the enhancement of nitric oxide (NO) production from HUVECs. Exogenous NO and NO from HUVECs enhanced ELAM-1 expression on HUVECs, HT1080 cell adhesion to HUVECs, permeability of the HUVEC monolayer, and HT1080 cell invasion through the HUVEC monolayer. These enhancements were not induced by NO synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME). These results suggest that NO expression induced by tumor cells via the CD15/ELAM-1 adhesion system may contribute to enhancement of tumor cell adhesion to endothelial cells and hyperpermeability of the endothelium, facilitating tumor cell invasion.     

Morphine attenuates endothelial cell adhesion molecules induced by the supernatant of LPS-stimulated colon cancer cells

A large reservoir of bacterial lipopolysaccharide (LPS) is available in the colon and this could promote colon cancer metastasis by enhancing tumor cell adhesion, intravasation, and extravasation. Furthermore, adhesion molecules like ICAM-1, VCAM-1, and E-selectin play important roles in the adhesion of tumor cells to endothelium. This study was designed to determine whether morphine can attenuate the expressions of adhesion molecules up-regulated by the supernatant of LPS-stimulated HCT 116 colon cancer cells (LPS-Sup). In this study, we divided to three groups by cell-growth medium of human umbilical vascular endothelial cells (HUVECs): the control group was incubated in growth factor-free endothelial medium, the Sup group was incubated in the supernatant of HCT 116 cells (Sup), and the LPS-Sup group was incubated in LPS-Sup. To observe effect of morphine to the adhesion molecules expressions in the LPS-Sup group, we co-treated morphine with LPS or added it to LPS-Sup. Adhesion molecule expressions on HUVECs in all three groups were measured during incubation period. Consquentially, ICAM-1, VCAM-1, and E-selectin expressions on HUVECs were significantly lower when morphine was co-treated with LPS than not co-treated. Thus, we suggest that morphine affects the expressions of adhesion molecules primarily by attenuating LPS stimuli on tumor cells.     

c-Kit mutation reduce intestinal epithelial cell proliferation and migration,but not influence intestinal permeability stimulated by lipopolysaccharide

The proto-oncogene c-kit, as a marker of interstitial cells of Cajal (ICCs) in the gastrointestinal tract, plays an important role in the ICCs. Although limited evidences showed c-kit is present in the colonic epithelium but its roles remain unclear. In the present study, we aimed to investigate the expression, location and function of c-kit in the intestinal epithelium. Immunofluorescence, western blotting, and RT-PCR were performed to detect the expression and location of c-kit in the intestinal mucosa of WT mice. We investigated intestinal epithelial proliferation and migration in vivo by performing 5-Bromodeoxyuridine (BrdU) incorporation and Ki-67 staining in WT and Wads ^m/m mice. An Ussing chamber with fluorescein-isothiocyanate dextran 4000 was used to detect the transepithelial electric resistance (TER), short circuit current (ISC) and permeability across ex vivo colon segments under control and endotoxaemia conditions. We demonstrated that c-kit was located and expressed in the gut crypt compartment in WT mice, which was demonstrated in the c-kit mutant mice (Wads ^m/m). In addition, both the number of proliferating cells and the percentage of the distance migrated were lower in the Wads ^m/m mice than those in the WT mice. Moreover, the intestinal permeability, TER and tight junction were unaltered in the Wads ^m/m mice under endotoxic conditions compared with those in both the control condition and the WT mice. Altogether, these observations imply that the expression of c-kit in the colonic epithelium is involved in the proliferation and permeability of the colonic epithelium.