血小板激活因子对内皮细胞形态参数影响的计算机定量分析

利用具有大容量、高运算速度和高分辨率的计算机图像分析系统建立了定量分析内皮细胞(EC)面积、形状因子、细胞间距及细胞间空隙面积占细胞单层面积百分比的软件,并分析了血小板激活因子(PAF)对EC形态     

血小板激活因子对内皮细胞形态参数的影响及其机制

内皮细胞收缩和细胞连接间隙开放是炎症介质引起血管壁通透性增高的主要机制。我们利用以APOLLODN 3500为主机的图像工作站建立了内皮细胞形态参数分析的软件,对血小板激活因子(PAF)引起的内皮细胞面积、形状因子、细胞间距及细胞间空隙面积占细胞单层面积百分比等形态参数变化进行定量分析,并研究了     

血小板激活因子对内皮细胞形态参数影响的计算机量分析

利用具有大容量、高运算速度和高分辨率的计算机图像分析系统建立量分析内皮细胞面积、形状因子、细胞间距及细胞间空隙面积占细胞单层面积百分比的软件，并分析血小板激活因子对ＥＣ形态参数的影响。结果表明，对照组ＥＣ相互连接紧密，细胞间隙较窄。ＰＡＦ作用６０ｍｉｎ，细胞明显回缩，膜表面有很多粗细不等的突起，细胞间隙增大。计算机定量分析表明，ＰＡＦ（１０＾８ｍｏｌ／Ｌ）作用１０ｍｉｎ，就可使细胞间距和细胞间空隙     

膀胱尿路上皮癌脱落细胞形态结构的定量分析

目的定量揭示膀胱尿路上皮癌脱落细胞(urothelial carcinoma exfoliated cell,UCC)巴氏染色的形态结构特征,筛选对膀胱尿路上皮细胞癌有重要诊断价值的几何参数。方法取107例尿液标本中500个UCC、439个尿路上皮结构不良脱落细胞(urothelium dysplastic exfoliated cell,UDC)和386个正常尿路上皮脱落细胞(uroepithelium normal exfoliated cell,UNC),巴氏染色后用计算机图像分析技术分别测试上述三类细胞质和细胞核的尺寸参数和形状参数,采用单因素方差和组间两两比较法(SNK法)分析各组细胞的测试结果。结果尺寸参数(Ac、Ap、An、Rnp、dmajc,、dmin,c、dmaj,n、dmin,n、Dc珚、D珚n、Cc、Cn)和形状参数(PEc、ARn、RFFc、RFFn、FIIn)在各类脱落细胞之间差异均有显著性(P<0.05)。结论尺寸参数和形状参数在UCC间差异有显著性,细胞形态、结构定量分析有助于这三类细胞的甄别。     

多视场多参数形态定量分析模块在神经研究领域中的应用

缺血性神经元损伤的性质是神经研究关注的热点。但目前用于区分凋亡与坏死细胞的ＴＵＮＥＬ技术,较易受主观因素的影响。本实验和图像分析技术对缺血动物脑的ＴＵＮＥＬ染色切片进行多场多参数形态定量分析,即细胞形态这亘参数（核面积、周长、直径、形状因子）和含 密度（平均光密度、积分光密度）。本实验结果显示图像分析能提高ＴＵＮＥＬ鉴别凋亡与坏死的灵敏度和精确性,并提供了一种简便快捷的形态定量方法,是在神经研究中     

TUNEL方法结合图像分析技术检测缺血诱导的神经元损伤

目的 缺血性神经元损伤的性质(凋亡或坏死)是神经研究领域的热点.但目前用于区分凋亡与坏死细胞的TUNEL技术,较易受主观因素的影响.方法 本实验运用图像分析技术对缺血大鼠脑的TUNEL染色切片进行多视场多参数形态定量分析,即细胞形态定量参数(核面积、周长、直径、形状因子)和含量光密度(平均光密度、积分光密度).结果 局灶性脑缺血可引起神经元凋亡和坏死.尾壳核以发生凋亡为主,且凋亡神经元的出现先于坏死神经元.缺血0.5h,尾壳核开始出现凋亡神经元.缺血3h达高峰.缺血6h,出现选择性细胞丢失.结论 图像分析技术能客观而准确地鉴别1:TUNEL染色切片上神经元损伤的性质,由此为神经领域的研究提供了一种敏感准确的形态学定量方法.     

血小板活化因子对培养人脐静脉血管内皮细胞损伤作用的探讨

目的:观察在培养条件下血小板活化因子(PAF)能否直接损伤原代人血管内皮细胞(HVEC)和ECV-304细胞系。方法:利用光镜和结晶紫比色的方法,观察HVEC、ECV-304细胞系与PAF孵育前后形态和生物活性变化。结果:PAF与HVEC孵育30min即发生细胞形态改变,生物活性则未见显著下降,与ECV-304细胞系孵育后的细胞形态和生物活性未见明显变化。结论:在培养条件下PAF可致HVEC损伤,对ECV-304细胞系则未见明显损伤。     

大鼠杏仁中央核神经紧张肽和强啡肽神经元的年龄变化及牛膝的干预作用-免疫组织化学及图像定量分析研究

采用免疫组织化学ABC法结合图像定量分析观察杏仁中央核内神经紧张肽和强啡肽标记神经元的年龄变化及牛膝的抗衰老作用.结果:神经紧张肽神经元随月龄增加呈现细胞数减少、细胞面积增加和灰度值增大(P<0.05),22月龄大鼠发现有形态改变.强啡肽神经元左右两侧不同步变化,左侧在9月龄呈现细胞数减少,细胞面积增加、灰度值增大(P<0.05),在22月龄还可见形态改变;右侧在22月才出现细胞数减少、细胞面积增加、灰度值增大(P<0.05),未见明显的形态改变.牛膝喂药组与同月龄大鼠相比,神经紧张肽和强啡肽神经元的细胞数减少、细胞面积增加和灰度值的增大均降低(P<0.05),未见明显的形态改变,证实牛膝有延缓衰老之功效.     

1-磷酸鞘氨醇对血小板活化因子所致大鼠肠系膜微血管通透性增高的影响

目的:探讨1-磷酸鞘氨醇(S1P)对血小板活化因子(PAF)引起的大鼠肠系膜微血管通透性增高的影响。方法:本研究拟采用大鼠在体肠系膜微血管灌注的方法,通过测定微静脉的静水传导性(Lp),观察S1P对外源性PAF引起的微血管通透性增高的影响;并利用激光共聚焦显微镜技术,观察S1P对PAF引起的微血管荧光强度变化以及血管内皮细胞钙粘蛋白(VE-cadherin)变化的影响。结果:给予10nmol/LPAF作用后,大鼠肠系膜微血管Lp值明显增高,而经1μmol/L S1P预处理后,再给予PAF并未引起Lp的明显变化;PAF作用微血管后可见微血管内皮细胞间隙打开,微血管荧光强度明显增加,大量红色荧光微球(FMs)分布于内皮细胞间隙之中,S1P预处理后并未见内皮细胞间隙打开及FMs的明显积聚,微血管荧光强度与正常对照值比较无显著差异。结论:PAF可增加微血管的通透性,改变内皮细胞VE-cadherin正常结构,导致粘附连接断裂,细胞间隙形成,血管通透性增加可能与此结构变化有关。S1P能改善PAF引起的血管通透性增高,其作用与加强内皮细胞间粘附连接,抑制细胞间隙打开有关。     

两种肝纤维化的组织学量化方法的实验研究

评价肝纤维化半定量计分及计算机图像分析肝纤维化面积百分比这两种组织学量化方法。分别采用半定量计分系统(Semiquantative scorning system,SSS)中纤维化程度计分及计算机图像分析纤维面积百分比两种方法,对温阳中药肝之福预防及治疗四氯化碳(CCl4)所致肝纤维化大鼠的肝脏组织共73份标本进行量化诊断,比较各组间差异,同时检测每份肝脏标本组织中的羟脯氨酸(Hyp)含量,分别与上述两种量化方法进行相关性分析。不论预防实验还是治疗实验,SSS计分、图像分析及Hyp含量三项指标结果均一致,SSS计分及图像分析与羟脯氨酸含量均有较好的相关性(r分别为0.604、0.630,P<0.01)。本研究表明SSS中纤维化程度计分法与计算机图像分析法均可作为比较可靠的肝纤维化的组织学量化诊断方法。     

Suppression by isoproterenol of endothelial cell morphology and barrier function changes induced by platelet-activating factor

Using a model to study vascular permeability on hydrostatically perfused bovine pulmonary artery endothelial cell (EC) monolayers and software to analyze cell morphological parameters automatically in a computer image workstation, we studied the effects of isoproterenol (IPN) on platelet-activating factor (PAF)-induced changes in EC monolayer permeability and cell morphological parameters. Albumin has fortifying effects on endothelial barrier function. As albumin concentration in the perfusate increased (0, 1, 5, 10, 20 mg/ml), EC monolayer hydraulic conductivity (Lp) decreased gradually while Lp of the filter membranes did not change. After treatment of the EC monolayer with PAF 10^–8 mol/liter for 30 min, transmonolayer fluid flow, protein clearance rate, and Lp value increased noticeably. At the same time, cell area decreased and intercellular distance and percentage of intercellular space area in total cell monolayer increased. Pretreatment with 10^–4 mol/liter IPN blocked PAF-induced EC permeability and morphological changes, suggesting that EC contraction and intercellular gap formation are important mechanisms for PAF-induced high vascular permeability. IPN inhibits the effects of PAF via stabilization of EC morphology, protection of intercellular junction, and blockade of intercellular gap formation.Grant support: National Natural Sciences Foundation 39200144.     

表阿霉素诱导血管通透性改变的机制

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。     

阿托伐他汀对高糖高脂环境下人脐静脉内皮细胞与人肾小球系膜细胞相互作用的影响

 目的 探讨高糖高脂环境下人脐静脉内皮细胞与人肾小球系膜细胞间的相互作用及阿托伐他汀对内皮细胞与系膜细胞相互作用的影响。方法人脐静脉内皮细胞与人肾小球系膜细胞共培养和人肾小球系膜细胞单独培养,分为对照组、甘露醇组、高糖高脂组、BN52021组、阿托伐他汀组。ELISA法检测细胞上清液纤维联接蛋白（Fn）、血小板活化因子（PAF）含量,实时荧光定量检测系膜细胞血小板活化因子受体（PAF-R）mRNA表达。结果 (1)高糖高脂条件下,共培养和单培养Fn、PAF升高（P <0.05）;高糖高脂条件下,共培养Fn、PAF较单培养升高（P <0.05）,BN52021和阿托伐他汀可抑制高糖高脂引起的Fn升高（P <0.05）,阿托伐他汀可抑制高糖高脂引起的PAF升高（P <0.05）;（2）高糖高脂可上调系膜细胞PAF-R mRNA表达（P <0.05）,阿托伐他汀可显著抑制PAF-R mRNA表达上调（P<0.05）。结论 (1) 高糖高脂环境下,系膜细胞和内皮细胞存在异常的相互作用。(2)阿托伐他汀可影响高糖高脂环境下内皮细胞与系膜细胞的相互作用。     

细胞相互作用对PAF、系膜细胞paf-R基因表达的影响及阿托伐他汀的干预作用

目的:探讨高糖高脂条件下内皮细胞与系膜细胞相互作用对PAF产生、系膜细胞paf-R基因表达的影响及阿托伐他汀的干预作用。方法:内皮细胞与系膜细胞共培养和系膜细胞单独培养后随机,分为对照组、甘露醇组、高糖高脂组、阿托伐他汀组,培养24h后,ELISA法检测各组细胞上清液PAF含量,实时荧光定量检测系膜细胞paf-RmRNA表达。结果:(1)高糖高脂促进共培养组和单培养组PAF升高(P0.05),共培养组PAF均较单培养组升高(P0.05);(2)高糖高脂可上调系膜细胞paf-R mRNA表达(P0.05);(3)共培养和单培养条件下,阿托伐他汀可抑制高糖高脂引起的PAF升高(P0.05),并可抑制高糖高脂引起的系膜细胞paf-RmRNA表达上调(P0.05)。结论:(1)高糖高脂环境下,系膜细胞和内皮细胞存在异常的相互作用,这种作用促进PAF产生;(2)高糖高脂促进系膜细胞paf-R基因表达,使PAF的生物效应进一步放大;(3)阿托伐他汀可影响高糖高脂条件下内皮细胞与系膜细胞之间的相互作用。     

Lymphocyte Binding to and Penetration through Vascular Endothelium is Stimulated by Platelet-Activating Factor

Lymphocytes have an important role in acute inflammatory reactions such as acute allograft rejection. Recirculating lymphocytes attach to vascular endothelium and penetrate through it into tissue parenchyma. Many recent studies have shown that different protein mediators, like gamma interferon, interleukin 1, and tumour necrosis factor enhance lymphocyte binding to and penetration through the endothelium, i.e. lymphocyte homing. We investigated the effect of platelet-activating factor (PAF) in lymphocyte binding and penetration in vitro. Treatment of rat heart microvascular endothelial monolayers with PAF (10(-6)-10(-10) M) increased the lymphocyte binding up to 1.6-fold. The effect is dose- and time-dependent. The PAF effect was reversible upon removal of the agonist: 60 min after removal of PAF no increase in the lymphocyte binding was detected. Pretreatment of endothelial cells, lymphocytes, or both of these cell types led to an increase in lymphocyte binding to endothelial monolayers. The effect of PAF in lymphocyte penetration through endothelium was investigated by using endothelial cell monolayers cultured on top of millipore filters. An optimal PAF dose (10(-8) M) for 10 min increased the number of lymphocytes penetrating through the endothelial cell monolayer into the filter by a factor of 3. These results suggest that PAF has no important role in lymphocyte homing, since it can activate the endothelial cells, the lymphocytes, or both cell types.     

Protective Effect of Intestinal Trefoil Factor on Injury of Intestinal Epithelial Tight Junction Induced by Platelet Activating Factor

Intestinal barrier dysfunction plays an important role in the pathogenesis of inflammatory bowel disease (IBD). To evaluate the effect of intestinal trefoil factor (ITF) on increased intestinal permeability and its association with tight junction proteins, an in vitro intestinal epithelia barrier model was established with Caco-2 cells and treated with platelet-activating factor (PAF). We found that exposing cells to 0.3 M ITF (30 min before or 30 min after PAF treatment) attenuated the PAF-induced changes in transepithelial electrical resistance and Lucifer yellow flux. A quantitative RT-PCR and western blot analysis revealed that ITF suppressed PAF-induced downregulation of tight junction proteins claudin-1 and ZO-1 expression; furthermore, an abnormal localization and distribution of these proteins was inhibited, as assessed by immunofluorescence staining. These results suggest that ITF decreases mucosal permeability and shows potential as a therapy for treating IBD.     

Effect of ozone on platelet-activating factor production in phorbol-differentiated HL60 cells, a human bronchial epithelial cell line (BEAS S6), and primary human bronchial epithelial cells.

Platelet-activating factor (PAF) is a phospholipid with a wide spectrum of pro-inflammatory properties. In the lung, PAF induces airway hyperresponsiveness, neutrophil sequestration, and increased vascular permeability. The alveolar macrophage and the bronchial epithelium are tissues that are exposed to inhaled ozone (O3). We studied the effect of an in vitro O3 exposure on PAF production in a macrophage-like HL60 human cell line (dHL60), a human bronchial epithelial cell line (BEAS S6), and also in primary human bronchial epithelial cells. PAF was quantified by thin-layer chromatographic separation of lipid extracts from cells radiolabeled with [3H]lysoPAF and by radioimmunoassay. In vitro exposure of dHL60 cells to 0.05 to 1.0 ppm O3 for 15 to 120 min was found to significantly increase PAF levels above air control values at all exposure levels and time points (average increase of 92%). Similarly, BEAS S6 cells grown on collagen-coated filter supports and exposed to 0.05 to 1.0 ppm O3 for 60 min released an average increase in PAF of 626% above control values. Primary human bronchial epithelial cells also demonstrated significant increases in [3H]PAF release (average increase of 289% after exposure to 1.0 ppm O3 for 60 min) compared with paired air controls. These findings suggest that some of the effects of O3 inhalation may be mediated by PAF.     

Functional implication of the hydrolysis of platelet endothelial cell adhesion molecule 1 (CD31) by gingipains of Porphyromonas gingivalis for the pathology of periodontal disease

Periodontitis is a response of highly vascularized tissues to the adjacent microflora of dental plaque. Progressive disease has been related to consortia of anaerobic bacteria, with the gram-negative organism Porphyromonas gingivalis particularly implicated. The gingipains, comprising a group of cysteine proteinases and associated hemagglutinin domains, are major virulence determinants of this organism. As vascular expression of leukocyte adhesion molecules is a critical determinant of tissue response to microbial challenge, the objective of this study was to determine the capacity of gingipains to modulate the expression and function of these receptors. Given the potential multifunctional role of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the vasculature, the effect of gingipains on PECAM-1 expression by endothelial cells was examined. Activated gingipains preferentially down-regulated PECAM-1 expression on endothelial cells compared with vascular cell adhesion molecule 1 and endothelial-leukocyte adhesion molecule 1, but the reduction in PECAM-1 expression was completely inhibited in the presence of the cysteine proteinase inhibitor TLCK (Nalpha-p-tosyl-l-lysine chloromethyl ketone). Endothelial monolayers treated with activated gingipains demonstrated progressive intercellular gap formation that correlated with reduced intercellular junctional PECAM-1 expression as determined by Western blotting and immunofluorescence microscopy. This was accompanied by enhanced transfer of both albumin and neutrophils across the monolayer. The results suggest that degradation of PECAM-1 by gingipains contributes to increased vascular permeability and neutrophil flux at disease sites.     

Effects of Cytotoxic Necrotizing Factor 1 and Lethal Toxin on Actin Cytoskeleton and VE-Cadherin Localization in Human Endothelial Cell Monolayers

Integrity of the vascular endothelium is largely dependent on endothelial cell shape and establishment of intercellular junctions. Certain pathogenic bacterial toxins alter the cytoskeletal architecture of intoxicated cells by modulating the GTPase activity of p21 Rho family proteins. In the present study we have analyzed the effect of Rho-directed toxins on the actin cytoskeleton and monolayer integrity of endothelial cells. We report here that Escherichia coli cytotoxic necrotizing factor 1 (CNF1) activates Rho in human umbilical vein endothelial cells (HUVEC). In confluent monolayers, CNF1 treatment induces prominent stress fiber formation without significantly modifying peripheral localization of VE-cadherin, a specific marker of vascular endothelial cell adherens junctions. Further, Rho activation with CNF1 blocks thrombin-induced redistribution of VE-cadherin staining and gap formation in HUVEC monolayers. Inhibition of Rho by prolonged treatment of cells with C3 exoenzyme (Clostridium botulinum) eliminates actin stress fibers without disrupting the continuity of VE-cadherin staining, indicating that Rho-dependent stress fibers are not required for maintaining this adhesion receptor at sites of intercellular contact. Lethal toxin (Clostridium sordellii), an inhibitor of Rac as well as Ras and Rap, potently disrupts the actin microfilament system and monolayer integrity in HUVEC cultures.