小檗碱、白头翁素对肠黏膜微血管内皮细胞E-selectin表达的影响

为了观察内毒素即细菌脂多糖(lipopolysaccharide,LPS)对肠黏膜微血管内皮细胞(RIMECs)表面E-selectin表达的影响及小檗碱和白头翁素对E-selectin表达的调控作用,试验以体外培养的肠黏膜微血管内皮细胞为模型,采用免疫组化SP法检测不同浓度LPS(10μg/mL、5μg/mL、1μg...     

LPS对大鼠肠黏膜微血管内皮细胞分泌NO的影响

一氧化氮（Nitric oxide，NO）是一种由血管内皮细胞分泌的内源性血管舒张因子。NO作为一种重要的信使分子具有舒张血管、神经传递以及杀灭微生物和肿瘤细胞等多种生物学功能。     

LLO对大鼠肠黏膜微血管内皮细胞分泌NO和ET-1的影响

为探讨李斯特菌溶血素(LLO)导致大鼠肠黏膜微血管内皮细胞(RIMVECs)损伤的作用机制,将体外培养的RIMVECs分为对照组和LLO组,观察细胞形态变化,测定细胞生长情况,并检测细胞培养上清液中NO、ET-1浓度变化。结果表明:LLO组RIMVECs的细胞间隙变大,有大量细胞碎片漂浮;当LLO浓度达到50ng/m L以上时,测得细胞增殖(OD值)均呈极显著下降(P0.01);12 h内,NO、ET-1分泌量高于正常水平。试验表明,LLO引起RIMVECs细胞因子NO、ET-1分泌量升高,导致细胞微环境紊乱,是LLO导致肠黏膜微血管内皮细胞损伤的机制之一。     

硫酸软骨素对LLO诱导大鼠肠黏膜微血管内皮细胞分泌NO、ET-1的影响

为研究李斯特菌溶血素(LLO)对大鼠肠黏膜微血管内皮细胞(RIMVECs)分泌NO、内皮素(ET-1)的影响,以及硫酸软骨素(CS)通过调节细胞微环境保护RIMVECs的作用,将体外培养的RIMVECs分为LLO+CS组、LLO组、CS组、空白组,经过MTT比色法测定细胞生长情况,硝酸还原酶法测定细胞上清液细胞因子NO含量以及酶联免疫法检测ET-1含量,并用原位杂交方法对结果进行验证。结果表明:LLO可抑制RIMVECs增殖活性,提高NO、ET-1分泌量且高于正常水平,并导致NO/ET-1比值失衡(下降);而CS可提高RIMVECs细胞的增殖活性,并显著上调NO/ET-1比值。由此可知,CS可通过改善LLO引起细胞因子NO和ET-1失衡而起到保护微血管内皮细胞的作用。     

小檗碱、绿原酸和黄芩苷对 LPS 损伤大鼠肠黏膜微血管内皮细胞表达ICAM-1的影响

为研究中药有效成分小檗碱、绿原酸和黄芩苷的抗炎机制,通过体外培养大鼠肠黏膜微血管内皮细胞（Rat intestinal mucosa microvascular endothelial cells,RIMMVECs）建立LPS损伤模型,采用免疫细胞化学方法观察了3种中药成分对LPS损伤RIMMVECs表达ICAM-1的影响,并对染色结果进行了图像灰度分析与统计学检验。研究发现,RIMVECs正常状态下低水平表达ICAM-1,用LPS刺激后ICAM—1的表达明显升高,而中药有效成分小檗碱、绿原酸和黄芩苷能够抑制LPS诱导的RIMVECs强阳性表达ICAM-1。结果表明,小檗碱、绿原酸和黄芩苷的抗炎作用与其下调ICAM-1的表达密切相关,为阐明其抗炎机制提供了数据。     

中药方剂五苓散对SLT-2e影响大鼠肠黏膜微血管内皮细胞分泌NO的观察

将中药方剂五苓散和志贺样毒素Ⅱ型变异体(SLT-2e)分别加入大鼠肠黏膜微血管内皮细胞(RIM-VEC)培养板中培养1、3、6和9h,观察五苓散及其抗SLT-2e对RIMVEC分泌NO的影响。结果:普通中药组中10μL/mL五苓散浓度在1-9h内使NO浓度升高;治疗组五苓散浓度0.1μL/mL、1μL/mL、10μL/mL在9h时内皮细胞分泌NO差异不显著;预防组0.1μL/mL在1-6h时变化不明显,在9h时NO升高;1μL/mL、10μL/mL在6h时NO量分泌最高,9h时下降。提示一定量的五苓散可使RIMVEC的NO分泌量升高(P<0.05);治疗组五苓散使NO分泌量不升高,预防组五苓散可使内皮细胞分泌NO量升高(P<0.05)。     

槲皮素对SLT-Ⅱe诱导大鼠肠黏膜微血管内皮细胞分泌细胞因子的影响

通过检测槲皮素对SLT-Ⅱe诱导的大鼠肠黏膜微血管内皮细胞分泌NO、ET-1、P-选凝素、sICAM-1及TNF-α的浓度变化,探索中药复方主要成分槲皮素对仔猪水肿痛的疗效机制.将培养的肠黏膜微血管内皮细胞分为对照组、SLT-Ⅱ e组、不同浓度槲皮素处理组等5个组,采用ELISA法测定了培养3、6、9和12 h时细胞培养上清液中NO、ET-1、P-选凝素、sICAM-1及TNF-α浓度的变化.结果,10 mg/L槲皮素可下调SLT-Ⅱ e诱导的大鼠肠黏膜微血管内皮细胞NO、ET-1及sICAM-1的分泌,12 h内使NO、ET-1及sICAM-1的分泌浓度接近正常水平;1、5、10 mg/L槲皮素不能下调SLT-Ⅱe诱导的大鼠肠黏膜微血管内皮细胞P-选凝素和TNF-α的分泌.结果表明,槲皮素通过抑制SLT-Ⅱ e诱导肠黏膜微血管内皮细胞NO、ET-1、sICAM-1的过量分泌,缓解肠道微循环障碍,减轻炎症反应,达到治疗水肿病的目的.     

黄芪多糖对SLT-Ⅱe诱导大鼠肠黏膜微血管内皮细胞分泌细胞因子的影响

通过检测黄芪多糖对SLT-Ⅱe诱导的大鼠肠黏膜微血管内皮细胞分泌NO、ET-1、PGI2、TXA2、P-选凝素及sICAM-1的浓度变化,探索中药复方主要成分黄芪多糖对仔猪水肿病的疗效机制。将培养的肠黏膜微血管内皮细胞分为对照组、SLT-Ⅱe组、不同浓度黄芪多糖处理组等5个组,采用ELISA法测定了培养3、6、9和12 h时细胞培养上清液中NO、ET-1、PGI2、TXA2、P-选凝素及sICAM-1浓度的变化。10 mg/L黄芪多糖可下调SLT-Ⅱe诱导的大鼠肠黏膜微血管内皮细胞NO、ET-1、TXA2及sICAM-1的分泌,12 h内使NO、ET-1、TXA2及sICAM-1的分泌浓度接近正常水平;5、10、15 mg/L黄芪多糖不能下调SLT-Ⅱe诱导的大鼠肠黏膜微血管内皮细胞PGI2及P-选凝素的分泌。黄芪多糖通过抑制SLT-Ⅱe诱导肠黏膜微血管内皮细胞NO、ET-1 TXA2及sICAM-1的过量分泌,缓解肠道微循环障碍,减轻局部炎症反应。     

LPS和LT对肠黏膜VEC内分泌NO和ET的影响

研究LPS和LT引起肠黏膜微血管内皮细胞(VEC)损伤的作用机制。将所培养的肠黏膜VEC 60个孔分为空白对照组、LPS(1μg/mL)组和LT(50μg/mL)组3个组,每个组又分4个不同时间段:3h、6h、9h、12h。结果LPS导致肠黏膜VEC分泌NO明显升高,3 h后达到高峰,随后逐渐降低,而ET分泌急剧升高,9 h后达到高峰。LT导致肠黏膜VEC分泌NO明显升高,3 h达到高峰,随后逐渐降低,而ET在6 h前明显降低,随后逐渐升高到正常水平。     

SLT-2e对大鼠肠黏膜微血管内皮细胞分泌PG12、TXA2和PAF的影响

志贺样毒素Ⅱ型变异体（SL T-2e）又叫猪水肿病毒素，能引起仔猪水肿病，是仔猪水肿病的主要毒力因子。SLT-2e作用于微血管能引起微血管内皮细胞损害而导致水肿，因此在猪水肿病的发生中起着决定性作用。Methiyapun等研究表明水肿病大肠杆菌对猪肠黏膜没有重要的病理变化，而靶细胞是微血管内皮细胞，表现为内皮细胞空泡化、血管周围水肿、微血栓形成等。     

大肠杆菌热敏肠毒素对肠黏膜微血管内皮细胞的影响及白头翁素对其保护作用的研究

大肠杆菌病是严重危害养殖业的疾病之一,可引起动物腹泻,导致死亡,造成了较大的经济损失.研究表明,细菌产生的热敏肠毒素(heat-labile enterotoxin,LT)是引起腹泻的主要毒素之一.肠黏膜微血管内皮细胞(RIMECs)不仅构成肠道局部的血液与组织细胞间的物理屏障,保证血管内外的物质交换,并且通过合成和释放各种血管活性物质,在调节正常的血液循环、维持周围细胞环境的稳定方面具有十分重要的作用.白头翁素是传统抗腹泻中药白头翁的主要成分.本试验在建立LT损伤RIMECs体外病理模型的基础上,研究了白头翁素对其作用,以进一步了解白头翁素的抗腹泻机制.     

唾液酸受体在大鼠肠黏膜微血管内皮细胞中的表达

肠黏膜微血管内皮细胞在肠道中广泛存在,为研究流感病毒唾液酸α2,3半乳糖(SAα2,3Gal)和α2,6半乳糖(SAα2,6Gal)两种受体在肠黏膜微血管内皮细胞表面的表达情况,本试验利用植物凝集素免疫荧光染色方法和流式细胞术分别进行了定性和定量分析。结果显示,大鼠肠黏膜微血管内皮细胞(RIM-MVECs)表面同时表达唾液酸α2,3-半乳糖和α2,6-半乳糖两种受体,即同时表达人流感病毒受体和禽流感病毒受体,并且唾液酸α2,6半乳糖的表达量显著高于α2,3半乳糖受体的表达量。该试验结果表明,RIM-MVECs可以被人源和禽源流感病毒感染,且更易被人源流感病毒感染。     

白头翁素对RIMEC分泌功能的影响

目的：探讨白头翁素对细菌脂多糖（LPS）诱导的肠黏膜微血管内皮细胞（RIMEC）内皮素（ET-Ⅰ）和一氧化氮（NO）分泌增加的影响.方法：以体外培养的大鼠肠黏膜微血管内皮细胞（RIMEC）为试验模型,ET-Ⅰ测定采用酶联免疫技术,NO测定采用还原酶法.结果：白头翁素10 μg/ml、5 μg/ml、1 μg/ml预孵2 h加入1 μg/ml的LPS刺激12 h后.白头翁素组1 μg/ml ET-Ⅰ分泌显著低于LPS组（P＜0.05）,5 μg/ml、10 μg/ml白头翁素组ET-Ⅰ分泌极显著低于LPS组（P＜0.01）;10 μg/ml、5 μg/ml、1 μg/ml白头翁素组NO分泌极显著低于LPS组（P＜0.01） 结论：白头翁素可以下调LPS诱导的ET-Ⅰ和NO分泌增加,这可能是白头翁的抗炎机制之一.     

Isolation and characterization of equine microvascular endothelial cells in vitro

The use of cultured tissue has not yet become widespread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consisted of Dulbecco modified Eagle medium with 10% whole fetal bovine serum (wFBS) and 20 micrograms of endothelial cell growth supplement/ml. The EMVE grew readily in culture, had the cobblestone morphologic feature at confluence, stained positively for factor VIII-related antigen, and metabolized acetylated low-density lipoprotein. Fibroblast and smooth muscle cell contamination was minimal in primary cell cultures, which were successfully passed and maintained in culture for 3 to 5 serial passages, using various media and substrates. Preliminary studies were undertaken to determine optimal growth conditions with a range of variables: serum concentration, extracellular matrix components, and growth factors, Optimal conditions were achieved with a minimum of 10% wFBS, and with either fibronectin or laminin as extracellular matrix substrates. The EMVE grew adequately in Dulbecco modified Eagle medium plus 10% wFBS, and the added growth factors or serum supplements did not appear necessary for growth of EMVE.     

铜对大鼠脑微血管内皮细胞的氧化应激损伤

选用浓度为30,60,120,180,240μmol/L的氯化铜(CuCl_2)作用于原代大鼠脑微血管内皮细胞(BMEC)12h,测定细胞活力、细胞内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和诱导型一氧化氮合酶(iNOS)活性的变化。结果显示:与对照组相比,细胞活力明显降低,30μmol/L浓度组SOD的活性变化不明显,60μmol/L和120μmol/L浓度组SOD的活性升高,而180μmol/L和240μmol/L浓度组SOD的活性降低,CAT的活性均明显降低,iNOS的活性均明显升高。结果表明:铜过量可致BMEC损伤,而氧化应激是其中一个重要因素。     

盐酸黄连素纳米微乳的制备及体外抗菌活性比较

黄连素(俗称为小檗碱Berberine)是从毛茛科黄连等植物根茎中提取的一种季胺类异喹啉生物碱,临床上多用其盐酸盐[1]。盐酸黄连素作为抗菌药在临床上已应用多年,其疗效确切,是一种广谱抗菌药物,对多种革兰氏阳性、阴性细菌以及真菌、霉菌、病毒、原虫、线虫具有抑制杀灭作用[2]。     

大肠杆菌热敏肠毒素对猪小肠黏膜微血管内皮细胞活力及细胞因子表达的影响

本试验旨在探究大肠杆菌热敏肠毒素处理(heat labile enterotoxin,LT)后,猪小肠黏膜微血管内皮细胞活力及细胞因子ET-1、IL-1β水平的变化。首先采用D-半乳糖凝胶树脂层析法提取猪E coli LT,利用SDS-PAGE验证蛋白纯度,并用Vero细胞检验提取物的生物学活性,最后采用CCK-8法检测LT对猪小肠黏膜微血管内皮细胞活力的剂量时间效应,ELISA法检测细胞培养上清中ET-1、IL-1β水平的变化。结果表明:所提蛋白为AB_5结构的LT,且具有良好的生物学活性;0.01 mg/L的提取蛋白作用3 h后便可显著降低小肠黏膜微血管内皮细胞的活性(P0.05);作用细胞6 h后,0.01,0.1和10 mg/L LT组细胞培养上清中ET-1的含量显著升高(P0.05),0.01和1 mg/LLT组IL-1β的含量显著升高(P0.05)。结果表明,E coli LT可以显著改变猪小肠黏膜微血管内皮细胞的活力及细胞因子的表达。     

In vitro effect of classical swine fever virus on a porcine aortic endothelial cell line

The effect of classical swine fever (CSF) virus on some phenotypic and functional features of an established porcine aortic endothelial cell (AOC) line was investigated. AOC cells show most of the characteristics of primary endothelial cells, avoiding the alterations and senescence that these cells undergo after a few passages in culture. AOC cells were susceptible to CSF virus infection to a high degree, reaching 90% of CSF virus positive cells after 24 h of infection; however as with other porcine susceptible cells, no cytopathic effect could be observed. In these conditions none of the surface molecules studied, including SLA-II MHC antigens, adhesion or co-stimulatory molecules, were altered by virus infection after 24 or 48 h. Functionally CSF virus infection induced a decrease in the pro-coagulant activity of the AOC cells, determined by the increase in the clot formation time shown by the lysates of these cells. This contrasts with the increase observed in the expression of mRNA corresponding to IL-1 alpha and IL-6, two proinflammatory and pro-coagulant cytokines, in CSF virus-infected AOC cells.     

In vivo and in vitro protective effect of arginine against intestinal inflammatory response induced by Clostridium perfringens in broiler chickens

Background: Necrotic enteritis is a widespread disease in poultry caused by Clostridium perfringens. We previously reported that dietary arginine supplementation protected the intestinal mucosa of broiler chickens with necrotic enteritis, but the related protective mechanisms remain unclear. The in vivo trial was designed as a 2 × 2 factorial arrangement to evaluated the effects of arginine supplementation on inflammatory responses, arginine transporters,arginine catabolism and JAK-STAT signalling pathway in broiler chickens challenged with C. perfringens or without C. perfringens. Furthermore, we validated the in vivo results using intestinal epithelial cells of chicken embryos.Results: C. perfringens infection markedly increased gut gross pathological and histopathological lesion scores,promoted liver C. perfringens invasion, reduced serum arginine levels, and elevated jejunal mucosal lysozyme activities(P 0.05), but these effects were significantly reversed by arginine supplementation in vivo(P 0.05). The challenge significantly increased serum procalcitonin levels, jejunal mucosal iNOS activities and jejunal IL-6, TGF-β3,cationic amino acid transporter(CAT)-1, and CAT-3 mRNA expression(P 0.05), whereas arginine supplementation significantly reduced jejunal IFN-γ, IL-1β, IL-6, IL-10, TGF-β3, and CAT-3 mRNA expression(P 0.05). Arginine supplementation significantly attenuated the C. perfringens challenge-induced increases in jejunal i NOS, arginase 2,arginine decarboxylase, arginine:glycine amidinotransferase, JAK1, JAK3, STAT1, and STAT6 mRNA expression(P 0.05).The in vitro experiment showed that C. perfringens challenge markedly increased cellular cytotoxicity and the mRNA expression of IL-1β, IL-8, IL-10, CAT-1 and CAT-3(P 0.05), which were significantly reversed by 50 μmol/L and/or400 μmol/L arginine pre-treatment(P 0.05).Conclusions: Arginine prevented C. perfringens challenge-induced circulated arginine deficiency, normalized intestinal arginine transport and catabolism, down-regulated JAK-STAT signalling pathway and attenuated the inflammatory response, which exerted protective effects on the intestine of broiler chickens.     

黄连素与其它抗菌药物联用对大肠杆菌体外抗菌活性研究

黄连素又名小檗碱,为毛莨科黄连属植物,是黄连、黄柏等中药中提取的一种生物碱,具有抗病原微生物、抗原虫、抗脂质过氧化作用,能增强白细胞、网状内皮系统的吞噬功能,对细菌毒素有明显的解毒功能,临床上主要用于治疗肠道细菌感染.文献报道[1～3],黄连素与甲氧苄氨嘧啶(TMP)、氟喹诺酮类药物联合,用于治疗志贺氏菌感染引起的肠道细菌感染,疗效明显增强.本试验研究了黄连素分别与氨苄西林、环丙沙星联合对禽大肠杆菌、氨苄西林、环丙沙星耐性鸭大肠杆菌的体外抗菌活性,对指导临床合理用药,减少细菌耐药性具有一定的生产实际意义.