脂多糖对ECV304细胞增殖、ESM-1表达和THCE细胞紧密连接表达的影响

发布时间：2018-01-06 20:28

本文关键词：脂多糖对ECV304细胞增殖、ESM-1表达和THCE细胞紧密连接表达的影响　出处：《安徽医科大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的通过脂多糖(lipopolysaccharide, LPS, 内毒素)对人脐静脉内皮细胞(human umbilical vein endothelial cell, HUVEC, ECV304)的生长、内皮细胞特异性分子-1(endothelial cell-specific molecule-1, ESM-1)表达和猴病毒40—永生性人角膜上皮[simian virus (SV) 40-immortalized human corneal epithelial, THCE]细胞中的紧密连接(tight junction, TJ)表达的影响来探讨其在炎症反应中的作用。方法在体外,用LPS在不同时间作用于ECV304。观察ECV304形态,用MTT法对ECV304增殖情况进行分析,用免疫细胞化学方法观察ESM-1、胞外信号调节激酶(external-signal regulated kinase, ERK)在ECV304中的表达情况,以及ERK的磷酸化情况。用免疫荧光检测LPS对occludin、ZO-1、ZO-2和claudin-1在THCE细胞中的分布影响,Western blot检测LPS对occludin、ZO-1和ZO-2在THCE细胞中表达影响,并用伏欧表测定LPS对THCE细胞的跨上皮阻抗(transepithelial electrical resistance, TER)的影响。结果加入LPS 24h后ECV304出现形态变化,48h时改变最为明显,细胞形态梭形明显,排列紊乱。加入LPS后0.5h时,1μl/ml和5μl/ml LPS作用的ECV304增殖大于正常ECV304(P0.05)。加入LPS后1h时,1μl/ml LPS作用的ECV304增殖大于正常ECV304(P0.05)。而2h后,三者无明显区别。与正常ECV304相比,LPS处理的ECV304的ESM-1表达明显增高,在0.5h时表达量增高最明显,1h时加入5μl/ml LPS的作用高于1μl/ml,在2h后表达量的增高有所下降。LPS对ERK在ECV304中的表达影响不显著。加入LPS 0.5h～4h期间,均能促进ECV304 ERK磷酸化,在1h和2h时,加入5μl/ml LPS的作用高于1μl/ml,8h后LPS对ERK磷酸化无影响。3μl/ml LPS处理6h的THCE细胞中,occludin、ZO-1和claudin-1在细胞边界几乎没有改变,而ZO-2在细胞边界几乎不可见。1μl/ml的LPS处理THCE细胞6h后,ZO-1和ZO-2
[Abstract]:Objective to lipopolysaccharide (lipopolysaccharide, LPS, endotoxin) on human umbilical vein endothelial cells (human umbilical vein endothelial cell, HUVEC, ECV304) on the growth of endothelial cell specific molecule -1 (endothelial cell-specific molecule-1, ESM-1) and the expression of simian virus 40 immortalized human corneal epithelial [simian virus (SV) 40-immortalized human corneal epithelial. Tight junctions in THCE] cells (tight, junction, TJ) expression to explore the influence of its role in inflammation. Methods in vitro, with LPS at different time in ECV304. observation of ECV304 morphology, proliferation of ECV304 was analyzed by MTT method, ESM-1 was observed by immunocytochemical method, extracellular signal regulated kinase (external-signal regulated kinase, ERK) expression in ECV304, and the phosphorylation of ERK. Occludin by immunofluorescence detection of LPS, ZO-1 Effect of distribution of ZO-2 and claudin-1, in THCE cells, Western blot detection of LPS of occludin, the expression of ZO-1 and ZO-2 in THCE cells, and determination of LPS on THCE cell transepithelial impedance voltohmmeter (transepithelial electrical resistance, TER). The effects of the addition of LPS into 24h ECV304 after morphological changes, 48h when the most obvious changes in cell morphology, spindle was disordered. After joining LPS 0.5h, 1 l/ml and 5 l/ml LPS ECV304 ECV304 (P0.05) proliferation is greater than normal. After joining LPS 1H, 1 l/ml LPS ECV304 ECV304 (P0.05) is higher than the normal proliferation and 2H., no significant difference between the three. Compared with the normal ECV304, LPS ECV304 ESM-1 expression was significantly increased, increased the most obvious expression in 0.5h, 1H added 5 l/ml LPS the role of higher than 1 l/ml, the rise of the amount of reduction of.LPS on ERK expression in ECV304 after 2H The expression has no significant effect. During the period of joining the LPS 0.5h ~ 4h, could promote the phosphorylation of ERK in ECV304, 1H and 2H, adding 5 l/ml LPS compared with 1 l/ml, LPS had no effect on ERK phosphorylation.3 l/ml LPS 6h in THCE cells, 8h occludin, ZO-1 and Claudin-1 in the cell boundary has hardly changed, while the ZO-2 in the cell boundary LPS THCE cells treated with 6h almost invisible.1 l/ml, ZO-1 and ZO-2

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R363

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