Notch信号通路对烧伤大鼠血清诱导的肺血管内皮细胞细胞间黏附分子-1的影响  

Effects of Notch signaling pathway on the intercellular adhesion molecule-1 of pulmonary vascular endothelial cell induced by serum in burned rats

作　　者：蔡维霞 曹涛 赵明 肖丹 贾艳慧 王璟 张月 王克甲 韩军涛 胡大海 Cai Weixia;Cao Tao;Zhao Ming;Xiao Dan;Jia Yanhui;Wang Jing;Zhang Yue;Wang Kejia;Han Juntao;Hu Dahai(Department of Burns and Cutaneous Surgery,Burn Center of PLA,First Affiliated Hospital of Air Force Medical University,Xi′an 710032,China)

机构地区：[1]空军军医大学第一附属医院全军烧伤中心,烧伤与皮肤外科,西安710032

出　　处：《中华损伤与修复杂志（电子版）》2022年第4期292-299,共8页Chinese Journal of Injury Repair and Wound Healing(Electronic Edition)

基　　金：国家自然科学基金青年项目(81601680);国家自然科学基金面上项目(81871561)。

摘　　要：目的探讨Notch信号通路对烧伤大鼠血清诱导的肺血管内皮细胞(PMVEC)细胞间黏附分子(ICAM)-1的影响。方法42只SD大鼠(6~8周龄)中,随机选取30只,按随机数字表法分为假伤组(n=15)和烧伤组(n=15),烧伤组大鼠于95℃热水浸浴背部18 s造成30%总体表面积Ⅲ度烧伤,假伤组大鼠于37℃水浴中浸浴背部18 s模拟致伤。伤后6、12、24、48、72 h,烧伤组随机分别取3只大鼠,腹主动脉采血,酶联免疫吸附试验(ELISA)法检测血清中ICAM-1含量,假伤组大鼠行相同检测。取剩下的12只大鼠(6~8周龄)中的6只,按前述方法造成30%总体表面积Ⅲ度烧伤,伤后24 h制备烧伤大鼠血清;剩下的6只大鼠不作处理,制备健康大鼠血清。切取10只出生3 d的SD大鼠的肺外边缘组织,组织块法培养大鼠PMVEC,倒置相差显微镜下观察原代细胞培养2、6 d后的形态特征,流式细胞术对原代培养7 d的细胞进行细胞鉴定。取处于对数生长期的第4代PMVEC进行实验,将细胞接种于6孔板中,待细胞生长至80%融合时按随机数字表法分为3组(每组设3个复孔):对照组(培养液中分别加入体积分数10%健康大鼠血清),二甲基亚砜(DMSO)+烧伤血清组、γ-分泌酶抑制剂(GSI)+烧伤血清组(分别加入3μL/mL DMSO、75μmol/L GSI,培养24 h后,加入体积分数10%烧伤大鼠血清刺激培养24 h),ELISA法检测PMVEC上清中ICAM-1的含量;流式细胞仪检测各组PMVEC中ICAM-1的含量;采用蛋白质印迹法检测ICAM-1蛋白表达水平,计算相对蛋白表达量;检测PMVEC的细胞黏附能力。数据比较采用单因素方差分析和LSD-t检验。结果(1)烧伤组大鼠伤后6、12、24、48、72 h血清中的ICAM-1含量分别为(19.77±3.03)、(22.09±3.65)、(22.44±4.04)、(25.40±2.51)、(26.37±3.07)pg/mL,显著高于假伤组的[(10.60±1.51)、(11.03±1.95)、(10.87±0.89)、(9.30±0.89)、(10.93±1.22)pg/mL],2组比较差异均有统计学意义(t=4.699、4.466、3.181、10.490、8.097,P<0.05)。(2)原代Objective To investigate the effect of Notch signaling pathway on the intercellular adhesion molecule(ICAM)-1 of pulmonary vascular endothelial cell(PMVEC)induced by serum in burned rats.Methods Among the 42 SD rats(6-8 weeks),30 were randomly selected and divided into sham injury group(n=15)and burn group(n=15)according to the random number table method.Rats in burn group were immeresed in hot water at 95℃ for 18 s,inflicted with 30%total body surface area full-thickness burn on back,and the rats in the sham injury group were immersed in a 37℃ water bath for 18 s to simulate injury.At 6,12,24,48,72 h after injury,3 rats were randomly selected from the burn group,and blood was collected from the abdominal aorta.Enzyme-linked immunoadsordent assay(ELISA)were used to detected the content of ICAM-1 in rats’serum.Rats in the sham injury group were subjected to the same test.Six rats of the remaining 12 rats(6-8 weeks)were taken,and 30%total body surface area full-thickness burn was made according to the aforementioned method,and the serum of the burned rats was prepared at 24 h after the injury;the remaining 6 rats were not treated,and the serum of healthy rats was prepared.Marginal pulmonary tissue was harvested from 3 days old 10 SD rats,and the rat PMVEC were culturned with tissue block method.The morphological characteristics of primary cells cultured for 2 and 6 d were observed under an inverted phase contrast microscope,and the cells in primary cultured for 7 d were identified by flow cytometry.The PMVEC cells of the 4th generation at logarithmic growth stage were taken for experiment.The cells were inoculated on 6-well plates,when the cells grew to 80%confluence,they were divided into 3 groups according to the random number table method:the control group,the dimethyl sulfoxide(DMSO)+burn serum group and the gamma-secretase inhibitor(GSI)+burn serum group,with 3 wells in each group.Cells in control group were cultured with 10%healthy rat serum,the latter 2 groups added with 3μL/mL DMSO and 75μmol/L GSI

关 键 词：烧伤 大鼠 血清 急性肺损伤 肺血管内皮细胞 细胞间黏附分子-1 NOTCH信号通路 

分 类 号：R644[医药卫生—外科学]