磷脂酰肌醇3激酶／蛋白激酶B通路在电烧伤大鼠血清诱导单核-内皮细胞黏附中的作用  

Role of phosphoinositide 3-kinase/protein kinase B signal pathway in monocyte-endothelial adhesion induced by serum of rats with electrical burn

作　　者：阮琼芳[1] 赵超莉[1] 叶子青[1] 张卫东[1] 谢琼慧[1] 谢卫国[1] 

机构地区：[1]武汉大学同仁医院暨武汉市第三医院烧伤研究所,430060

出　　处：《中华烧伤杂志》2014年第3期237-242,共6页Chinese Journal of Burns

基　　金：武汉市卫计委临床医学科研项目（WX13A09）;武汉市科技局重点攻关项目（20056007071）

摘　　要：目的 观察磷脂酰肌醇3激酶/蛋白激酶B（PI3 K/Akt）通路在电烧伤大鼠血清诱导的单核细胞中的变化,探讨其在单核细胞与血管内皮细胞黏附中的作用. 方法 取64只清洁级SD大鼠制作电烧伤模型,制备电烧伤大鼠血清;取24只大鼠不作处理,制备正常大鼠血清.（1）常规培养人单核细胞株THP-1细胞,按照随机数字表法将对数生长期细胞分为正常血清组（将细胞重新悬浮于含体积分数20％正常大鼠血清的RPMI 1640培养液中）和烧伤血清组（将细胞重新悬浮于含体积分数20％电烧伤大鼠血清的RPMI 1640培养液中）,每组设6个复孔.正常血清组于培养24 h,烧伤血清组于培养3、6、24 h,观察THP-1细胞形态,双抗体夹心ELISA法测定2组细胞上清液中TNF-α含量.2组均于培养3、6、24 h收集细胞,蛋白质印迹法检测Akt活化状态.（2）另取THP-1细胞按照随机数字表法分为4组：正常血清组、烧伤血清组,2组各自培养方法同前;正常血清＋阻断剂组、烧伤血清＋阻断剂组,在正常血清组、烧伤血清组的培养液中加入渥曼青霉素100 nmol/L,余培养方法同前.每组设6个复孔.取培养3、6h的THP-1细胞,加入单层人血管内皮细胞株EA.hy926细胞,行单核-内皮细胞黏附检测.对数据行单因素方差分析、LSD-￡检验. 结果 （1）正常血清组培养24 h,THP-1细胞呈悬浮生长、形态一致.烧伤血清组培养3h,细胞膜完整,细胞形态不规则;培养6h细胞大小不一,细胞膜与细胞质膨胀;培养24 h细胞膜破裂,细胞死亡.正常血清组培养24 h和烧伤血清组培养3、6、24 h,THP-1细胞上清液中TNF-α含量分别为（38.5±1.4）、（75.1±1.5）、（91.5±1.8）、（117.0±1.4）pg/mL,总体比较差异明显（F=1 415.306,P＜0.01）.烧伤血清组培养3、6、24 h THP-1细胞上清液中TNF-α含量均明显高于正常血清组培养24 h（￡值分别为29.614、42.852、63.485,P值均小于0.01）.烧伤血清组培养3�Objective To observe the change in phosphoinositide 3-kinase/protein kinase B （PI3K/Akt） signal pathway in monocytes as induced by serum of rats with electrical burn,and to explore the effects of PI3K/Akt pathway on monocyte-endothelial cell adhesion.Methods Sixty-four SD rats of clean grade were inflicted with electrical burn for the collection of serum of rats with electrical burn ; another group of twenty-four SD rats were used to obtain normal serum without treatment.（1） Human monocyte line THP-1 was routinely cultured.The THP-1 cells in logarithmic phase were divided into normal serum group （resuspended in RPMI 1640 medium with 20% normal rat serum） and burn serum group （resuspended with RPMI 1640 medium with 20% serum of rats with electrical burn） according to the random number table,with 6 wells in each group.Morphology of THP-1 cells in normal serum group was observed at post culture hour （PCH） 24,and that in burn serum group at PCH 3,6,24.The contents of TNF-α in culture supernatant were determined by double-antibody sandwich ELISA at the corresponding time point in each group.The state of Akt activation was determined by Western blotting at PCH 3,6,24.（2） Another portion of THP-1 cells were divided into 4 groups according to the random number table,with 6 wells in each group.Cells in normal serum group and burn serum group were given with the same culture condition as above; cells in normal serum ＋ inhibitor group and burn serum ＋ inhibitor group were cultured with the same culture conditions as in the former two groups correspondingly with addition of 100 nmol/L wortmannin in the nutrient solution.At PCH 3 and 6,THP-1 cells were added into the well with a monolayer of endothelial cell line EA.hy926 to observe the monocyte-endothelial cell adhesion.Data were processed with one-way analysis of variance and LSD-t test.Results （1） In normal serum group,THP-1 cells showed growth in suspension,with uniform shape at PCH 24.In burn serum group,the cell shape became irregular th

关 键 词：烧伤 电 磷脂酰肌醇3-激酶 蛋白激酶类 血清 单核细胞 内皮细胞 黏附作用 

分 类 号：R644[医药卫生—外科学]