非受体酪氨酸激酶Tec在内毒素/脂多糖诱导人肺泡上皮细胞A549白细胞介素8产生中的作用及机制  

Role and mechanism of nonreceptor tyrosine kinase Tec in endotoxin/lipopolysaccharide-induced interleukin-8 production in human alveolar epithelial cells A549

作　　者：王艺[1,2] 胡迎 王飞[1] 刘晟[1] 王永杰[1] 陈旭林[1] Wang Yi;Hu Ying;Wang Fei;Liu Sheng;Wang Yongjie;Chen Xulin(Department of Burns, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China, is working at the First Affiliated Hospital of Tsinghua University, Beijing 100020, China)

机构地区：[1]安徽医科大学第一附属医院烧伤科,合肥230022 [2]清华大学第一附属医院,北京100020

出　　处：《中华烧伤杂志》2019年第8期580-586,共7页Chinese Journal of Burns

基　　金：国家自然科学基金(81671877).

摘　　要：目的探讨非受体酪氨酸激酶Tec在内毒素/脂多糖(LPS)诱导人肺泡上皮细胞A549促炎性细胞因子白细胞介素8(IL-8)产生中的作用及机制。方法将人肺泡上皮细胞A549用含体积分数10%胎牛血清的RPMI-1640培养液常规培养传代,取第2或第3代细胞进行后续实验。(1)取细胞,按随机数字表法分为6组,每组4孔。空白对照组细胞常规培养2h;单纯LPS组细胞常规培养1h后加入1μg/mL的LPS刺激1h;单纯LFM-A13组细胞于常规培养液中加入75μmol/L的LFM-A13处理1h后更换培养液常规培养1h;25μmol/LLFM-A13+LPS组、75μmol/LLFM-A13+LPS组、100μmol/LLFM-A13+LPS组细胞分别于常规培养液中加入25、75、100μmol/L的LFM-A13处理1h后,均更换培养液并加入1μg/mL的LPS刺激1h。采用蛋白质印迹法检测各组细胞内Tec的蛋白表达情况,采用酶联免疫吸附测定法检测各组细胞培养上清液中IL-8的含量。(2)取细胞,按随机数字表法分为5组,每组4孔。空白对照组细胞常规培养2h;小干扰RNA(siRNA)对照+LPS组细胞用空的慢病毒转染10h后,于常规培养液中加入1μg/mL的LPS刺激2h;Tecmus-298RNAi+LPS组、Tecmus-299RNAi+LPS组、Tecmus-300RNAi+LPS组细胞分别用搭载Tecmus-298RNAi、Tecmus-299RNAi、Tecmus-300RNAi的慢病毒转染10h后,均于常规培养液中加入1μg/mL的LPS刺激2h。采用蛋白质印迹法检测各组细胞内Tec的蛋白表达情况,筛选沉默Tec基因效果最佳的Tec-siRNA。(3)取细胞,按随机数字表法分为4组,每组4孔。空白对照组细胞常规培养2h;病毒对照组细胞转染空的慢病毒10h后,常规培养2h;单纯LPS组细胞于常规培养液中加入1μg/mL的LPS刺激2h;Tec-siRNA+LPS组细胞转染搭载沉默Tec基因效果最佳的Tec-siRNA的慢病毒10h后,于常规培养液中加入1μg/mL的LPS刺激2h。采用蛋白质印迹法检测各组细胞内p38丝裂原活化蛋白激酶(MAPK)和细胞外信号调节激酶(ERK)MAPK的蛋白表达。对数据行单因素方差分析和LSD-t检�Objective To investigate the role and mechanism of nonreceptor tyrosine kinase Tec in the production of pro-inflammatory cytokine interleukin-8 (IL-8) induced by endotoxin/lipopolysaccharide (LPS) in human alveolar epithelial cells A549. Methods Human alveolar epithelial cells A549 were routinely cultured and passaged in Roswell Park Memorial Institute-1640 medium containing 10% fetal bovine serum. The second or third passage of cells were collected for subsequent experiments.(1) Cells were collected and divided into 6 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in simple LPS group were routinely cultured for 1 h and then stimulated by 1 μg/mL LPS for 1 h. Cells in simple LFM-A13 group were cultured with conventional culture medium adding 75 μmol/L LFM-A13 for 1 h and then cultured with replaced conventional culture medium for 1 h. Cells in 25 μmol/L LFM-A13+ LPS group, 75 μmol/L LFM-A13+ LPS group, and 100 μmol/L LFM-A13+ LPS group were cultured with conventional culture medium adding 25, 75, and 100 μmol/L LFM-A13 respectively for 1 h and then all stimulated by 1 μg/mL LPS added into the replaced conventional culture medium for 1 h. The protein expression of Tec in cells of each group was detected by Western blotting, and the content of IL-8 in cell culture supernatant of each group was determined by enzyme-linked immunosorbent assay.(2) Cells were collected and divided into 5 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in small interfering RNA (siRNA) control+ LPS group were transfected with empty lentivirus for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec mus-298 RNA interference (RNAi)+ LPS group, Tec mus-299 RNAi+ LPS group, and Tec mus-300 RNAi+ LPS group were transfected with lentivirus loaded with Tec mus-298 RNAi, Tec mus-299 RNAi, and Tec mus-300

关 键 词：脂多糖类 白细胞介素8 P38丝裂原活化蛋白激酶类 细胞外信号调节MAP激酶类 人肺泡上皮细胞 非受体酪氨酸激酶Tec 

分 类 号：R459.7[医药卫生—急诊医学]