X染色体骨髓酪氨酸激酶在内毒素／脂多糖诱导RAW264．7小鼠单核巨噬细胞促炎性细胞因子产生中的作用及机制  

Role of bone marrow tyrosine kinase on chromosome X in the production of pro-inflammatory cyto- kines from mouse mononuclear-macrophages RAW264.7 induced by endotoxin/lipopolysaccharide and its mechanism

作　　者：方旭[1] 胡迎[1] 王艺[1] 刘晟[1] 王飞[1] 陈旭林[1] 

机构地区：[1]安徽医科大学第一附属医院烧伤科,合肥230022

出　　处：《中华烧伤杂志》2017年第4期211-216,共6页Chinese Journal of Burns

基　　金：国家自然科学基金（81372050）

摘　　要：目的 探讨X染色体骨髓酪氨酸激酶（BMX）在LPS诱导小鼠单核巨噬细胞产生TNF－α和IL－1β中的作用及相关机制。 方法 将RAW264.7小鼠单核巨噬细胞接种于6孔板中，常规培养，用于以下实验。（1）取细胞，按随机数字表法分为空白对照组、LPS对照组及75、750、7 500、75 000 nmol/L BMX－IN－1预处理组，每组8孔。空白对照组细胞常规培养25 h；LPS对照组细胞常规培养24 h后，用终质量浓度（下同）为0.1 μg/mL LPS刺激1 h；后4组细胞分别用终物质的量浓度（下同）为75、750、7 500、75 000 nmol/L的BMX－IN－1处理24 h后，用0.1 μg/mL LPS刺激1 h。实时荧光定量RT－PCR法检测各组细胞中TNF－α mRNA表达量，筛选BMX－IN－1最佳作用浓度。（2）取细胞，按随机数字表法分为LPS对照组及BMX－IN－1预处理2、4、8、12、18 h组，每组8孔。LPS对照组细胞用0.1 μg/mL LPS刺激1 h；后5组细胞用最佳作用浓度的BMX－IN－1分别处理2、4、8、12、18 h后，用0.1 μg/mL LPS刺激1 h。实时荧光定量RT－PCR法检测各组细胞中TNF－α mRNA表达量，筛选BMX－IN－1最佳作用时间。（3）取细胞，按随机数字表法分为空白对照组、BMX－IN－1对照组、LPS对照组和BMX－IN－1＋LPS组，每组16孔。空白对照组细胞常规培养最佳作用时间加1 h；BMX－IN－1对照组细胞用最佳作用浓度BMX－IN－1处理最佳作用时间后，常规培养1 h；LPS对照组细胞常规培养最佳作用时间后，用0.1 μg/mL LPS刺激1 h；BMX－IN－1＋LPS组细胞用最佳作用浓度BMX－IN－1处理最佳作用时间后，用0.1 μg/mL LPS刺激1 h。采用实时荧光定量RT－PCR法检测各组细胞中TNF－α和IL－1β mRNA表达量，蛋白质印迹法检测各组细胞中BMX和p38MAPK活性，样本数均为8。对数据行单因素方差分析和LSD检验。结果 （1）与空白对照组比较，其余5组细胞中TNF－α mRNA表达量显著增高（P值均小于0.01�Objective To investigate the role of bone marrow tyrosine kinase on chromosome X （BMX） in the production of tumor necrosis factor α （TNF-α） and interleukin-1β （IL-1β） from mouse mononuclear-macrophages induced by endotoxin/lipopolysaccharide （LPS） and its related mechanism.Methods Mouse mononuclear-macrophages RAW264.7 were inoculated in 6-well plates and routinely cultured for the following experiments. （1） Cells were collected and divided into blank control group, LPS control group, and 75, 750, 7 500, 75 000 nmol/L BMX-IN-1 pretreatment groups according to the random number table, with 8 wells in each group. Cells in blank control group were routinely cultured for 25 h. Cells in LPS control group were routinely cultured for 24 h and stimulated by LPS in the final mass concentration （the same below） of 0.1 μg/mL for 1 h. Cells in the latter 4 groups were pretreated with BMX-IN-1 in the final molarity （the same below） of 75, 750, 7 500, 75 000 nmol/L for 24 h and stimulated by 0.1 μg/mL LPS for 1 h. The mRNA expression of TNF-α of cells in each group was determined by real-time fluorescent quantitative reverse transcription polymerase chain reaction （RT-PCR） to screen the optimum concentration of BMX-IN-1. （2） Cells were collected and divided into LPS control group and 2, 4, 8, 12, 18 h BMX-IN-1 pretreatment groups according to the random number table, with 8 wells in each group. Cells in LPS control group were stimulated by 0.1 μg/mL LPS for 1 h. Cells in the latter 5 groups were pretreated with optimum concentration of BMX-IN-1 for 2, 4, 8, 12, 18 h respectively and stimulated by 0.1 μg/mL LPS for 1 h. The mRNA expression of TNF-α of cells in each group was determined by real-time fluorescent quantitative RT-PCR to screen the optimum time for BMX-IN-1 pre-treatment. （3） Cells were collected and divided into blank control group, BMX-IN-1 control group, LPS control group, and BMX-IN-1＋ LPS group according to the random number table, with 16 wells in each group.

关 键 词：脂多糖类 巨噬细胞 细胞因子类 P38丝裂原活化蛋白激酶类 X染色体骨 髓酪氨酸激酶 

分 类 号：R459.7[医药卫生—急诊医学]