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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 被引量:5

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
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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)。与LPS对照组比较,各BMX-IN-1预处理组细胞中TNF-α mRNA表达量均下降,但仅75 000 nmol/L BMX-IN-1预处理组细胞中TNF-α mRNA表达量显著降低(P〈0.05)。BMX-IN-1最佳作用浓度为75 000 nmol/L。(2)与LPS对照组比较,BMX-IN-1预处理2、4 h组细胞中TNF-α mRNA表达量无明显变化(P值均大于0.05),BMX-IN-1预处理8、12、18 h组细胞中TNF-α mRNA表达量显著降低(P〈0.05或P〈0.01)。其中,BMX-IN-1预处理12 h组细胞中TNF-α mRNA表达量最低。BMX-IN-1最佳作用时间为12 h。(3)BMX-IN-1对照组细胞中TNF-α和IL-1β mRNA表达量分别为0.97±0.13、0.98±0.06,与空白对照组的1.00相近(P值均大于0.05)。LPS对照组细胞中TNF-α和IL-1β mRNA表达量分别为2.97±0.17和3.07±0.60,BMX-IN-1+LPS组细胞中TNF-α和IL-1β mRNA表达量分别为2.31±0.94和2.55±0.73,均显著高于空白对照组(P值均小于0.01)。BMX-IN-1+LPS组细胞中TNF-α和IL-1β mRNA表达量明显低于LPS对照组(P值均小于0.05)。BMX-IN-1对照组细胞中BMX和p38MAPK活性分别为0.95±0.19和0.98±0.18,均与空白对照组的1.00±0.14和1.00±0.22相近(P值均大于0.05);LPS对照组细胞中BMX和p38MAPK活性分别为1.98±0.33和2.05±0.34,均显著高于空白对照组(P值均小于0.01)。BMX-IN-1+LPS组细胞中BMX和p38MAPK活性分别为1.00±0.17和1.67±0.27,明显低于LPS对照组(P〈0.05或P〈0.01)。结论 BMX可促进LPS诱导小鼠单核巨噬细胞促炎性细胞因子TNF-α和IL-1β的产生,该作用可能与BMX活化p38MAPK途径有关。 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. Cells in blank control group were routinely cultured for the optimum time plus 1 h. Cells in BMX-IN-1 control group were pretreated with BMX-IN-1 in the optimum concentration for the optimum time and then routinely cultured for 1 h. Cells in LPS control group were routinely cultured for the optimum time and then stimulated by 0.1 μg/mL LPS for 1 h. Cells in BMX-IN-1+ LPS group were pretreated with BMX-IN-1 in the optimum concentration for the optimum time and then stimulated by 0.1 μg/mL LPS for 1 h. The mRNA expressions of TNF-α and IL-1β were determined by real-time fluorescent quantitative RT-PCR, and the activity of BMX and p38 mitogen-activated protein kinase (MAPK) were determined by Western blotting, with 8 samples in each determination. Data were processed with one-way analysis of variance and LSD test.Results (1) Compared with that in blank control group, the mRNA expression of TNF-α of cells was significantly increased in the other 5 groups (with P values below 0.01). Compared with that in LPS control group, the mRNA expression of TNF-α of cells was decreased in each BMX-IN-1 pretreatment group, but only the mRNA expression of TNF-α of cells in 75 000 nmol/L BMX-IN-1 pretreatment group was significantly decreased (P〈0.05). The optimum concentration of BMX-IN-1 was 75 000 nmol/L. (2) Compared with that in LPS control group, the mRNA expression of TNF-α of cells was not significantly changed in 2 and 4 h BMX-IN-1 pretreatment groups (with P values above 0.05) but significantly decreased in 8, 12, and 18 h BMX-IN-1 pretreatment groups (P〈0.05 or P〈0.01). The mRNA expression of TNF-α of cells in 12 h BMX-IN-1 pretreatment group was the lowest. The optimum time for BMX-IN-1 pre-treatment was 12 h. (3) The mRNA expressions of TNF-α and IL-1β of cells in BMX-IN-1 control group were 0.97±0.13 and 0.98±0.06, respectively, which were similar to 1.00 of blank control group (with P values above 0.05). The mRNA expressions of TNF-α and IL-1β of cells in LPS control group were 2.97±0.17 and 3.07±0.60, respectively, while those in BMX-IN-1+ LPS group were 2.31±0.94 and 2.55±0.73, respectively, with the 4 values significantly higher than those in blank control group (with P values below 0.01). The mRNA expressions of TNF-α and IL-1β of cells in BMX-IN-1+ LPS group were significantly lower than those in LPS control group (with P values below 0.05). The activity values of BMX and p38MAPK of cells in BMX-IN-1 control group were 0.95±0.19 and 0.98±0.18, respectively, which were close to 1.00±0.14 and 1.00±0.22 of blank control group (with P values above 0.05). The activity values of BMX and p38MAPK of cells in LPS control group were 1.98±0.33 and 2.05±0.34, respectively, which were significantly higher than those of blank control group (with P values below 0.01). The activity values of BMX and p38MAPK of cells in BMX-IN-1+ LPS group were 1.00±0.17 and 1.67±0.27, respectively, which were obviously lower than those of LPS control group (P〈0.05 or P〈0.01).Conclusions BMX can increase the production of pro-inflammatory cytokines TNF-α and IL-1β from mouse mononuclear-macrophages induced by LPS, which may be associated with the activation of the p38MAPK pathway by BMX.
出处 《中华烧伤杂志》 CAS CSCD 北大核心 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)
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 Lipopolysaccharides Macrophages Cytokines p38 mitogen-activated protein kinases Bone marrow tyrosine kinase on chromosome X
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