miR-21在脂多糖诱导的肺泡巨噬细胞中的动态表达及其与TNF-α的相关性分析

发布时间：2019-06-17 19:24

【摘要】：目的： 观察在脂多糖（LPS）诱导的NR8383肺泡巨噬细胞炎症反应中miR-21及肿瘤坏死因子-α（TNF-α）的动态表达情况，分析它们的表达变化相关性，探讨miR-21在肺泡巨噬细胞炎性反应中的作用。 方法： 按1×106个/mL把体外培养的肺泡巨噬细胞接种于六孔板，90min贴壁后加入终浓度1μg/mL的脂多糖，在刺激之后的0h、3h、6h、12h时间点离心后分别收集培养上清液与细胞团块。Western Blot检测细胞核中NF-κB p65蛋白表达，实时荧光定量PCR（RT-qPCR）测定细胞中TNF-α mRNA与miR-21的表达情况，酶联免疫吸附法（ELISA）测定上清液中TNF-α蛋白表达水平。双变量相关性分析法分析miR-21和TNF-α mRNA的相关性。 结果： （1）NR8383肺泡巨噬细胞中的TNF-α mRNA表达在脂多糖刺激细胞3h后达到顶峰（61.57±11.42倍，P＜0.01），6h下降至27.72±5.16倍（P＜0.05），12h时为12.02±2.61倍（P＜0.01）；培养上清液中的TNF-α蛋白在脂多糖刺激3h后出现升高（367.98±45.23pg/mL，P＜0.01），6h继续升高（591.73±48.18pg/mL），12h到达表达高峰（769.92±57.73pg/mL，P＜0.01）； （2）脂多糖刺激NR8383肺泡巨噬细胞后，，细胞中的miR-21表达水平逐渐增高；3h与0h miR-21表达量差异无统计学意义、6h（3.27±0.54倍P＜0.01）和12h（6.28±0.95倍P＜0.01） miR-21的表达量逐渐升高。 （3）脂多糖刺激NR8383肺泡巨噬细胞后诱导NF-κB活化，在3h、6h、12h时间点细胞核中NF-κB p65蛋白较0h组均表达上调。 （4）NR8383肺泡巨噬细胞中miR-21的表达水平与TNF-α mRNA的含量呈负相关（r=-0.895，P＜0.01）。 结论： 脂多糖刺激NR8383肺泡巨噬细胞后，miR-21表达逐渐上调而TNF-αmRNA表达逐渐下调，即细胞中miR-21的表达水平与TNF-α mRNA的表达水平呈负相关，推测miR-21可能负性调控了NR8383肺泡巨噬细胞的炎症反应。
[Abstract]:Aim: to observe the dynamic expression of miR-21 and tumor necrosis factor-伪 (TNF- 伪) in NR8383 alveolar macrophages induced by lipopolysaccharide (LPS), to analyze the correlation between their expression and to explore the role of miR-21 in alveolar macrophage inflammation. Methods: alveolar macrophages cultured in vitro were inoculated on six-well plate according to 1 脳 10 ~ 6 / mL. The final concentration of lipopolysaccharide was 1 渭 g / mL after 90min adherent. The culture medium and cell mass were collected at 0 h, 6 h and 12 h after stimulation. The expression of NF- 魏 B p65 protein in nucleus was detected by Western Blot, and the expression of TNF- 伪 mRNA and miR-21 in cells was detected by real time fluorescence quantitative PCR (RT-qPCR). The expression of TNF- 伪 protein in the culture medium was determined by enzyme-linked immunosorbent assay (Elisa). The correlation between miR-21 and TNF- 伪 mRNA was analyzed by bivariate correlation analysis. Results: (1) the expression of TNF- 伪 mRNA in NR8383 alveolar macrophages reached its peak at 3 h after lipopolysaccharide stimulation (61.57 卤11.42 times, P < 0.01), decreased to 27.72 卤5.16 times at 6 h and 12.02 卤2.61 times at 12 h. The expression of TNF- 伪 protein in the culture medium increased 3 hours after lipopolysaccharide stimulation (367.98 卤45.23 PG / mL, P < 0.01), and continued to increase at 6 h (591.73 卤48.18 PG / mL), 12 h, P < 0.01); (2). The expression level of miR-21 in alveolar macrophages stimulated by lipopolysaccharide increased gradually. There was no significant difference in the expression of miR-21 between 3 h and 0 h, but the expression of miR-21 increased gradually at 6 h (3. 27 卤0. 54 times P < 0. 01) and 12 h (6. 28 卤0. 95 times P < 0. 01). (3) lipopolysaccharide stimulated NR8383 alveolar macrophages and induced NF- 魏 B activation. At 3 h, 6 h, 12 h, the expression of NF- 魏 B p65 protein in nucleus was up-regulated compared with that in 0 h group. (4) the expression of miR-21 in NR8383 alveolar macrophages was negatively correlated with the content of TNF- 伪 mRNA (r 鈮

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