【摘要】：目的： 觀察在脂多糖（LPS）誘導(dǎo)的NR8383肺泡巨噬細(xì)胞炎癥反應(yīng)中miR-21及腫瘤壞死因子-α（TNF-α）的動態(tài)表達(dá)情況，分析它們的表達(dá)變化相關(guān)性，探討miR-21在肺泡巨噬細(xì)胞炎性反應(yīng)中的作用。 方法： 按1×106個/mL把體外培養(yǎng)的肺泡巨噬細(xì)胞接種于六孔板，90min貼壁后加入終濃度1μg/mL的脂多糖，在刺激之后的0h、3h、6h、12h時間點離心后分別收集培養(yǎng)上清液與細(xì)胞團(tuán)塊。Western Blot檢測細(xì)胞核中NF-κB p65蛋白表達(dá)，實時熒光定量PCR（RT-qPCR）測定細(xì)胞中TNF-α mRNA與miR-21的表達(dá)情況，酶聯(lián)免疫吸附法（ELISA）測定上清液中TNF-α蛋白表達(dá)水平。雙變量相關(guān)性分析法分析miR-21和TNF-α mRNA的相關(guān)性。 結(jié)果： （1）NR8383肺泡巨噬細(xì)胞中的TNF-α mRNA表達(dá)在脂多糖刺激細(xì)胞3h后達(dá)到頂峰（61.57±11.42倍，P＜0.01），6h下降至27.72±5.16倍（P＜0.05），12h時為12.02±2.61倍（P＜0.01）；培養(yǎng)上清液中的TNF-α蛋白在脂多糖刺激3h后出現(xiàn)升高（367.98±45.23pg/mL，P＜0.01），6h繼續(xù)升高（591.73±48.18pg/mL），12h到達(dá)表達(dá)高峰（769.92±57.73pg/mL，P＜0.01）； （2）脂多糖刺激NR8383肺泡巨噬細(xì)胞后，，細(xì)胞中的miR-21表達(dá)水平逐漸增高；3h與0h miR-21表達(dá)量差異無統(tǒng)計學(xué)意義、6h（3.27±0.54倍P＜0.01）和12h（6.28±0.95倍P＜0.01） miR-21的表達(dá)量逐漸升高。 （3）脂多糖刺激NR8383肺泡巨噬細(xì)胞后誘導(dǎo)NF-κB活化，在3h、6h、12h時間點細(xì)胞核中NF-κB p65蛋白較0h組均表達(dá)上調(diào)。 （4）NR8383肺泡巨噬細(xì)胞中miR-21的表達(dá)水平與TNF-α mRNA的含量呈負(fù)相關(guān)（r=-0.895，P＜0.01）。 結(jié)論： 脂多糖刺激NR8383肺泡巨噬細(xì)胞后，miR-21表達(dá)逐漸上調(diào)而TNF-αmRNA表達(dá)逐漸下調(diào)，即細(xì)胞中miR-21的表達(dá)水平與TNF-α mRNA的表達(dá)水平呈負(fù)相關(guān)，推測miR-21可能負(fù)性調(diào)控了NR8383肺泡巨噬細(xì)胞的炎癥反應(yīng)。
[Abstract]:Aim: to observe the dynamic expression of miR-21 and tumor necrosis factor-偽 (TNF- 偽) 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- 偽 mRNA and miR-21 in cells was detected by real time fluorescence quantitative PCR (RT-qPCR). The expression of TNF- 偽 protein in the culture medium was determined by enzyme-linked immunosorbent assay (Elisa). The correlation between miR-21 and TNF- 偽 mRNA was analyzed by bivariate correlation analysis. Results: (1) the expression of TNF- 偽 mRNA in NR8383 alveolar macrophages reached its peak at 3 h after lipopolysaccharide stimulation (61.57 鹵11.42 times, P < 0.01), decreased to 27.72 鹵5.16 times at 6 h and 12.02 鹵2.61 times at 12 h. The expression of TNF- 偽 protein in the culture medium increased 3 hours after lipopolysaccharide stimulation (367.98 鹵45.23 PG / mL, P < 0.01), and continued to increase at 6 h (591.73 鹵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 鹵0. 54 times P < 0. 01) and 12 h (6. 28 鹵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- 偽 mRNA (r 鈮

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