miR-146a在脂多糖诱导的大鼠急性肺损伤中的作用及机制研究

发布时间：2018-05-12 04:23

  本文选题：微小RNA-146a + 肺泡巨噬细胞　； 参考：《南昌大学》2012年博士论文

【摘要】：研究背景和目的 急性肺损伤(Acute Lung Injury, ALI)/急性呼吸窘迫综合征(Acute Respiratory Distress Syndrome, ARDS)指由心源性以外的各种肺内外致病因素导致的肺部炎性反应,使肺泡-毛细血管上皮细胞及肺部屏障结构受损,肺气体交换功能发生障碍,是一种以急性、进行性、顽固性低氧血症为特征的临床综合征,是目前导致危重病人死亡的主要原因。尽管不断出现新的治疗ALI/ARDS的方法,包括肺保护性通气策略、体外膜肺氧合、高容量血液滤过等,但其死亡率依然高达30%-50%。在ALI/ARDS发生发展过程中,肺泡巨噬细胞介导的固有免疫反应发挥着关键作用。当肺组织受到肺内外各种因素刺激时,可以活化肺泡巨噬细胞膜上的Toll受体(Toll-like receptors, TLRs),再经信号蛋白的转导活化核转录因子-KappaB (nuclear factor-KappaB, NF-κB),诱导大量炎症因子的表达释放,促使炎症反应的级联放大,形成肺内炎症“瀑布”反应,导致ALI/ARDS的发生。因此,抑制肺泡巨噬细胞过度活化、降低肺内炎症反应是治疗ALI/ARDS的根本措施。microRNA-146a (miR-146a)在固有免疫反应的调控中具有重要作用,它通过靶向沉默TLRs/NF-κB信号通路中的两个关键信号转导蛋白,能够阻断NF-κB活化,从而抑制炎症因子的释放,有望成为抑制肺泡巨噬细胞过度活化,降低肺内炎症反应的有效措施。本实验首先通过体外构建肺泡巨噬细胞炎症反应模型,观察miR-146a和炎症因子的表达变化,分析miR-146a在肺泡巨噬细胞炎症反应中的调控作用,然后经体外转染miR-146a mimic上调肺泡巨噬细胞内miR-146a的表达,观察miR-146a对炎症因子和炎症信号通路关键蛋白表达的影响,分析它在肺泡巨噬细胞炎症反应中的调控机制,最后于体内构建大鼠ALI模型,观察miR-146a在肺损伤组织中的表达变化,并结合体外研究结果,探讨其在大鼠ALI模型中的调控作用及作用机制,为临床治疗ALI提供新的治疗思路。 研究内容和方法： 1体外实验 (1)将体外培养的大鼠肺泡巨噬细胞(NR8383)按2×106个/孔接种于六孔板,细胞贴壁90min,加入终浓度1μg/ml LPS处理细胞,于刺激0h、3h、6h、12h的各个时间点收集细胞和细胞上清液。用实时荧光定量PCR (RT-qPCR)分别检测细胞中TNF-α mRNA和miR-146a的表达变化,用酶联免疫吸附实验(ELISA)检测细胞培养上清液中TNF-α蛋白的表达变化； (2)将体外培养的NR8383细胞按1.5×106个/孔接种于六孔板,细胞贴壁90min,加入50nM浓度的miR-146a mimic转染NR8383细胞24h后收集细胞。用RT-qPCR检测细胞中miR-146a上调的表达倍数和miR-146a两个靶基因IRAK-1mRNA和TRAF-6mRNA的表达变化,用Western blot检测细胞内IRAK-1和TRAF-6的表达变化,验证miR-146a转染的有效性以及验证miR-146a的靶基因； (3)将体外培养的NR8383细胞按1.5×106个/孔接种于六孔板,细胞贴壁90min,加入50nM浓度的miR-146a mimic转染NR8383细胞24h,再加入终浓度1μg/ml LPS处理细胞6h后收集细胞和细胞上清液。用RT-qPCR检测细胞中TNF-a mRNA和miR-146a的表达变化,用ELISA检测细胞培养上清液中TNF-a蛋白的表达变化,用免疫荧光及Western blot检测细胞内NF-κB p65的核移位变化,明确miR-146a对炎症反应的调控作用及作用机制。 2体内实验 选择雄性SPF级8～10周龄SD大鼠,按7.5mg/kg的脂多糖,经气道滴入大鼠肺部诱导ALI模型,造模6小时后处死大鼠,检测肺损伤程度。留取右肺行肺泡灌洗,用ELISA检测灌洗液中TNF-a蛋白的表达变化；留取左肺用RT-qPCR检测肺组织中miR-146a以及TNF-a mRNA的表达变化、用Western blot检测NF-κB p65含量变化。 结果： 1.体外实验结果 (1)LPS刺激NR8383细胞后,诱导了细胞内炎症因子TNF-α的表达变化,其中3、6、12h时间点的TNF-α mRNA表达量分别是0h对照组的67.48±24.52倍(P0.01)、29.53±4.26倍(P0.01)、11.37±2.52倍(P0.01)；细胞上清液中TNF-α蛋白的含量于LPS刺激Oh后逐渐上升,0h为29.53±7.80pg/ml、3h为359.80±57.54pg/ml (P0.01)、6h为586.22±30.57pg/ml (P0.01)、12h为729.22±50.40pg/ml (P0.01)。 (2)LPS刺激NR8383细胞后,诱导了细胞内炎症因子TNF-α的表达变化,同时也诱导了miR-146a的表达变化,3h与0h miR-146a表达量差异无统计学意义、6h和12h miR-146a的表达量逐渐升高,分别是0h的5.33±0.81倍(P0.01)和8.21±1.19倍(P0.01)。 (3)转染50nM浓度的miR-146a mimic入NR8383细胞24h后,与control相比较,miR-146a的表达量上调至24.55±6.14倍(P0.01),miR-146a的靶基因IRAK-1mRNA和TRAF-6mRNA表达量无明显变化,而IRAK-1和TRAF-6蛋白表达水平分别下降至0.73±0.05倍(P0.05)和0.64±0.09倍(P0.05)。 (4)转染50nM浓度的miR-146a mimic入NR8383细胞24h后,给予LPS刺激6h后,相对control组,转染miR-146a mimic组TNF-α mRNA表达下降至0.47±0.06倍(P0.05),细胞上清液中TNF-α蛋白从616.6±42.28pg/ml下降至211.5±30.39pg/ml (P0.01),细胞浆中NF-κB p65蛋白含量增加至1.74±0.11倍(P0.05),细胞核中NF-κB p65蛋白含量下降至0.56±0.12倍(P0.05),NF-κBp65核移位明显受到抑制。 2.体内实验结果： LPS气管滴入大鼠肺部6h后,ALI模型构建成功：氧和指数300mmHg、肺病理损伤严重、模型组肺泡灌洗液中TNF-α蛋白为506.4±40.3pg/ml,明显高于对照组48.51±12.11pg/ml(P0.01);相对于对照组,模型组肺组织细胞浆中NF-κB p65蛋白含量下降至0.37±0.14倍(P0.05),细胞核中NF-κB p65蛋白含量增加至2.43±0.12倍(P0.05)、TNF-a mRNA表达上调了3.61±1.03倍(P0.05),miR-146a的表达上升2.07±0.26倍(P0.05)。 结论： 在LPS诱导的肺泡巨噬细胞炎症反应中,miR-146a通过靶向作用于TLRs/NF-κB信号通路中的两个关键信号转导蛋白IRAK-1和TRAF-6,从而阻断NF-κB的活化,抑制炎症因子TNF-a的释放；在LPS诱导的ALI模型中,LPS同样诱导了miR-146a的表达上调。因此,可以推测,在ALI发生过程中,通过上调肺泡巨噬细胞内miR-146a的表达,可以阻断肺泡巨噬细胞内TLRs/NF-κB信号通路的活化,从而减少肺部炎症因子的释放,减轻肺部炎症反应,有望提高ALI/ARDS成功救治率。
[Abstract]:Background and purpose of research
Acute lung injury (Acute Lung Injury, ALI) / acute respiratory distress syndrome (Acute Respiratory Distress Syndrome, ARDS) refers to the pulmonary inflammatory response caused by various pulmonary and internal pathogenic factors other than cardiogenic. The pulmonary alveolar capillary epithelial cells and lung barrier structures are damaged and the pulmonary gas exchange function is impaired. Acute, progressive, refractory hypoxemia, characterized by clinical syndrome, is the main cause of death in critically ill patients. Although new methods of treating ALI/ARDS, including pulmonary protective ventilation, extracorporeal membrane oxygenation, and high volume hemofiltration, the mortality rate is still as high as 30%-50%. in ALI/ARDS. During the process, the intrinsic immune response mediated by alveolar macrophages plays a key role. When the lung tissue is stimulated by various factors inside and outside the lung, it can activate the Toll receptor (Toll-like receptors, TLRs) on the membrane of the alveolar macrophage, and then activate the nuclear factor -KappaB (nuclear factor-KappaB, NF- kappa B) through the transduction of signal protein. The expression of inflammatory factors is released to promote the cascade of inflammatory reactions and to form a "waterfall" reaction in lung inflammation, resulting in the occurrence of ALI/ARDS. Therefore, inhibition of pulmonary alveolar macrophage activation and the reduction of pulmonary inflammatory response are the fundamental measures for the treatment of ALI/ARDS,.MicroRNA-146a (miR-146a), which plays an important role in the regulation of the inherent immune response. By targeting two key signal transduction proteins in the TLRs/NF- kappa B signaling pathway, it can block the activation of NF- kappa B and inhibit the release of inflammatory factors. It is expected to be an effective measure to inhibit the excessive activation of alveolar macrophages and reduce the inflammatory response in the lung. First, this experiment first constructed the alveolar macrophage inflammatory response model in vitro. The expression of miR-146a and inflammatory factors was observed and the regulation of miR-146a in the inflammatory response of alveolar macrophages was analyzed. Then, the expression of miR-146a in alveolar macrophages was up-regulated by miR-146a mimic transfected in vitro, and the effects of miR-146a on the expression of key proteins of inflammatory factors and inflammatory signaling pathways were observed, and the macrophage in alveolar macrophages was analyzed. The regulatory mechanism of cellular inflammatory response was established in vivo. Finally, the rat ALI model was constructed in vivo, and the expression of miR-146a in the lung injury tissues was observed. In addition, the regulatory role and mechanism in the rat ALI model were investigated in vitro, and a new therapeutic idea was provided for the clinical treatment of ALI.
Research contents and methods:
1 in vitro experiment
(1) the rat alveolar macrophages (NR8383) cultured in vitro were inoculated with 2 x 106 / pores on the six pore plate, the cells were adhered to the cell wall 90min, and the final concentration 1 mu g/ml LPS were added to the cells. The cells and cell supernatants were collected at every time point of 0h, 3h, 6h and 12h. The TNF- alpha mRNA and the table of TNF- alpha mRNA and the cells were detected by real-time fluorescence determination PCR (RT-qPCR). The expression of TNF- alpha protein in cell culture supernatant was detected by enzyme linked immunosorbent assay (ELISA).
(2) NR8383 cells cultured in vitro were inoculated with 1.5 x 106 / holes on six orifice plates, cell adhered to 90min, and NR8383 cell 24h was transfected with 50nM concentration. NR8383 cells were transfected to 24h to collect cells. The expression of miR-146a up-regulation and miR-146a two target genes IRAK-1mRNA and TRAF-6mRNA were detected by RT-qPCR. The expression of IRAK-1 and TRAF-6 in the cells verified the validity of miR-146a transfection and verified the target gene of miR-146a.
(3) NR8383 cells cultured in vitro were inoculated with 1.5 x 106 / pores on six pore plates, cells adhered to 90min, and NR8383 cells 24h were transfected with 50nM concentration of miR-146a mimic, then the cells and cell supernatants were collected after the final concentration of 1 mu g/ml LPS, and the cells and cell supernatants were detected by RT-qPCR. The expression of TNF-a protein in the supernatant of cell culture was changed. The changes of nuclear shift of NF- kappa B p65 were detected by immunofluorescence and Western blot, and the regulatory role and mechanism of miR-146a on the inflammatory reaction were determined.
2 in vivo experiment
The male SPF 8~10 weeks old SD rats were selected to induce the ALI model in the lungs of rats by 7.5mg/kg lipopolysaccharide (LPS). The rats were killed and the degree of lung injury was detected after 6 hours of model building. The right lung was left with alveolar lavage, and the expression of TNF-a protein in the lavage fluid was detected by ELISA; RT-qPCR in the left lung was used to detect miR-146a and TNF-a in the lung tissue. The expression of mRNA was detected by Western blot and NF- kappa B p65 content was detected.
Result锛，

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