本文選題：微小RNA-146a + 肺泡巨噬細(xì)胞　； 參考：《南昌大學(xué)》2012年博士論文

【摘要】：研究背景和目的 急性肺損傷(Acute Lung Injury, ALI)/急性呼吸窘迫綜合征(Acute Respiratory Distress Syndrome, ARDS)指由心源性以外的各種肺內(nèi)外致病因素導(dǎo)致的肺部炎性反應(yīng),使肺泡-毛細(xì)血管上皮細(xì)胞及肺部屏障結(jié)構(gòu)受損,肺氣體交換功能發(fā)生障礙,是一種以急性、進(jìn)行性、頑固性低氧血癥為特征的臨床綜合征,是目前導(dǎo)致危重病人死亡的主要原因。盡管不斷出現(xiàn)新的治療ALI/ARDS的方法,包括肺保護(hù)性通氣策略、體外膜肺氧合、高容量血液濾過(guò)等,但其死亡率依然高達(dá)30%-50%。在ALI/ARDS發(fā)生發(fā)展過(guò)程中,肺泡巨噬細(xì)胞介導(dǎo)的固有免疫反應(yīng)發(fā)揮著關(guān)鍵作用。當(dāng)肺組織受到肺內(nèi)外各種因素刺激時(shí),可以活化肺泡巨噬細(xì)胞膜上的Toll受體(Toll-like receptors, TLRs),再經(jīng)信號(hào)蛋白的轉(zhuǎn)導(dǎo)活化核轉(zhuǎn)錄因子-KappaB (nuclear factor-KappaB, NF-κB),誘導(dǎo)大量炎癥因子的表達(dá)釋放,促使炎癥反應(yīng)的級(jí)聯(lián)放大,形成肺內(nèi)炎癥“瀑布”反應(yīng),導(dǎo)致ALI/ARDS的發(fā)生。因此,抑制肺泡巨噬細(xì)胞過(guò)度活化、降低肺內(nèi)炎癥反應(yīng)是治療ALI/ARDS的根本措施。microRNA-146a (miR-146a)在固有免疫反應(yīng)的調(diào)控中具有重要作用,它通過(guò)靶向沉默TLRs/NF-κB信號(hào)通路中的兩個(gè)關(guān)鍵信號(hào)轉(zhuǎn)導(dǎo)蛋白,能夠阻斷NF-κB活化,從而抑制炎癥因子的釋放,有望成為抑制肺泡巨噬細(xì)胞過(guò)度活化,降低肺內(nèi)炎癥反應(yīng)的有效措施。本實(shí)驗(yàn)首先通過(guò)體外構(gòu)建肺泡巨噬細(xì)胞炎癥反應(yīng)模型,觀察miR-146a和炎癥因子的表達(dá)變化,分析miR-146a在肺泡巨噬細(xì)胞炎癥反應(yīng)中的調(diào)控作用,然后經(jīng)體外轉(zhuǎn)染miR-146a mimic上調(diào)肺泡巨噬細(xì)胞內(nèi)miR-146a的表達(dá),觀察miR-146a對(duì)炎癥因子和炎癥信號(hào)通路關(guān)鍵蛋白表達(dá)的影響,分析它在肺泡巨噬細(xì)胞炎癥反應(yīng)中的調(diào)控機(jī)制,最后于體內(nèi)構(gòu)建大鼠ALI模型,觀察miR-146a在肺損傷組織中的表達(dá)變化,并結(jié)合體外研究結(jié)果,探討其在大鼠ALI模型中的調(diào)控作用及作用機(jī)制,為臨床治療ALI提供新的治療思路。 研究?jī)?nèi)容和方法： 1體外實(shí)驗(yàn) (1)將體外培養(yǎng)的大鼠肺泡巨噬細(xì)胞(NR8383)按2×106個(gè)/孔接種于六孔板,細(xì)胞貼壁90min,加入終濃度1μg/ml LPS處理細(xì)胞,于刺激0h、3h、6h、12h的各個(gè)時(shí)間點(diǎn)收集細(xì)胞和細(xì)胞上清液。用實(shí)時(shí)熒光定量PCR (RT-qPCR)分別檢測(cè)細(xì)胞中TNF-α mRNA和miR-146a的表達(dá)變化,用酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)檢測(cè)細(xì)胞培養(yǎng)上清液中TNF-α蛋白的表達(dá)變化； (2)將體外培養(yǎng)的NR8383細(xì)胞按1.5×106個(gè)/孔接種于六孔板,細(xì)胞貼壁90min,加入50nM濃度的miR-146a mimic轉(zhuǎn)染NR8383細(xì)胞24h后收集細(xì)胞。用RT-qPCR檢測(cè)細(xì)胞中miR-146a上調(diào)的表達(dá)倍數(shù)和miR-146a兩個(gè)靶基因IRAK-1mRNA和TRAF-6mRNA的表達(dá)變化,用Western blot檢測(cè)細(xì)胞內(nèi)IRAK-1和TRAF-6的表達(dá)變化,驗(yàn)證miR-146a轉(zhuǎn)染的有效性以及驗(yàn)證miR-146a的靶基因； (3)將體外培養(yǎng)的NR8383細(xì)胞按1.5×106個(gè)/孔接種于六孔板,細(xì)胞貼壁90min,加入50nM濃度的miR-146a mimic轉(zhuǎn)染NR8383細(xì)胞24h,再加入終濃度1μg/ml LPS處理細(xì)胞6h后收集細(xì)胞和細(xì)胞上清液。用RT-qPCR檢測(cè)細(xì)胞中TNF-a mRNA和miR-146a的表達(dá)變化,用ELISA檢測(cè)細(xì)胞培養(yǎng)上清液中TNF-a蛋白的表達(dá)變化,用免疫熒光及Western blot檢測(cè)細(xì)胞內(nèi)NF-κB p65的核移位變化,明確miR-146a對(duì)炎癥反應(yīng)的調(diào)控作用及作用機(jī)制。 2體內(nèi)實(shí)驗(yàn) 選擇雄性SPF級(jí)8～10周齡SD大鼠,按7.5mg/kg的脂多糖,經(jīng)氣道滴入大鼠肺部誘導(dǎo)ALI模型,造模6小時(shí)后處死大鼠,檢測(cè)肺損傷程度。留取右肺行肺泡灌洗,用ELISA檢測(cè)灌洗液中TNF-a蛋白的表達(dá)變化；留取左肺用RT-qPCR檢測(cè)肺組織中miR-146a以及TNF-a mRNA的表達(dá)變化、用Western blot檢測(cè)NF-κB p65含量變化。 結(jié)果： 1.體外實(shí)驗(yàn)結(jié)果 (1)LPS刺激NR8383細(xì)胞后,誘導(dǎo)了細(xì)胞內(nèi)炎癥因子TNF-α的表達(dá)變化,其中3、6、12h時(shí)間點(diǎn)的TNF-α mRNA表達(dá)量分別是0h對(duì)照組的67.48±24.52倍(P0.01)、29.53±4.26倍(P0.01)、11.37±2.52倍(P0.01)；細(xì)胞上清液中TNF-α蛋白的含量于LPS刺激Oh后逐漸上升,0h為29.53±7.80pg/ml、3h為359.80±57.54pg/ml (P0.01)、6h為586.22±30.57pg/ml (P0.01)、12h為729.22±50.40pg/ml (P0.01)。 (2)LPS刺激NR8383細(xì)胞后,誘導(dǎo)了細(xì)胞內(nèi)炎癥因子TNF-α的表達(dá)變化,同時(shí)也誘導(dǎo)了miR-146a的表達(dá)變化,3h與0h miR-146a表達(dá)量差異無(wú)統(tǒng)計(jì)學(xué)意義、6h和12h miR-146a的表達(dá)量逐漸升高,分別是0h的5.33±0.81倍(P0.01)和8.21±1.19倍(P0.01)。 (3)轉(zhuǎn)染50nM濃度的miR-146a mimic入NR8383細(xì)胞24h后,與control相比較,miR-146a的表達(dá)量上調(diào)至24.55±6.14倍(P0.01),miR-146a的靶基因IRAK-1mRNA和TRAF-6mRNA表達(dá)量無(wú)明顯變化,而IRAK-1和TRAF-6蛋白表達(dá)水平分別下降至0.73±0.05倍(P0.05)和0.64±0.09倍(P0.05)。 (4)轉(zhuǎn)染50nM濃度的miR-146a mimic入NR8383細(xì)胞24h后,給予LPS刺激6h后,相對(duì)control組,轉(zhuǎn)染miR-146a mimic組TNF-α mRNA表達(dá)下降至0.47±0.06倍(P0.05),細(xì)胞上清液中TNF-α蛋白從616.6±42.28pg/ml下降至211.5±30.39pg/ml (P0.01),細(xì)胞漿中NF-κB p65蛋白含量增加至1.74±0.11倍(P0.05),細(xì)胞核中NF-κB p65蛋白含量下降至0.56±0.12倍(P0.05),NF-κBp65核移位明顯受到抑制。 2.體內(nèi)實(shí)驗(yàn)結(jié)果： LPS氣管滴入大鼠肺部6h后,ALI模型構(gòu)建成功：氧和指數(shù)300mmHg、肺病理?yè)p傷嚴(yán)重、模型組肺泡灌洗液中TNF-α蛋白為506.4±40.3pg/ml,明顯高于對(duì)照組48.51±12.11pg/ml(P0.01);相對(duì)于對(duì)照組,模型組肺組織細(xì)胞漿中NF-κB p65蛋白含量下降至0.37±0.14倍(P0.05),細(xì)胞核中NF-κB p65蛋白含量增加至2.43±0.12倍(P0.05)、TNF-a mRNA表達(dá)上調(diào)了3.61±1.03倍(P0.05),miR-146a的表達(dá)上升2.07±0.26倍(P0.05)。 結(jié)論： 在LPS誘導(dǎo)的肺泡巨噬細(xì)胞炎癥反應(yīng)中,miR-146a通過(guò)靶向作用于TLRs/NF-κB信號(hào)通路中的兩個(gè)關(guān)鍵信號(hào)轉(zhuǎn)導(dǎo)蛋白IRAK-1和TRAF-6,從而阻斷NF-κB的活化,抑制炎癥因子TNF-a的釋放；在LPS誘導(dǎo)的ALI模型中,LPS同樣誘導(dǎo)了miR-146a的表達(dá)上調(diào)。因此,可以推測(cè),在ALI發(fā)生過(guò)程中,通過(guò)上調(diào)肺泡巨噬細(xì)胞內(nèi)miR-146a的表達(dá),可以阻斷肺泡巨噬細(xì)胞內(nèi)TLRs/NF-κB信號(hào)通路的活化,從而減少肺部炎癥因子的釋放,減輕肺部炎癥反應(yīng),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錛，

本文編號(hào)：1877082