右美托咪定调控miRNA-146a对COPD肺损伤的保护作用及机制

发布时间：2018-07-10 09:24

本文选题：右美托咪定 + COPD　；参考：《南方医科大学》2017年博士论文

【摘要】：研究背景及目的慢性阻塞性肺疾病(Chronic obstructive pulmonary disease,COPD)是一种以气道、肺组织为损伤部位,以持续性气流受限为特征的慢性支气管炎和(或)肺气肿,可进展为肺源性心脏病和呼吸衰竭的常见慢性肺部疾病,与气道和肺组织对有毒气体或有害颗粒产生异常炎性反应有关,具有较高发病率、致残率和致死率,造成严重社会经济负担。右美托咪定(Dexmedetomidine,Dex)是一种新型高选择性α2肾上腺素能受体激动剂,是临床常用麻醉辅助药物。越来越多临床研究表明,右美托咪定可通过抑制细胞凋亡、减轻炎症反应和细胞氧化应激反应等机制对大脑、心脏、肾脏、肝脏及肺脏等多种器官起保护作用,其中,尤以肺脏保护作用最为明显。miRNA-146a是近年来研究热点,其可靶定TLR4/依赖MyD88途径重要组成部分TNF受体相关因子6(TRAF6)和白细胞介素1受体相关激酶1(IRAK1),激活TLR4下游信号分子NF-κB,诱导大量炎症因子,如IL-6、IL-1β、TNF-α等表达释放,促进炎症反应级联放大,参与COPD炎症相关发病过程,可能是COPD治疗新靶点。由此,我们提出假设,右美托咪定可调控miRNA-146a对COPD肺损伤起到保护作用,其明确机制尚不清楚,是本课题研究重点,也是创新之处。本研究拟建立COPD大鼠模型,明确COPD大鼠肺损伤的病理基础;阐明右美托咪定调控miRNA-146a对COPD肺损伤的保护作用及机制。第一部分COPD大鼠动物模型的建立及肺功能检测与组织学检查目的建立COPD肺损伤大鼠动物模型,通过观察大鼠一般状况、肺功能检测、动脉血气分析、支气管肺泡灌洗液检测以及肺组织病理学检查,明确COPD肺损伤的病理基础。方法选取16只SD大鼠并随机分为2组,空白对照组(8只)和COPD组(8只),利用烟雾熏吸法建立大鼠COPD肺损伤模型,检测肺功能指标如潮气量(Tidal volume,TV)、最大呼气流量(Peak expiratory flow,PEF)、50%肺活量最大呼气流量(EF 50)、0.3秒用力呼气容积(FEV0.3)和FEV0.3与用力肺活量(FVC)比值(FEV0.3/FVC);分析大鼠动脉血氧分压(Pa02)和二氧化碳分压(PaC02);收取大鼠支气管肺泡灌洗液,并进行细胞计数、分类及蛋白质含量检测;取大鼠肺组织,行湿/干重比检测和组织病理学检查。结果1.一般状况:空白对照组大鼠实验过程中饮食、饮水正常,体重增长正常,毛发光滑,无明显呼吸道症状;COPD组大鼠逐渐出现厌食、体重减轻、毛发暗黄脱落、活动减少,并出现喷嚏、喘息以及呼吸频率增快等呼吸道症状。2.肺功能检测:空白对照组大鼠TV为2.65±0.21 mL,PEF为38.55±0.24 mL/s,EF50 为 1.81±0.06 mL/s,FEV0.3 为 4.44±0.26 mL,而 FEV0.3/FVC 为 88.45±0.34%;COPD 组大鼠 TV 为 1.26±0.17 mL,PEF 为 17.61±0.35 mL/s,EF50 为 1.20±0.14 mL/s,FEV0.3 为 2.52±0.28 mL,FEV0.3/FVC 为 63.39±0.22%。COPD 组大鼠TV、PEF、EF 50、FEV0.3和FEV0.3/FVC均显著低于空白对照组大鼠(0.05)。3.动脉血气分析:空白对照组大鼠动脉血Pa02为89.35±4.30 mmHg,而COPD组动脉血Pa02为73.12±5.11 mmHg,COPD组大鼠动脉血Pa02显著低于空白对照组大鼠动脉血Pa02(p0.05)。空白对照组大鼠动脉血PaCO2为43.22±5.19 mmHg,而 COPD 组动脉血 PaCO2 为 56.36±6.71 mmHg,COPD 组大鼠动脉血PaC02显著高于空白对照组大鼠动脉血PaC02(p0.05)。4.支气管肺泡灌洗液(BALF)细胞计数及蛋白浓度检测:相比于空白对照组,COPD组大鼠BALF白细胞总数显著增加(2.33±1.19×1 08/L vs.1.45±0.41 × 108/L,p0.05),且中性粒细胞比例显著增高(17.4±7.2%vs.8.6±3.4%,p0.05),单核巨噬细胞比例显著减低(73.3±2.6%vs.83.4±1.1%,p0.05),淋巴细胞比例无显著性差异(8.1±2.0%vs.7.8±2.7%,p0.05)。空白对照组大鼠BALF蛋白含量为193.19±33.21 mg/L,COPD组大鼠BALF蛋白含量为363.93±41.38 mg/L,COPD组大鼠BALF蛋白含量显著高于空白对照组大鼠BALF 蛋白含量(p0.05)。5.大鼠肺脏湿/干重比(W/D):空白对照组大鼠肺脏整体观呈粉红色,W/D为4.02±0.39,而COPD组大鼠肺脏整体观色泽苍白,肺表面可见大小不等肺大疱,W/D为5.41±1.03,COPD组大鼠肺脏W/D显著高于空白对照组大鼠肺脏W/D(p0.05)。6.肺组织病理学变化:空白对照组肺组织切片镜下可见支气管上皮纤毛丰富、排列整齐、未见脱落,支气管管壁规整、未见增厚及炎性细胞浸润,管腔内未见炎性渗出物,肺泡腔结构完整、未见明显病理性扩大。COPD组肺组织切片镜下可见支气管纤毛柱状上皮呈锯齿样增生增厚,纤毛脱落倒伏,粘膜下腺体增生肥大,炎性细胞广泛浸润,杯状细胞增生,支气管管腔内粘液蓄积,管壁结缔组织增生,平滑肌增厚,可见单核细胞和淋巴细胞浸润,肺泡大小不等、结构紊乱,肺泡壁变薄、断裂,肺泡腔扩大,部分融合成较大的囊腔。结论烟雾熏吸法可有效建立COPD肺损伤大鼠模型,可明确典型COPD肺损伤的病理基础。第二部分右美托咪定降低miRNA-146a表达抑制COPD大鼠肺泡上皮细胞凋亡目的研究右美托咪定对具有介导COPD大鼠肺泡上皮细胞凋亡作用的miRNA-146a表达的影响,利用流式细胞仪、实时定量PCR检测miRNA-146a以及凋亡相关因子p53和Bcl-2表达水平改变,以期明确右美托咪定对COPD肺损伤的保护机制。方法选取24只SD大鼠并随机分为3组:空白对照组(8只)、COPD未给药组(8只)和COPD右美托咪定给药组(8只)。分离、纯化、培养各组大鼠肺泡上皮细胞。空白对照组肺泡上皮细胞取自正常大鼠肺组织,COPD未给药组和COPD右美托咪定给药组肺泡上皮细胞取自COPD大鼠模型肺组织,COPD右美托咪定给药组加入5 μM右美托咪定培养3天,空白对照组和COPD未给药组将给予等量生理盐水培养3天,进行细胞凋亡检测和实时定量PCR检测,明确细胞凋亡情况和miRNA-146a以及凋亡相关因子p53和Bcl-2的表达水平。结果1.肺泡上皮细胞凋亡流式细胞仪检测结果显示:COPD未给药组和COPD右美托咪定给药组损伤细胞比例、坏死细胞比例和凋亡细胞比例均较正常对照组显著升高(p0.05),而正常存活细胞比例显著降低(p0.05);COPD右美托咪定给药组损伤细胞比例和凋亡细胞比例较COPD未给药组显著降低(p0.05),正常存活细胞比例则显著增高(p0.05)。COPD右美托咪定给药组肺泡上皮细胞凋亡率较COPD未给药组显著降低(11.15±0.51vs30.19±1.61%,p0.05)。2.右美托咪定抑制miRNA-146a基因表达情况实时定量PCR检测结果显示:COPD未给药组和COPD右美托咪定给药组miRNA-146a表达明显高于空白对照组(p0.05),但COPD右美托咪定给药组miRNA-146a表达明显低于COPD未给药组(p0.05)。3.右美托咪定抑制p53基因表达情况实时定量PCR检测结果显示:COPD未给药组和COPD右美托咪定给药组p53表达明显高于空白对照组(p0.05),但COPD右美托咪定给药组p53表达明显低于COPD未给药组(p0.05)。4.右美托咪定抑制Bcl-2基因表达情况实时定量PCR检测结果显示:COPD未给药组和COPD右美托咪定给药组Bcl-2表达明显高于空白对照组(p0.05),但COPD右美托咪定给药组Bcl-2表达明显低于COPD未给药组(p0.05)。结论miRNA-146a通过p53和Bcl-2介导肺泡上皮细胞凋亡,右美托咪定可降低miRNA-146a表达抑制COPD大鼠肺泡上皮细胞凋亡从而有效保护肺组织。
[Abstract]:Background and objective Chronic obstructive pulmonary disease (COPD) is a chronic bronchitis and / or emphysema characterized by airway and lung tissue injury, characterized by persistent airflow limitation, and can be developed as a common chronic pulmonary disease of pulmonary heart disease and respiratory failure, with airway and lung tissue. Dexmedetomidine (Dex) is a new type of high selective alpha 2 adrenergic receptor agonist, which is a kind of high selective alpha 2 adrenergic receptor agonist. More and more clinical studies have shown that Dexmedetomidine can protect the brain, heart, kidney, liver, lung and other organs by inhibiting apoptosis, alleviating inflammatory reaction and cell oxidative stress. Especially, the most obvious.MiRNA-146a of lung protection is the research heat point in recent years, and the target TLR4/ depends on the important component of the MyD88 pathway, T NF receptor related factor 6 (TRAF6) and interleukin 1 receptor related kinase 1 (IRAK1) activate NF- kappa B of the downstream signal molecules of TLR4, inducing a large number of inflammatory factors, such as IL-6, IL-1 beta, and TNF- alpha, to release the expression of IL-6, IL-1 beta, and TNF- alpha, to promote the cascade of inflammatory reactions and to participate in the pathogenesis related pathogenesis of COPD, which may be a new target for COPD treatment. Amidazine can regulate the protective effect of miRNA-146a on COPD lung injury. Its clear mechanism is not clear, it is the focus of this study, and it is also an innovation. This study is to establish a COPD rat model to clarify the pathological basis of lung injury in COPD rats, and to clarify the protective effect and mechanism of right metoimidin on COPD lung injury by miRNA-146a. The establishment of COPD rat model and lung function detection and histological examination were established to establish the rat model of COPD lung injury. The pathological basis of COPD lung injury was confirmed by observing the general condition of the rat, the lung function test, the arterial blood gas analysis, the bronchoalveolar lavage fluid and the pathological examination of the lung tissue. Methods 16 SD were selected. Rats were randomly divided into 2 groups, blank control group (8 rats) and COPD group (8 rats). COPD lung injury model was established by smoke fumigation. The lung function indexes such as tidal volume (Tidal volume, TV), maximum expiratory flow (Peak expiratory flow, PEF), 50% vital expiratory flow (EF 50), 0.3 second forced expiratory volume (FEV0.3) and FEV0.3 and exertion were measured. Lung activity (FVC) ratio (FEV0.3/FVC); analysis of rat arterial oxygen pressure (Pa02) and carbon dioxide partial pressure (PaC02); collection of rat bronchoalveolar lavage fluid, cell count, classification and protein content detection; rat lung tissue, wet / dry weight ratio test and histopathology examination. Results 1. general condition: blank control group rats Diet, normal drinking water, normal weight growth, smooth hair and no obvious respiratory symptoms; group COPD rats gradually appeared anorexia, weight loss, dark yellow and shedding of hair, reduced activity, and respiratory symptoms of respiratory symptoms, such as sneezing, wheezing and increasing respiratory rate, and.2. lung function test: the TV in the blank control group was 2.65 + 0.21 mL, PEF was 38.5. 5 + 0.24 mL/s, EF50 is 1.81 + 0.06 mL/s, FEV0.3 is 4.44 + 0.26 mL, and FEV0.3/FVC is 88.45 + 0.34%, TV in COPD group is 1.26 + 0.17 mL, PEF is 17.61 + 0.35 mL/s, EF50 is 1.20. The arterial blood gas analysis in the white control group (0.05).3. artery blood gas analysis: the arterial blood of the blank control group was 89.35 + 4.30 mmHg, and the arterial blood of the COPD group was 73.12 + 5.11 mmHg, and the arterial blood Pa02 of the COPD group was significantly lower than that of the blank control group (P0.05). The arterial blood of the blank control group was 43.22 + 5.19 mmHg, while the arterial blood of the control group was 43.22 + 5.19. PaCO2 was 56.36 + 6.71 mmHg, and the arterial blood PaC02 of group COPD rats was significantly higher than that of PaC02 (P0.05).4. bronchoalveolar lavage (BALF) cell count and protein concentration in the arterial blood of the blank control group. Compared with the blank control group, the total number of BALF leucocytes in the COPD group rats increased significantly (2.33 + 1.19 * 1 08/L vs.1.45 0.41 * 0.41), and The proportion of neutrophils increased significantly (17.4 + 7.2%vs.8.6 + 3.4%, P0.05), the proportion of mononuclear macrophages decreased significantly (73.3 + 2.6%vs.83.4 + 1.1%, P0.05), and there was no significant difference in the proportion of lymphocytes (8.1 + 2.0%vs.7.8 2.7%, P0.05). The content of BALF protein in the blank control group was 193.19 + 33.21 mg/L, and the BALF protein content of COPD group was 363.93 + 41. The content of BALF protein in.38 mg/L, group COPD rats was significantly higher than that of BALF protein content (P0.05) in the blank control group (P0.05), the lung wet / dry weight ratio (W/D) in.5. rats: the whole view of lung in the blank control group was pink, W/D was 4.02 + 0.39, while the lungs of the COPD group were pale with the whole lung, and the lung surface showed the size of different lung bullus, W/D 5.41 + 1.03. COPD The lung W/D in the rats of the group was significantly higher than that in the lungs of the blank control group. The lung tissue of W/D (P0.05).6. lung tissue was found in the blank control group. The lung tissue section of the blank control group showed that the bronchoepithelial cilium was rich and orderly. The bronchial tube wall was not shedding, the bronchial tube wall was regular, no thickening and inflammatory cell infiltration, no inflammatory exudation was found in the lumen, and the alveolar cavity was intact. There was no obvious pathological enlargement of.COPD group in the lung tissue section, which showed that the bronchial ciliated columnar epithelium was thickened with serrated hyperplasia, ciliated and lodged, the submucous gland hyperplasia and hypertrophy, extensive infiltration of inflammatory cells, goblet cell proliferation, mucus accumulation in the bronchus cavity, hyperplasia of connective tissue in the tube wall, thickening of smooth muscle, and monocyte and monocyte. Lymphocyte infiltration, alveolar size, structure disorder, alveolar wall thinning, fracture, alveolar cavity enlargement, partial fusion into larger capsule cavity. Conclusion smoke fumigation can effectively establish a rat model of COPD lung injury, and can clarify the pathological basis of typical COPD lung injury. Second right metoimidin reduces miRNA-146a expression and inhibits the alveoli of COPD rats Objective to investigate the effect of dexmedetomidin on the expression of miRNA-146a expression in the apoptosis of alveolar epithelial cells in COPD rats, using flow cytometry, real-time quantitative PCR detection of miRNA-146a and the changes of p53 and Bcl-2 expression levels of apoptosis related factors, in order to clarify the protective mechanism of right metoimidin on COPD lung injury. 24 SD rats were randomly divided into 3 groups: blank control group (8), COPD non administration group (8) and COPD right metomomidine administration group (8). Isolated, purified and cultured rat alveolar epithelial cells. Alveolar epithelial cells in blank control group were derived from normal rats' lung tissue, COPD non administration group and COPD right metomomidin group's alveolar epithelium fine The cells were taken from the COPD rat model lung tissue, and the COPD right metomomomidine administration group was added to 5 u M dexmeimidin for 3 days. The blank control group and the COPD non administration group were given the same amount of normal saline for 3 days. The apoptosis detection and real-time quantitative PCR detection were carried out to determine the cell apoptosis and the table of miRNA-146a and apoptosis related factors p53 and Bcl-2. Results 1. the results of the 1. alveolar epithelial cell apoptosis flow cytometry showed that the proportion of damaged cells, the proportion of necrotic cells and the proportion of apoptotic cells in the group of COPD and right metomomidin increased significantly (P0.05), while the proportion of normal living cells decreased significantly (P0.05); COPD right metoimidin administration group The ratio of damaged cells and apoptotic cells was significantly lower than that of COPD (P0.05), and the proportion of normal living cells increased significantly (P0.05), the apoptosis rate of alveolar epithelial cells in.COPD dexmeimidine group was significantly lower than that in COPD group (11.15 + 0.51vs30.19 + 1.61%, P0.05).2. right metomomidin inhibition of miRNA-146a gene expression The results of quantitative PCR detection showed that the expression of miRNA-146a in the COPD group and COPD dexmedetomidine administration group was significantly higher than that in the blank control group (P0.05), but the miRNA-146a expression in the COPD right metoimidin group was significantly lower than that of the COPD non administration group (P0.05) (P0.05).3. right metomomidin The expression of p53 in the administration group and the COPD dexmedetomidine administration group was significantly higher than that in the blank control group (P0.05), but the expression of p53 in the COPD dexmedetomidin group was significantly lower than that in the COPD group (P0.05). The real-time quantitative PCR detection results of the inhibition of the Bcl-2 gene expression in the right metomomidin group (P0.05) showed that COPD was not given to the drug group and the right metoimidine administration group was expressed. It was significantly higher than that in the blank control group (P0.05), but the expression of Bcl-2 in the COPD dexmedetomidine administration group was significantly lower than that in the COPD group (P0.05). Conclusion miRNA-146a mediated the apoptosis of alveolar epithelial cells through p53 and Bcl-2, and dexmedetomidine could reduce the miRNA-146a expression to inhibit the apoptosis of pulmonary alveolar cells in COPD rats and effectively protect the lung tissue.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R614

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