PARP-1基因沉默对机械牵张诱导的人支气管上皮细胞炎性细胞因子表达的研究

发布时间：2018-05-11 04:32

本文选题：支气管哮喘 + 机械牵张　；参考：《山东大学》2014年博士论文

【摘要】：研究背景：最常见的气道炎症性疾病是支气管哮喘,其发生率在世界范围内逐年增加,严重危害公众健康,增加了社会经济负担。在发达国家,成人支气管哮喘的发生率将近10%,在儿童中更高。在发展中国家,发生率稍低,但是增长迅速。支气管哮喘是一种气道反应性疾病,炎症反应在其病理机制中起到非常重要的作用。目前关于哮喘病理机制的研究集中在变应原和刺激剂引发的炎性反应过程。除了生物化学刺激,物理的力学作用在调节肺的结构、功能和代谢方面亦发挥了重要的作用。支气管哮喘的病人,肺组织和正常呼吸时相比遭受了更多的牵张,并且牵张加重了支气管哮喘时的气道阻力,机械牵张可以诱发哮喘中的炎症反应,但是具体的机制仍不清楚。支气管上皮细胞是产花粉植物最初的切触点,并且目前成为许多研究感兴趣的靶点。支气管上皮细胞不仅是物理屏障,而且在气道炎症反应中有积极作用。支气管上皮细胞能合成炎症细胞因子,从而来影响局部组织免疫细胞的募集和活化,并且创造一个微环境使免疫细胞参与炎性反应过程。多聚腺苷二磷酸核糖聚合酶-1(PARP-1)富含在真核生物细胞核,占据多聚腺苷二磷酸核糖聚合酶(PARP)在细胞内将近90%的活性,它可以调节许多炎症细胞因子表达。在细胞核内,PARP-1能被DNA断裂、氧化应激、环境刺激等活化,并且参与许多反应,催化NAD+成烟酰胺和ADP核糖,在目标蛋白上形成ADP核糖聚合物,包括转录因子和自身。然而,PARP-1过度活化导致细胞内NAD+和ATP耗竭,导致细胞能量代谢危机和细胞死亡。PARP-1激活和许多疾病的发病机制有关,如心血管疾病、糖尿病、类风湿关节炎、内毒素休克和中风等。此外,PARP-1已被证实调控许多重要的炎症基因的表达,像VCAM-1,其是被核转录因子-κB(NF-κB)调节。PARP-1已被证实和支气管哮喘的发病机制密切相关。PARP-1拮抗剂阻止了变应原暴露后炎症细胞在肺组织的浸润。在变应原诱导的支气管哮喘模型,PARP-1缺失阻止了IL-5的表达,通过calpain依赖的通路分解STAT-6蛋白。但是支气管哮喘时机械牵张对PARP-1的影响仍不明确。 NF-κB是炎性反应放大环路的重要转录因子,其活化和移位入细胞核能够和许多基因启动子的固定序列结合而启动多种炎性介质的转录,在炎性疾病的发生发展过程中起到非常重要的作用。越来越多的证据表明NF-κB在支气管哮喘炎症介质的分泌过程中起到非常重要的作用。研究发现PARP-1的抑制剂3-AB能阻止H202引起的NF-κB活化和IL-8分泌。PARP-1可以通过转录因子调节炎症细胞因子基因的表达。研究目的：在本实验中,研究PARP-1在机械牵张诱导的人支气管上皮细胞炎症反应中的作用和机制。研究方法：在实验中,人支气管上皮细胞(HBEpiCs)通过机械牵张而被刺激,分别刺激细胞4、8、12、16、24小时,静止状态的细胞作为对照组。PARP-1的表达被siRNA干扰。通过实时定量RT-PCR、蛋白印迹、ELISA、小干扰RNA的转染以及免疫荧光等方法,分别检测牵张刺激组及阴性对照组中炎症因子相关基因、蛋白的表达量。氧化应激通过对超氧化物敏感的荧光染料-DHE来检测超氧化物的含量水平。DNA损伤通过彗星分析法(单细胞凝胶电泳)评估。研究结果： 1.IL-8和血管细胞粘附分子-1(VCAM-1)的表达受到机械牵张调节,并呈时间依赖性。给予牵张力刺激人支气管上皮细胞不同的时间点,0、4、8、12、16、24小时。牵张刺激呈时间依赖性调控IL-8和VCAM-1的表达。和静止状态的细胞比较,牵张刺激24小时会明显增加VCAM-1mRNA和蛋白表达量,ELISA法也表明IL-8分泌因牵张刺激而上调(P0.05)。 2.机械牵张可能通过上调PARP-1的表达和活性来诱导超氧化物产生和DNA损伤。在人支气管上皮细胞被牵张24小时后,我们检测了牵张对PARP-1表达和活性的影响。和静止状态细胞相比,牵张使PARP-1表达明显增加,而且PARP-1活性亦明显上调(P0.05)。静止状态的细胞几乎没有超氧化物产生。然而牵张刺激会导致明显的氧化应激(P0.05)。在静止状态的细胞,在尾部几乎没有DNA损伤,但是牵张刺激会明显增加细胞尾部DNA百分含量,这表明牵张刺激会导致人支气管上皮细胞DNA损伤。 3. PARP-1基因沉默减轻了机械牵张导致的IL-8和VCAM-1的表达上调,并减轻了单核细胞的粘附,这和阻止了核转录因子-κB (NF-κB)的移位有关。和静止状态细胞相比,牵张刺激明显增加了IL-8分泌和VCAM-1表达。然而,PARP-1基因沉默后,IL-8分泌和VCAM-1表达明显减少(P0.05),PARP-1siRNA阴性对照对炎症细胞因子表达没有明显影响。和静止状态的细胞相比,牵张刺激人支气管上皮细胞导致单核细胞(THP-1细胞)的粘附性明显增加,然而在牵张刺激前用PARP-1siRNA预处理导致单核细胞的粘附性明显下降。 NF-κB p65在牵张刺激前主要位于胞浆,但是在刺激后迅速发生位置变化,移位到细胞核内。PARP-1基因沉默后,虽给予牵张刺激p65仍位于细胞浆。研究结论： 1.机械牵张会诱发人支气管上皮细胞炎症反应、超氧化物产生和DNA损伤。 2. PARP-1基因沉默减轻了IL-8和VCAM-1的表达,部分是因为阻止了NF-κB的核移位。 3. PARP-1在机械牵张诱发的炎症反应中起到重要作用。 4. PARP-1基因沉默在逆转哮喘支气管上皮炎症反应中可能会是一个潜在的治疗途径。
[Abstract]:Background of Study :

The most common inflammatory diseases of the airways are bronchial asthma , the incidence of which increases year by year in the world , poses a serious threat to the health of the public and increases the socio - economic burden . In developed countries , the incidence of adult bronchial asthma is almost 10 per cent and is higher among children . In developing countries , the incidence is slightly lower , but growth is rapid .

Bronchial epithelial cells are an important role in regulating the structure , function and metabolism of lung . In addition to biochemical stimulation , physical and mechanical effects play an important role in regulating the structure , function and metabolism of lung .

Poly ( ADP - ribose ) polymerase - 1 ( parp - 1 ) is rich in eukaryotic cell nuclei , which occupies nearly 90 % of the activity of poly ( ADP - ribose polymerase ) in cells . It can regulate the expression of NAD + and ATP in cells . It is involved in many reactions , such as cardiovascular disease , diabetes , rheumatoid arthritis , endotoxic shock , apoplexy , etc .

NF - 魏B is an important transcription factor of inflammatory response amplification loop . Its activation and translocation into the nucleus can initiate transcription of various inflammatory mediators and play a very important role in the development of inflammatory diseases .

Purpose of study :

In this experiment , the role and mechanism of parp - 1 in the inflammatory response of human bronchial epithelial cells induced by mechanical stretch was studied .

Study method :

In the experiment , human bronchial epithelial cells ( HBEPCs ) were stimulated by mechanical tension , and cells 4 , 8 , 12 , 16 , 24 hours were stimulated respectively , and the cells in quiescent state were used as control groups . The expression of parp - 1 was disrupted by siRNA .

By means of real - time quantitative RT - PCR , Western blot , ELISA , transfection of small interfering RNA and immunofluorescence , the expression of inflammatory factor related genes and proteins in the control group and negative control group were detected respectively . Oxidative stress was assessed by comet assay ( single cell gel electrophoresis ) .

Results of the study :

1 . The expression of IL - 8 and VCAM - 1 was mechanically regulated and time - dependent .

The expression of VCAM - 1 mRNA and VCAM - 1 was significantly increased in 24 hours , and the level of VCAM - 1 mRNA and protein expression was significantly increased in 24 hours . ELISA method also showed that IL - 8 secretion was up - regulated by the stimulation ( P0.05 ) .

2 . Mechanical drafting may induce superoxide generation and DNA damage by up - regulation of the expression and activity of parp - 1 .

After 24 hours in human bronchial epithelial cells , we examined the influence of the stretch on the expression and activity of parp - 1 . Compared with the resting state cells , the stretch increased the expression of parp - 1 , and the activity of parp - 1 was also increased significantly ( P0.05 ) .

Cells in the quiescent state were almost free of superoxide generation , however , the stimulus could lead to significant oxidative stress ( P0.05 ) .

In the quiescent state cells , there was little DNA damage at the tail , but the stretch increase significantly increased the percentage of DNA in the tail of the cell , suggesting that the stimulus would lead to DNA damage to the human bronchial epithelial cells .

3 . The expression of IL - 8 and VCAM - 1 induced by mechanical tension was reduced by the silencing of the gene silencing by the gene silencing , and the adhesion of monocytes was reduced , which was related to the inhibition of the translocation of nuclear transcription factor - kappa B ( NF - 魏B ) .

However , IL - 8 secretion and VCAM - 1 expression were significantly decreased ( P0.05 ) .

As compared with the cells in the quiescent state , the stretch - stimulating human bronchial epithelial cells resulted in a significant increase in adhesion of monocytes ( THP - 1 cells ) , however pre - treatment with poly - 1 siRNA prior to stretching resulted in a significant decrease in adhesion of monocytes .

NF - 魏B was mainly located in the cytoplasm before the stimulation , but after the stimulation , the position changed rapidly and shifted to the nucleus . After the silencing of the gene , it was still in the cytoplasm , although it was given the stretch - stimulated P65 .

Conclusions of the study :

1 . Mechanical drawing can induce inflammatory reaction , superoxide generation and DNA damage in human bronchial epithelial cells .

2 . The expression of IL - 8 and VCAM - 1 was reduced in part by silencing of the gene silencing of NF - 魏B in part because of the inhibition of nuclear translocation of NF - 魏B .

3 . parp - 1 plays an important role in the inflammatory response induced by mechanical tension .

4 . The silencing of parp - 1 gene may be a potential therapeutic approach in the reversal of bronchial epithelial inflammatory response in asthma .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R562.25

【共引文献】