纳米氧化锌致大鼠呼吸道炎症中血清外泌体差异miRNAs的筛选

发布时间：2018-05-13 19:50

本文选题：纳米氧化锌 + 呼吸道炎症　；参考：《郑州大学》2017年硕士论文

【摘要】：背景及目的纳米氧化锌(Zinc oxide nanoparticles,ZnO-NPs)是一种粒径在1~100 nm的金属氧化物,具有纳米材料的宏观量子隧道效应、体积效应、表面效应等特性,可用于水泥、轮胎、颜料、纺织品、食品药品、油漆、润滑剂等工业生产中。随着纳米氧化锌的广泛应用,职业人群的暴露几率也大大增加。研究表明,纳米氧化锌能够通过呼吸道进入人体,沉积于支气管及肺泡中,引起炎性介质及细胞因子释放,炎性细胞浸润,诱发呼吸道炎症,但具体机制尚不清楚。外泌体(exosomes)是由细胞分泌的膜性囊泡,含有丰富的蛋白质、核酸物质,参与细胞通讯、免疫调节、氧化应激、炎症及肿瘤的发生发展。研究显示,外泌体主要是通过将携带的miRNAs传递给受体细胞,调控受体细胞靶基因的表达,改变细胞因子及炎症因子的表达水平,促进炎症的发生发展。那么纳米氧化锌是否会改变外泌体中的miRNAs呢?目前尚未有相关研究,有待进一步的探究证实。为探究纳米氧化锌所致的呼吸道炎症中外泌体miRNAs的表达,本实验通过超速离心法和试剂盒法分离血清外泌体,利用高通量基因芯片检测技术,筛选差异miRNAs,以期找到与纳米氧化锌所致呼吸道炎症密切相关的miRNAs。方法1.纳米氧化锌致呼吸道炎症:将SD大鼠随机分为4组:低(2 mg/ml)、中(4 mg/ml)、高(8 mg/ml)剂量纳米氧化锌染毒组和生理盐水对照组,采用气管滴注法连续染毒3 d,每天染毒1次,染毒结束后24 h,麻醉大鼠后经腹主动脉采血,检测血液白细胞分类,ELISA法测定各组血清IL-8、IL-1β、TNF-α;收集肺泡灌洗液,检测BALF中总蛋白及乳酸脱氢酶(LDH)活性;通过肺组织HE染色观察病理改变。2.外泌体的提取与鉴定:采用超高速离心法和试剂盒法提取血清外泌体,通过透射电镜观察外泌体形态结构,Western blot方法对外泌体特征性蛋白CD63、Alix进行鉴定。3.外泌体差异miRNAs的生物学信息检测及分析:用外泌体RNA提取试剂盒提取外泌体总RNA,通过去磷酸化、样品变性、连接标记、样品纯化,使用Agilent芯片杂交,芯片洗涤、扫描后对结果进行标准化处理,筛选差异miRNAs。结果1.纳米氧化锌诱发大鼠呼吸道炎症:(1)染毒后大鼠的基本情况:染毒结束后,纳米氧化锌染毒组大鼠出现精神萎靡,摄食量减少;染毒前后,生理盐水组大鼠体重增加,而各染毒组体重均减轻。(2)肺泡灌洗液中总蛋白及乳酸脱氢酶活性:与生理盐水对照组相比,低、中、高剂量染毒组乳酸脱氢酶活性、总蛋白含量均升高,并呈现一定的剂量依赖效应,差异具有统计学意义(P0.05)。(3)纳米氧化锌对大鼠外周血细胞分类的影响:与生理盐水对照组相比,低、中、高剂量纳米氧化锌染毒组的外周血中白细胞总数、淋巴细胞数目、中性粒细胞及中性粒细胞百分比,差异具有统计学意义(P0.05)。(4)大鼠血清细胞因子IL-8、IL-1β、TNF-α的变化:低、中、高剂量纳米氧化锌染毒组大鼠血清中IL-8、IL-1β、TNF-α水平均高于生理盐水对照组(P0.05)。(5)肺组织病理学改变:生理盐水组肺泡结构清晰,少有结构破坏;低剂量组伴轻微炎细胞浸润和少量渗出物形成;中剂量组炎性细胞浸润和渗出物明显增多,肺实变面积增加;高剂量组肺泡壁充血水肿,肺组织完全实变。2.大鼠血清来源外泌体的提取与鉴定:超高速离心法和试剂盒法均能提取到血清外泌体,通过透射电镜观察到外泌体大小均匀,呈椭圆形或圆形,并表达外泌体特征性蛋白CD63和Alix。3.外泌体miRNAs芯片分析:miRNA芯片共筛选出23种差异表达的miRNAs,其中16种表达上调,7种表达下调,其中miR-134-5p、miR-290、miR-130a-3p、miR-19a-3p、miR-223-3p在炎症发生发展中发挥重要作用。结论1.纳米氧化锌诱发了大鼠呼吸道炎症,并改变其血清外泌体miRNA的表达。2.纳米氧化锌诱发的呼吸道炎症中,外泌体可能通过miR-223-3p、miR-134-3p、miR-130a-3p、miR-30b-5p调控炎症反应。
[Abstract]:Background and purpose Zinc oxide nanoparticles (ZnO-NPs) is a metal oxide with a particle size of 1~100 nm, with the macroscopic quantum tunneling effect, volume effect, surface effect and other properties of nanomaterials, which can be used in the industrial production of cement, tires, pigments, textiles, food drugs, paints, lubricants and so on. With nanoscale Zinc Oxide The study shows that nano Zinc Oxide can enter the human body through the respiratory tract, deposit in the bronchi and alveoli through the respiratory tract, cause the release of inflammatory mediators and cytokines, inflammatory cells infiltrating, and induced respiratory inflammation, but the body mechanism is not clear. The exocrine (exosomes) is secreted by the secretory cells. Membranous vesicles, rich in protein, nucleic acid substances, involved in cell communication, immunomodulation, oxidative stress, inflammation and tumor development. Studies show that exocrine mainly transfers the miRNAs to receptor cells, regulates the expression of receptor cell target genes, changes the expression level of cytokines and inflammatory factors, and promotes the expression of cytokines and inflammatory factors. The occurrence and development of inflammation. Will nano Zinc Oxide change the miRNAs in the exocrine? There is no relevant research yet to be further explored. To explore the expression of miRNAs in the respiratory tract of the respiratory tract caused by nanoscale Zinc Oxide, this experiment uses high pass centrifugation and kit method to separate the serum exosbodies and use high pass. Quantitative gene chip detection technique was used to screen differential miRNAs, in order to find the respiratory inflammation caused by miRNAs. method 1. nanoscale Zinc Oxide, which is closely related to respiratory inflammation caused by nano Zinc Oxide. The SD rats were randomly divided into 4 groups: low (2 mg/ml), medium (4 mg/ml), high (8 mg/ml) dose nanoscale Zinc Oxide dye group and saline control group, using trachea drop 3 D were continuously poisoned, 1 times a day and 24 h after the end of the poison. After the anesthesia rats were collected through the abdominal aorta, the blood leucocyte classification was detected. The serum IL-8, IL-1 beta, TNF- alpha were measured by ELISA method, and the pulmonary alveolar lavage fluid was collected to detect the activity of total protein and lactate dehydrogenase (LDH) in BALF, and the HE staining of lung tissue was used to observe the pathological changes of.2. exudate by HE staining in the lung tissue. Extraction and identification: ultra high speed centrifugation and kit method were used to extract serum exocrine. The morphological structure of exocrine was observed by transmission electron microscope. Western blot method was used to detect and analyze the biological information of.3. exocrine differential miRNAs, Alix was used to detect and analyze the biological information of.3. exocrine differential miRNAs. External secretory RNA extraction kit was used to extract the total RNA of external secretory. Through dephosphorylation, sample denaturation, connection markers, sample purification, Agilent chip hybridization, chip washing, and scanning, the results were standardized, and the results of differential miRNAs. were screened by 1. nano Zinc Oxide to induce respiratory inflammation in rats: (1) the basic situation of rats after poisoning: after the poisoning was finished, the spirit of nanoscale Zinc Oxide exposed rats appeared spirit The total protein and lactate dehydrogenase activity in the alveolar lavage solution: compared with the normal saline control group, the activity of lactate dehydrogenase and the total protein content in the low, middle and high dose groups were increased, and the dose dependent effect was found in the low, middle and high dose groups. The difference was statistically significant (P0.05). (3) the effect of nano Zinc Oxide on the classification of peripheral blood cells in rats: compared with the normal saline control group, the total number of white blood cells in the peripheral blood, the number of lymphocytes, the percentage of neutrophils and neutrophils in the low, middle and high dose group of nanoscale Zinc Oxide were statistically significant (4). Changes in serum cytokine IL-8, IL-1 beta, TNF- alpha in rat serum: low, medium, high dose nano Zinc Oxide infected rats serum IL-8, IL-1 beta, TNF- alpha levels were higher than the normal saline control group (P0.05). (5) pathological changes in lung tissue: the alveolar structure of the saline group was clear, less structural damage, low dose group with mild inflammatory cell infiltration and a small amount of exudation. The infiltration and exudation of inflammatory cells in the middle dose group increased significantly and the area of pulmonary consolidation increased; the alveolar wall congestion and edema in the high dose group, the extraction and identification of the serum sources of the.2. rats in the lung tissue completely changed: the ultra high speed centrifugation and the kit method could extract the serum exocrine, and the size of the exosecrete was observed through transmission electron microscope. Homogeneous, oval or circular, and expression of exosecrete characteristic protein CD63 and Alix.3. exosecrete miRNAs chip analysis: miRNA chip has screened 23 differentially expressed miRNAs, 16 of which are up regulated and 7 down regulated, of which miR-134-5p, miR-290, miR-130a-3p, miR-19a-3p, miR-223-3p play an important role in the development of inflammation. On 1. nanoscale Zinc Oxide induced respiratory inflammation in rats and changing the expression of its serum exosomatic miRNA expression of.2. nanoscale Zinc Oxide induced respiratory inflammation, exosbodies may regulate the inflammatory response through miR-223-3p, miR-134-3p, miR-130a-3p, and miR-30b-5p.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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