ATP水解酶对实验性矽肺小鼠的干预研究
发布时间：2018-09-05 16:26
【摘要】：目的:矽肺是由长期慢性吸入二氧化硅(silicon dioxide,Si O2)粉尘引起的职业性肺疾病。由于起病隐匿、持续进展且没有有效的治疗手段,矽肺已经成为威胁发展中国家、特别是我国相关从业人员健康的非常严重的公共卫生问题。目前普遍认为,肺部巨噬细胞可以吞噬Si O2颗粒,释放一系列的炎症介质,在矽肺的发生发展过程发挥着重要的作用。而巨噬细胞具有异质性,本课题组前期研究表明,干预巨噬细胞表型偏移可以减轻矽肺早期炎症及晚期的纤维化表现。而胞外三磷酸腺苷(extracellular adenosine triphosphate,e ATP)作为机体各个系统细胞之间信息交流的重要分子和内环境的重要组成部分,可以影响巨噬细胞的表型极化。使用三磷酸腺苷双磷酸酶(apyrase,Apy)降解呼吸道ATP能否减轻矽肺小鼠的炎症和纤维化尚未明确。因此,本研究做出假设:使用Apy局部耗竭e ATP可以通过调节单核巨噬细胞表型偏移,对矽肺的炎症和纤维化起到干预作用。方法:1 200只雄性C57BL/6J小鼠,随机分为矽肺造模组(silica组),生理盐水组(NS组),矽肺造模+溶剂对照组(Silica+NS组)及矽肺造模+Apy组(Silica+Apy),每组均为50只。2%异氟烷轻度麻醉小鼠后,silica组经口咽吸入Si O2悬浊液建立小鼠矽肺模型,NS组给予等体积生理盐水;Silica+Apy组在造模同时给予0.8μL 0.5mg/m L Apy,并在造模后4h重复给药一次,Silica+NS组给予等量的生理盐水。造模后3h、7d、14d、28d、56d按标准方法取材。灌洗组小鼠进行经气管肺泡灌洗,取肺泡灌洗液(Bronchoalveolar Lavage Fluid,BALF)进行灌洗液细胞计数、细胞分类计数及相关细胞因子的ELISA检测,用流式细胞术检测外周血单核细胞、肺泡巨噬细胞、肺间质巨噬细胞表型;对非灌洗组右上肺进行病理学检测,分别进行HE染色,苦味酸-天狼星红染色,α-SMA、NLRP3+F4/80免疫荧光染色,左肺提取总RNA,进行IL-1β、CCL2、NLRP3、COLⅠ、COLⅢ表达量检测,右下中肺进行ATP含量检测。2取5只雄性C57BL/6J小鼠,颌下静脉丛取外周静脉血,分选流式分选出外周血单核细胞;小鼠经2%异氟烷麻醉后进行经气管肺泡灌洗,将肺泡灌洗液细胞及外周血单核细胞进行甩片、固定及破膜后进行P2X_7R免疫荧光染色。结果:1肺组织ATP检测表明,矽肺造模后肺组织ATP水平明显升高,给予Apy治疗后可以显著减低肺组织ATP的含量,达到局部耗竭ATP的目的。2 7d肺组织HE染色及相应的炎症评分表明,与NS组相比,Silica组小鼠肺组织表现为大量炎症细胞浸润,肺泡间隔增宽、破坏及炎症评分增高。BALF细胞计数表明,Silica组炎症细胞的总数、巨噬细胞、中性粒细胞数均显著升高,BALF上清IL-1β、CCL2蛋白水平及肺组织IL-1β、CCL2、NLRP3基因水平的表达量均显著增高,肺组织NLRP3+F4/80免疫荧光染色表明,矽肺小鼠肺组织NLRP3阳性巨噬细胞明显增多。而Silica+Apy组与Silica+NS组相比,肺组织炎症表现、炎症细胞的浸润明显缓解,IL-1β、CCL2、NLRP3基因蛋白水平的表达均下降。3 28d肺组织苦味酸-天狼星红染色表明,Silica组小鼠肺组织在暗场可以观察到大量的粗大的红色胶原及细小的黄绿色胶原,基于苦味酸-天狼星红染色的半定量分析表明矽肺组胶原容积分数显著升高,肺组织α-SMA免疫荧光染色表明矽肺组α-SMA阳性细胞百分数显著增高,肺组织COLⅠ、COLⅢ的表达量增高;而Silica+Apy组与Silica+NS组相比,肺组织红色及黄绿色胶原区域、胶原容积分数、α-SMA阳性细胞百分数显著下降,COLⅠ、COLⅢ基因水平的表达量明显下调。4 7d、14d造模后,相对于NS组,Silica组小鼠外周血单核细胞Ly6Chi亚群比例显著提高,肺泡巨噬细胞、肺间质巨噬细胞M2表型比例增高,而Apy干预可以显著逆转矽肺造模导致的上述单核巨噬细胞表型的偏移。5用流式细胞术成功分选出外周血单核细胞,对分选出的细胞进行的P2X_7R免疫荧光染色表明,外周血单核细胞存在P2X_7R的表达,对肺泡灌洗液的免疫荧光染色表明F4/80阳性巨噬细胞均存在P2X_7R的表达。结论:Apy可以逆转矽肺小鼠外周血单核细胞、肺巨噬细胞表型偏移,减轻小鼠的早期炎症、晚期纤维化表现,该作用有可能是通过阻断ATP-P2X_7R-NLRP3通路实现的。
[Abstract]:Objective: Silicosis is an occupational lung disease caused by long-term chronic inhalation of silicon dioxide (Si O2) dust. Silicosis has become a very serious public health problem threatening the health of developing countries, especially in China, because of its concealed onset, continuous progress and lack of effective treatment. In order to release a series of inflammatory mediators, lung macrophages can phagocytose silica particles and play an important role in the development of silicosis. Macrophages are heterogeneous. Previous studies in this group showed that interfering with macrophage phenotypic migration can alleviate early inflammation and late fibrosis of silicosis. Extracellular adenosine triphosphate (e-ATP), as an important molecule of information exchange between cells in various systems of the body and an important part of the internal environment, can affect the phenotypic polarization of macrophages. Can the degradation of respiratory ATP by adenosine triphosphate (Apy) alleviate inflammation and fibrosis in silicosis mice Therefore, this study hypothesized that local depletion of e-ATP with Apy could interfere with inflammation and fibrosis of silicosis by regulating the phenotypic shift of monocytes and macrophages. Methods: 1 200 male C57BL/6J mice were randomly divided into silicosis model group (silica group), normal saline group (NS group), silicosis model + solvent control group (Silica group). Silica + Apy group and Silica + Apy group were all 50 mice under mild isoflurane anesthesia. Silica + NS group was given 0.8 mu L 0.5 mg/m L Apy and Silica + NS group was given 0.8 mu L 0.5 mg/m L Apy at the same time. Silica + NS group was given Silica + Apy 4 hours after modeling. The mice in the lavage group were given bronchoalveolar lavage and the bronchoalveolar lavage fluid (BALF) was taken for cell counting, cell classification and related cytokines detection by ELISA. Peripheral blood mononuclear cells and lung were detected by flow cytometry. Alveolar macrophages, pulmonary interstitial macrophages phenotype; non-lavage group right upper lung pathological examination, respectively, HE staining, picric acid-Sirius red staining, alpha-SMA, NLRP3+F4/80 immunofluorescence staining, left lung extract total RNA, IL-1 beta, CCL2, NLRP3, COL I, COL III expression detection, right lower and middle lung ATP content detection. In 7BL/6J mice, peripheral venous blood was collected from the submandibular venous plexus and the peripheral blood mononuclear cells were sorted out by flow cytometry. After anesthesia with 2% isoflurane, the mice were lavaged by trachea and alveolar lavage, then the alveolar lavage fluid cells and peripheral blood mononuclear cells were smeared, fixed and broken membranes were stained by P2X_7R immunofluorescence. After treatment with Apy, the ATP content in lung tissue was significantly decreased and the ATP content was locally depleted. HE staining and corresponding inflammation score showed that compared with NS group, the lung tissue of Silica group showed a large number of inflammatory cells infiltration, widened alveolar septum, increased damage and inflammation score. ALF cell count showed that the total number of inflammatory cells, macrophages and neutrophils were significantly increased in Silica group, and the levels of IL-1 beta, CCL2 protein in BALF supernatant and IL-1 beta, CCL2 and NLRP3 gene expression in lung tissue were significantly increased. NLRP3+F4/80 immunofluorescence staining showed that NLRP3 positive macrophages in lung tissue of silicosis mice were significantly increased. The expression of IL-1 beta, CCL2 and NLRP3 gene protein decreased in the Silica+Apy group compared with the Silica+NS group. Picric acid-Sirius red staining showed that a large amount of red collagen and small yellow could be observed in the lung tissue of the Silica group in the dark field. Semi-quantitative analysis of green collagen based on picric acid-Sirius red staining showed that the volume fraction of collagen in Silicosis group was significantly increased, the percentage of positive cells in lung tissue was significantly increased, and the expression of COL I and COL III in lung tissue was increased in silicosis group, while that in Silica+Apy group was red and yellow compared with Silica+NS group. The expression levels of COL I and COL III were significantly down-regulated. After modeling for 47 days and 14 days, the proportion of Ly6Chi subsets in peripheral blood mononuclear cells, alveolar macrophages and interstitial macrophages M2 phenotypes in Silica group were significantly higher than those in NS group, while that in Apy group was significantly lower. Pre-treatment could significantly reverse the phenotypic shift of monocytes and macrophages induced by silicosis. 5 Peripheral blood monocytes were successfully separated by flow cytometry. P2X_7R immunofluorescence staining showed that the expression of P2X_7R was present in peripheral blood monocytes and F4/80 was positive in alveolar lavage fluid. Conclusion: Apy can reverse the phenotypic shift of peripheral blood mononuclear cells and pulmonary macrophages in silicosis mice, alleviate early inflammation and late fibrosis in mice, which may be achieved by blocking ATP-P2X_7R-NLRP3 pathway.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R135.2
本文编号：2224817
[Abstract]:Objective: Silicosis is an occupational lung disease caused by long-term chronic inhalation of silicon dioxide (Si O2) dust. Silicosis has become a very serious public health problem threatening the health of developing countries, especially in China, because of its concealed onset, continuous progress and lack of effective treatment. In order to release a series of inflammatory mediators, lung macrophages can phagocytose silica particles and play an important role in the development of silicosis. Macrophages are heterogeneous. Previous studies in this group showed that interfering with macrophage phenotypic migration can alleviate early inflammation and late fibrosis of silicosis. Extracellular adenosine triphosphate (e-ATP), as an important molecule of information exchange between cells in various systems of the body and an important part of the internal environment, can affect the phenotypic polarization of macrophages. Can the degradation of respiratory ATP by adenosine triphosphate (Apy) alleviate inflammation and fibrosis in silicosis mice Therefore, this study hypothesized that local depletion of e-ATP with Apy could interfere with inflammation and fibrosis of silicosis by regulating the phenotypic shift of monocytes and macrophages. Methods: 1 200 male C57BL/6J mice were randomly divided into silicosis model group (silica group), normal saline group (NS group), silicosis model + solvent control group (Silica group). Silica + Apy group and Silica + Apy group were all 50 mice under mild isoflurane anesthesia. Silica + NS group was given 0.8 mu L 0.5 mg/m L Apy and Silica + NS group was given 0.8 mu L 0.5 mg/m L Apy at the same time. Silica + NS group was given Silica + Apy 4 hours after modeling. The mice in the lavage group were given bronchoalveolar lavage and the bronchoalveolar lavage fluid (BALF) was taken for cell counting, cell classification and related cytokines detection by ELISA. Peripheral blood mononuclear cells and lung were detected by flow cytometry. Alveolar macrophages, pulmonary interstitial macrophages phenotype; non-lavage group right upper lung pathological examination, respectively, HE staining, picric acid-Sirius red staining, alpha-SMA, NLRP3+F4/80 immunofluorescence staining, left lung extract total RNA, IL-1 beta, CCL2, NLRP3, COL I, COL III expression detection, right lower and middle lung ATP content detection. In 7BL/6J mice, peripheral venous blood was collected from the submandibular venous plexus and the peripheral blood mononuclear cells were sorted out by flow cytometry. After anesthesia with 2% isoflurane, the mice were lavaged by trachea and alveolar lavage, then the alveolar lavage fluid cells and peripheral blood mononuclear cells were smeared, fixed and broken membranes were stained by P2X_7R immunofluorescence. After treatment with Apy, the ATP content in lung tissue was significantly decreased and the ATP content was locally depleted. HE staining and corresponding inflammation score showed that compared with NS group, the lung tissue of Silica group showed a large number of inflammatory cells infiltration, widened alveolar septum, increased damage and inflammation score. ALF cell count showed that the total number of inflammatory cells, macrophages and neutrophils were significantly increased in Silica group, and the levels of IL-1 beta, CCL2 protein in BALF supernatant and IL-1 beta, CCL2 and NLRP3 gene expression in lung tissue were significantly increased. NLRP3+F4/80 immunofluorescence staining showed that NLRP3 positive macrophages in lung tissue of silicosis mice were significantly increased. The expression of IL-1 beta, CCL2 and NLRP3 gene protein decreased in the Silica+Apy group compared with the Silica+NS group. Picric acid-Sirius red staining showed that a large amount of red collagen and small yellow could be observed in the lung tissue of the Silica group in the dark field. Semi-quantitative analysis of green collagen based on picric acid-Sirius red staining showed that the volume fraction of collagen in Silicosis group was significantly increased, the percentage of positive cells in lung tissue was significantly increased, and the expression of COL I and COL III in lung tissue was increased in silicosis group, while that in Silica+Apy group was red and yellow compared with Silica+NS group. The expression levels of COL I and COL III were significantly down-regulated. After modeling for 47 days and 14 days, the proportion of Ly6Chi subsets in peripheral blood mononuclear cells, alveolar macrophages and interstitial macrophages M2 phenotypes in Silica group were significantly higher than those in NS group, while that in Apy group was significantly lower. Pre-treatment could significantly reverse the phenotypic shift of monocytes and macrophages induced by silicosis. 5 Peripheral blood monocytes were successfully separated by flow cytometry. P2X_7R immunofluorescence staining showed that the expression of P2X_7R was present in peripheral blood monocytes and F4/80 was positive in alveolar lavage fluid. Conclusion: Apy can reverse the phenotypic shift of peripheral blood mononuclear cells and pulmonary macrophages in silicosis mice, alleviate early inflammation and late fibrosis in mice, which may be achieved by blocking ATP-P2X_7R-NLRP3 pathway.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R135.2
【参考文献】
相关期刊论文 前1条
1 Silvia Speca;Ilaria Giusti;Florian Rieder;Giovanni Latella;;Cellular and molecular mechanisms of intestinal fibrosis[J];World Journal of Gastroenterology;2012年28期
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