ATP水解酶對實驗性矽肺小鼠的干預(yù)研究
發(fā)布時間：2018-09-05 16:26
【摘要】：目的:矽肺是由長期慢性吸入二氧化硅(silicon dioxide,Si O2)粉塵引起的職業(yè)性肺疾病。由于起病隱匿、持續(xù)進展且沒有有效的治療手段,矽肺已經(jīng)成為威脅發(fā)展中國家、特別是我國相關(guān)從業(yè)人員健康的非常嚴重的公共衛(wèi)生問題。目前普遍認為,肺部巨噬細胞可以吞噬Si O2顆粒,釋放一系列的炎癥介質(zhì),在矽肺的發(fā)生發(fā)展過程發(fā)揮著重要的作用。而巨噬細胞具有異質(zhì)性,本課題組前期研究表明,干預(yù)巨噬細胞表型偏移可以減輕矽肺早期炎癥及晚期的纖維化表現(xiàn)。而胞外三磷酸腺苷(extracellular adenosine triphosphate,e ATP)作為機體各個系統(tǒng)細胞之間信息交流的重要分子和內(nèi)環(huán)境的重要組成部分,可以影響巨噬細胞的表型極化。使用三磷酸腺苷雙磷酸酶(apyrase,Apy)降解呼吸道ATP能否減輕矽肺小鼠的炎癥和纖維化尚未明確。因此,本研究做出假設(shè):使用Apy局部耗竭e ATP可以通過調(diào)節(jié)單核巨噬細胞表型偏移,對矽肺的炎癥和纖維化起到干預(yù)作用。方法:1 200只雄性C57BL/6J小鼠,隨機分為矽肺造模組(silica組),生理鹽水組(NS組),矽肺造模+溶劑對照組(Silica+NS組)及矽肺造模+Apy組(Silica+Apy),每組均為50只。2%異氟烷輕度麻醉小鼠后,silica組經(jīng)口咽吸入Si O2懸濁液建立小鼠矽肺模型,NS組給予等體積生理鹽水;Silica+Apy組在造模同時給予0.8μL 0.5mg/m L Apy,并在造模后4h重復給藥一次,Silica+NS組給予等量的生理鹽水。造模后3h、7d、14d、28d、56d按標準方法取材。灌洗組小鼠進行經(jīng)氣管肺泡灌洗,取肺泡灌洗液(Bronchoalveolar Lavage Fluid,BALF)進行灌洗液細胞計數(shù)、細胞分類計數(shù)及相關(guān)細胞因子的ELISA檢測,用流式細胞術(shù)檢測外周血單核細胞、肺泡巨噬細胞、肺間質(zhì)巨噬細胞表型;對非灌洗組右上肺進行病理學檢測,分別進行HE染色,苦味酸-天狼星紅染色,α-SMA、NLRP3+F4/80免疫熒光染色,左肺提取總RNA,進行IL-1β、CCL2、NLRP3、COLⅠ、COLⅢ表達量檢測,右下中肺進行ATP含量檢測。2取5只雄性C57BL/6J小鼠,頜下靜脈叢取外周靜脈血,分選流式分選出外周血單核細胞;小鼠經(jīng)2%異氟烷麻醉后進行經(jīng)氣管肺泡灌洗,將肺泡灌洗液細胞及外周血單核細胞進行甩片、固定及破膜后進行P2X_7R免疫熒光染色。結(jié)果:1肺組織ATP檢測表明,矽肺造模后肺組織ATP水平明顯升高,給予Apy治療后可以顯著減低肺組織ATP的含量,達到局部耗竭ATP的目的。2 7d肺組織HE染色及相應(yīng)的炎癥評分表明,與NS組相比,Silica組小鼠肺組織表現(xiàn)為大量炎癥細胞浸潤,肺泡間隔增寬、破壞及炎癥評分增高。BALF細胞計數(shù)表明,Silica組炎癥細胞的總數(shù)、巨噬細胞、中性粒細胞數(shù)均顯著升高,BALF上清IL-1β、CCL2蛋白水平及肺組織IL-1β、CCL2、NLRP3基因水平的表達量均顯著增高,肺組織NLRP3+F4/80免疫熒光染色表明,矽肺小鼠肺組織NLRP3陽性巨噬細胞明顯增多。而Silica+Apy組與Silica+NS組相比,肺組織炎癥表現(xiàn)、炎癥細胞的浸潤明顯緩解,IL-1β、CCL2、NLRP3基因蛋白水平的表達均下降。3 28d肺組織苦味酸-天狼星紅染色表明,Silica組小鼠肺組織在暗場可以觀察到大量的粗大的紅色膠原及細小的黃綠色膠原,基于苦味酸-天狼星紅染色的半定量分析表明矽肺組膠原容積分數(shù)顯著升高,肺組織α-SMA免疫熒光染色表明矽肺組α-SMA陽性細胞百分數(shù)顯著增高,肺組織COLⅠ、COLⅢ的表達量增高;而Silica+Apy組與Silica+NS組相比,肺組織紅色及黃綠色膠原區(qū)域、膠原容積分數(shù)、α-SMA陽性細胞百分數(shù)顯著下降,COLⅠ、COLⅢ基因水平的表達量明顯下調(diào)。4 7d、14d造模后,相對于NS組,Silica組小鼠外周血單核細胞Ly6Chi亞群比例顯著提高,肺泡巨噬細胞、肺間質(zhì)巨噬細胞M2表型比例增高,而Apy干預(yù)可以顯著逆轉(zhuǎn)矽肺造模導致的上述單核巨噬細胞表型的偏移。5用流式細胞術(shù)成功分選出外周血單核細胞,對分選出的細胞進行的P2X_7R免疫熒光染色表明,外周血單核細胞存在P2X_7R的表達,對肺泡灌洗液的免疫熒光染色表明F4/80陽性巨噬細胞均存在P2X_7R的表達。結(jié)論:Apy可以逆轉(zhuǎn)矽肺小鼠外周血單核細胞、肺巨噬細胞表型偏移,減輕小鼠的早期炎癥、晚期纖維化表現(xiàn),該作用有可能是通過阻斷ATP-P2X_7R-NLRP3通路實現(xiàn)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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R135.2
本文編號：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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R135.2
【參考文獻】
相關(guān)期刊論文 前1條
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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