嗜酸粒细胞性支气管炎小鼠模型的建立及嗜酸粒细胞激活对气道反应性的影响

发布时间：2018-05-29 09:08

本文选题：支气管炎 + 嗜酸粒细胞增多　；参考：《北京协和医学院》2017年博士论文

【摘要】：研究目的第一部分建立嗜酸粒细胞性支气管炎小鼠模型。第二部分探讨嗜酸粒细胞激活对气道反应性的影响。研究方法第一部分无特定病原体(SPF)级雌性BALB/c小鼠80只,分为多黏菌素B组、生理盐水组、多黏菌素B+N-甲酰-蛋氨酸-亮氨酸-苯丙氨酸(fMLP)组、生理盐水+fMLP组。各组小鼠给予对应的点鼻液点鼻,共21 d。其中多黏菌素B组和生理盐水组分别以12 μ1多黏菌素B(5 mg/ml,溶于双蒸水中)和等量生理盐水点鼻,1次/d;多黏菌素B+fMLP组、生理盐水+fMLP组除以上对应处理外,分别于第21天点鼻后3 h再予10 μ1 fMLP(0.05 mg/ml,溶于乙酸中)点鼻1次。每组随机抽取10只小鼠测定气道反应性,以吸气阻力、呼气阻力和动态肺顺应性作为检测指标;测定时间多黏菌素B组、生理盐水组为末次点鼻后3 h,多黏菌素B+fMLP组、生理盐水+fMLP组为末次点鼻后24 h。末次点鼻后24 h内搜集各组余10只小鼠支气管肺泡灌洗液(BALF)进行细胞总数及分类计数;肺组织行HE染色、变色酸2R染色观察气道周围炎症细胞浸润程度。第二部分SPF级雌性BALB/c小鼠80只,实验动物分组及模型构建同第一部分,每组20只。末次点鼻后24 h内通过心脏采血的方式获取小鼠血清,通过气管插管下支气管肺泡灌洗的方法获取各组小鼠BALF,并制作小鼠肺组织匀浆上清液,采用酶联免疫吸附法(ELISA)测定血清、BALF、肺组织匀浆中嗜酸粒细胞过氧化物酶(EPX)、嗜酸粒细胞阳离子蛋白(ECP)、嗜酸粒细胞趋化因子(ECF)水平。免疫组织化学染色检测各组小鼠肺组织中EG2的表达,结果以平均光密度(optical density,OD)值表示。透射电镜下观察低密度嗜酸粒细胞。研究结果第一部分多黏菌素B组BALF细胞总数显著高于生理盐水组[29.50(3.25)×104/ml比15.25(2.25)×104/ml,p0.001],以嗜酸粒细胞最明显[11.76(6.02)×104/ml 比0.12(1.08)×104/ml,P0.001],而两组肺泡巨噬细胞、淋巴细胞和中性粒细胞差异均无统计学意义。多黏菌素B+fMLP组细胞总数及嗜酸粒细胞计数显著高于生理盐水+fMLP 组[分别为 27.00(3.50)×104/ml 比 14.00(2.50)×104/ml、10.17(6.15)×104/ml0.17(0.96)×104/ml,均P0.001]。而多黏菌素B+fMLP组与多黏菌素B组细胞总数和分类计数差异均无统计学意义。多黏菌素B组和生理盐水组小鼠静注乙酰甲胆碱(Ach)的剂量-反应曲线比较,吸气阻力、呼气阻力和肺顺应性差异均无统计学意义。生理盐水+fMLP组与前两组相似,未见对倍增浓度乙酰甲胆碱的吸气阻力、呼气阻力增高,肺顺应性降低。但是随着注射Ach浓度的逐渐升高,多黏菌素B+fMLP组小鼠吸气阻力、呼气阻力均显著高于其他3组,肺顺应性均显著低于其他3组(均P0.001)。肺组织切片HE染色、变色酸2R染色示多黏菌素B组和多黏菌素B+fMLP组小鼠炎症反应显著,大量炎症细胞浸润气道周围,浸润的炎症细胞以嗜酸粒细胞为主。而生理盐水组和生理盐水+fMLP组未见上述炎症反应。第二部分多黏菌素B组和生理盐水组小鼠血清、BALF、肺组织中EPX、ECP、ECF水平差异均无统计学意义。生理盐水+fMLP组血清、BALF、肺组织中EPX、ECP、ECF水平与前两组相似,无统计学差异。而与其他3组相比,多黏菌素B+fMLP组血清、BALF、肺组织中EPX、ECP和ECF水平均显著升高(均P0.001)。免疫组化染色结果示生理盐水组、多黏菌素组、生理盐水+fMLP组和多黏菌素B+fMLP 组小鼠 OD 值分别为(0.0608±0.0211)、(0.1298±0.0268)、(0.0618±0.0201)和(0.1876±0.0329)。多黏菌素B+fMLP组小鼠表达较多EG2蛋白,显著高于其他3组(均P0.001);多黏菌素组小鼠可见EG2表达,明显高于生理盐水组和生理盐水+fMLP组(均P0.001);而生理盐水+fMLP组和生理盐水组小鼠均未见明显EG2蛋白表达,无显著差异(P=1.000)。肺组织电镜观察结果:生理盐水组、生理盐水+fMLP组未见嗜酸粒细胞浸润,多黏菌素B组、多黏菌素B+fMLP组见较多嗜酸粒细胞浸润;其中多黏菌素B组嗜酸粒细胞胞膜完整,颗粒密度一致,中心键清晰,偶见脱颗粒后形成的空泡而成为低密度嗜酸粒细胞;多黏菌素B+fMLP组见较多激活的低密度嗜酸粒细胞,细胞胞膜崩解,颗粒中心键脱失、密度减低,部分颗粒脱离细胞,胞质出现空泡。研究结论第一部分应用多黏菌素B点鼻可建立嗜酸粒细胞性支气管炎小鼠模型第二部分嗜酸粒细胞激活对气道高反应性的发生有重要作用
[Abstract]:The first part of the study was to establish a mouse model of eosinophilic bronchitis. Second, the second part explored the effect of eosinophil activation on airway responsiveness. The first part of the study was 80 female BALB/c mice without specific pathogen (SPF), divided into the polymyxin B group, the physiological salt water group, the polymyxin B+N- methyl methionine - leucine - benzene Alanine (fMLP) group and normal saline +fMLP group. The mice in each group were given the corresponding point nasal nasal point, 21 D. in which the polymyxin B group and normal saline group were treated with 12 mu 1 polymyxin B (5 mg/ml, dissolved in double steam water) and equal amount of saline point nose, 1 /d, polymyxin B+ fMLP group, and normal saline +fMLP group except for the above corresponding treatment, respectively Twenty-first days after the nasal 3 h were given to 10 mu 1 fMLP (0.05 mg/ml, dissolved in acetic acid) point nose 1 times. 10 mice in each group were randomly selected to determine the airway responsiveness, with inhalation resistance, expiratory resistance and dynamic lung compliance as the test index; the time of the determination of the polymyxin B group, the saline group at the last point of the nose, the group of polymyxin B+fMLP, and the physiological saline +fML In group P, the total number and classification count of 10 mice bronchoalveolar lavage fluid (BALF) were collected within 24 h of each group after the end of nose at the last point of the end of the nose at the end of the 24 h.. The lung tissue was stained with HE, and the degree of infiltration of inflammatory cells around the airway was observed by the chromotropic acid 2R staining. 80 female BALB/c mice of SPF grade were found in second parts of the mice, and the group and model of experimental animals were constructed in the same way. In the first part, 20 rats in each group were obtained in 24 h after the end of the nose. The mouse serum was obtained through the blood collection by the heart. The BALF of the mice was obtained through the bronchoalveolar lavage under the tracheal intubation, and the lung tissue homogenate supernatant was produced. The serum, BALF, and the eosinophil peroxy in the lung homogenate were measured by ELISA. Chemical enzyme (EPX), eosinophil cationic protein (ECP) and eosinophil chemotactic factor (ECF) level. Immunohistochemical staining was used to detect the expression of EG2 in lung tissues of mice in each group. The results were expressed as optical density (OD). Under transmission electron microscopy, the low density eosinophil was observed under transmission electron microscope. The first part of the study was the BA of polymyxin B group BA The total number of LF cells was significantly higher than that in the saline group [29.50 (3.25) x 104/ml ratio 15.25 (2.25) x 104/ml, p0.001], the most obvious [11.76 (6.02) x 104/ml ratio of eosinophil to 0.12 (1.08) x 104/ml, P0.001], and two groups of alveolar macrophages, lymphocytes and neutrophils were not statistically significant. The total number of cells and eosinophilia in the group of polymyxin B+fMLP group and the eosinophil The granulocyte count was significantly higher than that of the normal saline +fMLP Group [27 (3.50) * 104/ml ratio 14 (2.50) x 104/ml, 10.17 (6.15) x 104/ml0.17 (0.96) x 104/ml, all P0.001]. and the difference of total cell count and classification count of polymyxin B+fMLP group and polymyxin B group and taxonomy count. There was no significant difference in the dose response curve of methacholine (Ach), and there was no significant difference in the inhalation resistance, expiratory resistance and lung compliance. The physiological saline +fMLP group was similar to the first two groups, and no inhalation resistance to the multiplier concentration of methacholine was found, the expiratory resistance increased, and the lung compliance decreased. But with the increase of Ach concentration, polymyxin B The inhalation resistance and expiratory resistance of the +fMLP group were significantly higher than those of the other 3 groups, and the lung compliance was significantly lower than the other 3 groups (all P0.001). The lung tissue sections were stained with HE, and the chromotropic acid 2R staining showed that the inflammatory reaction in the group of polymyxin B and the polymyxin B+fMLP group was significant. A large number of inflammatory cells infiltrated the airway, and the infiltrating inflammatory cells were eosinophils. There was no significant difference in the levels of serum, BALF, EPX, ECP, ECF in the second part of the group of polymyxin B and the saline group, and the levels of serum, BALF, and lung tissue, EPX, ECP and ECF were similar to those of the first two groups, but there was no statistical difference between the second parts of the normal saline group and the normal saline group. In the other 3 groups, the serum levels of polymyxin B+fMLP, BALF, EPX, ECP and ECF in the lung were significantly higher (P0.001). The immunohistochemical staining results showed that the normal saline group, the polymyxin group, the saline +fMLP group and the polymyxin B+fMLP group were (0.0608 + 0.0211), (0.1298 + 0.0268), (0.0618 + 0.0201) and (0.1876 + 0) (0.1876 + 0), respectively. 329). The expression of more EG2 protein in the mice of the polymyxin B+fMLP group was significantly higher than the other 3 groups (all P0.001). The expression of EG2 in the mice of the polymyxin group was significantly higher than that of the saline group and the +fMLP group of normal saline (all P0.001), but no significant EG2 protein expression was found in the normal saline +fMLP group and the normal saline group, and no significant difference (P=1.000). The results of electron microscope observation: no eosinophilic granulocyte infiltration, group B of polymyxin and group B+fMLP of polymyxin B+fMLP were found in the physiological saline group, and the group B+fMLP of polymyxin was more eosinophil infiltration, and the membrane of the eosinophils in group B of polymyxin was intact, the density of the particles was consistent, the central key was clear, and the vacuoles formed after the degranulation became low density eosinophils. Cells in the group of polymyxin B+fMLP saw more activated low density eosinophils, cell membrane disintegration, particle central key loss, density reduction, partial particles detached from cells and vacuoles in cytoplasm. Conclusion the first part of the study was to use polymyxin B point nose to establish eosinophilic bronchitis model in second parts of eosinophilic granulocytic excitation Activity plays an important role in the occurrence of hyperresponsiveness in the airway
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R562.21;R-332

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