本文選題：支氣管炎 + 嗜酸粒細(xì)胞增多��； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：研究目的第一部分建立嗜酸粒細(xì)胞性支氣管炎小鼠模型。第二部分探討嗜酸粒細(xì)胞激活對氣道反應(yīng)性的影響。研究方法第一部分無特定病原體(SPF)級雌性BALB/c小鼠80只,分為多黏菌素B組、生理鹽水組、多黏菌素B+N-甲酰-蛋氨酸-亮氨酸-苯丙氨酸(fMLP)組、生理鹽水+fMLP組。各組小鼠給予對應(yīng)的點(diǎn)鼻液點(diǎn)鼻,共21 d。其中多黏菌素B組和生理鹽水組分別以12 μ1多黏菌素B(5 mg/ml,溶于雙蒸水中)和等量生理鹽水點(diǎn)鼻,1次/d;多黏菌素B+fMLP組、生理鹽水+fMLP組除以上對應(yīng)處理外,分別于第21天點(diǎn)鼻后3 h再予10 μ1 fMLP(0.05 mg/ml,溶于乙酸中)點(diǎn)鼻1次。每組隨機(jī)抽取10只小鼠測定氣道反應(yīng)性,以吸氣阻力、呼氣阻力和動態(tài)肺順應(yīng)性作為檢測指標(biāo);測定時間多黏菌素B組、生理鹽水組為末次點(diǎn)鼻后3 h,多黏菌素B+fMLP組、生理鹽水+fMLP組為末次點(diǎn)鼻后24 h。末次點(diǎn)鼻后24 h內(nèi)搜集各組余10只小鼠支氣管肺泡灌洗液(BALF)進(jìn)行細(xì)胞總數(shù)及分類計數(shù);肺組織行HE染色、變色酸2R染色觀察氣道周圍炎癥細(xì)胞浸潤程度。第二部分SPF級雌性BALB/c小鼠80只,實(shí)驗(yàn)動物分組及模型構(gòu)建同第一部分,每組20只。末次點(diǎn)鼻后24 h內(nèi)通過心臟采血的方式獲取小鼠血清,通過氣管插管下支氣管肺泡灌洗的方法獲取各組小鼠BALF,并制作小鼠肺組織勻漿上清液,采用酶聯(lián)免疫吸附法(ELISA)測定血清、BALF、肺組織勻漿中嗜酸粒細(xì)胞過氧化物酶(EPX)、嗜酸粒細(xì)胞陽離子蛋白(ECP)、嗜酸粒細(xì)胞趨化因子(ECF)水平。免疫組織化學(xué)染色檢測各組小鼠肺組織中EG2的表達(dá),結(jié)果以平均光密度(optical density,OD)值表示。透射電鏡下觀察低密度嗜酸粒細(xì)胞。研究結(jié)果第一部分多黏菌素B組BALF細(xì)胞總數(shù)顯著高于生理鹽水組[29.50(3.25)×104/ml比15.25(2.25)×104/ml,p0.001],以嗜酸粒細(xì)胞最明顯[11.76(6.02)×104/ml 比0.12(1.08)×104/ml,P0.001],而兩組肺泡巨噬細(xì)胞、淋巴細(xì)胞和中性粒細(xì)胞差異均無統(tǒng)計學(xué)意義。多黏菌素B+fMLP組細(xì)胞總數(shù)及嗜酸粒細(xì)胞計數(shù)顯著高于生理鹽水+fMLP 組[分別為 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組與多黏菌素B組細(xì)胞總數(shù)和分類計數(shù)差異均無統(tǒng)計學(xué)意義。多黏菌素B組和生理鹽水組小鼠靜注乙酰甲膽堿(Ach)的劑量-反應(yīng)曲線比較,吸氣阻力、呼氣阻力和肺順應(yīng)性差異均無統(tǒng)計學(xué)意義。生理鹽水+fMLP組與前兩組相似,未見對倍增濃度乙酰甲膽堿的吸氣阻力、呼氣阻力增高,肺順應(yīng)性降低。但是隨著注射Ach濃度的逐漸升高,多黏菌素B+fMLP組小鼠吸氣阻力、呼氣阻力均顯著高于其他3組,肺順應(yīng)性均顯著低于其他3組(均P0.001)。肺組織切片HE染色、變色酸2R染色示多黏菌素B組和多黏菌素B+fMLP組小鼠炎癥反應(yīng)顯著,大量炎癥細(xì)胞浸潤氣道周圍,浸潤的炎癥細(xì)胞以嗜酸粒細(xì)胞為主。而生理鹽水組和生理鹽水+fMLP組未見上述炎癥反應(yīng)。第二部分多黏菌素B組和生理鹽水組小鼠血清、BALF、肺組織中EPX、ECP、ECF水平差異均無統(tǒng)計學(xué)意義。生理鹽水+fMLP組血清、BALF、肺組織中EPX、ECP、ECF水平與前兩組相似,無統(tǒng)計學(xué)差異。而與其他3組相比,多黏菌素B+fMLP組血清、BALF、肺組織中EPX、ECP和ECF水平均顯著升高(均P0.001)。免疫組化染色結(jié)果示生理鹽水組、多黏菌素組、生理鹽水+fMLP組和多黏菌素B+fMLP 組小鼠 OD 值分別為(0.0608±0.0211)、(0.1298±0.0268)、(0.0618±0.0201)和(0.1876±0.0329)。多黏菌素B+fMLP組小鼠表達(dá)較多EG2蛋白,顯著高于其他3組(均P0.001);多黏菌素組小鼠可見EG2表達(dá),明顯高于生理鹽水組和生理鹽水+fMLP組(均P0.001);而生理鹽水+fMLP組和生理鹽水組小鼠均未見明顯EG2蛋白表達(dá),無顯著差異(P=1.000)。肺組織電鏡觀察結(jié)果:生理鹽水組、生理鹽水+fMLP組未見嗜酸粒細(xì)胞浸潤,多黏菌素B組、多黏菌素B+fMLP組見較多嗜酸粒細(xì)胞浸潤;其中多黏菌素B組嗜酸粒細(xì)胞胞膜完整,顆粒密度一致,中心鍵清晰,偶見脫顆粒后形成的空泡而成為低密度嗜酸粒細(xì)胞;多黏菌素B+fMLP組見較多激活的低密度嗜酸粒細(xì)胞,細(xì)胞胞膜崩解,顆粒中心鍵脫失、密度減低,部分顆粒脫離細(xì)胞,胞質(zhì)出現(xiàn)空泡。研究結(jié)論第一部分應(yīng)用多黏菌素B點(diǎn)鼻可建立嗜酸粒細(xì)胞性支氣管炎小鼠模型第二部分嗜酸粒細(xì)胞激活對氣道高反應(yīng)性的發(fā)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
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R562.21;R-332

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