支气管哮喘α防御素的表达及P2Y2受体对其调节作用的研究
发布时间：2018-10-17 17:27
【摘要】：背景：中性粒细胞在支气管哮喘中受到广泛重视，其通过脱颗粒及释放炎性因子等参与气道炎症反应，但机制仍不完全清楚。α防御素是主要存在于中性粒细胞内的一类阳离子抗菌肽。研究报道，作用于中性粒细胞膜表面P2Y2受体促进中性粒细胞脱颗粒参与炎症反应。 目的：探讨α防御素在支气管哮喘中表达情况及P2Y2受体对其影响。 方法：实验共分两部分。 动物实验部分：72只健康雄性豚鼠随机分为对照组（C）、哮喘组（A）；卵蛋白致敏复制哮喘豚鼠模型，用生理盐水替代卵蛋白作为对照组；再将对照组和哮喘组分成：生理盐水组（C1/A1）、ATP干预组（C2/A2）、苏拉明干预组（C3/A3），分别腹腔注射生理盐水、ATP25mg/kg及苏拉明7.5mg/kg，20小时后获取标本，检测支气管肺泡灌洗液（BALF）细胞总数并对嗜酸性粒细胞及中性粒细胞计数；检测豚鼠血浆中白介素-8（IL-8）和α防御素（RNP）及肺组织中P2Y2和RNPmRNA表达水平。 细胞实验部分：选取支气管哮喘急性发作期患者和健康志愿者外周血分离中性粒细胞作为标本，，分别进行干预后培养（P2Y2受体诱导剂ATP和拮抗剂苏拉明）；检测细胞上清液中IL-8，HNP1-3以及中性粒细胞中P2Y2受体蛋白及mRNA表达水平。 结果： （一）动物实验部分结果 （1）A1组BALF中细胞总数、嗜酸性粒细胞及中性粒细胞数目显著多于C1组(P0.05)；A1组BALF中细胞总数及中性粒细胞数目与A3组比较亦增多，较A2组降低(P0.05)；C组组内比较，无统计学差异(P0.05）。 （2）A1组豚鼠血浆和肺组织中RNP蛋白及mRNA表达较C1组增高，与A2组比较降低，较A3组增高(P0.05）；C1组较C2组比较降低，与C3组比较增高（P0.05）。 （3）A1组豚鼠血浆中IL-8表达较C1组增高，与A2组比较降低，较A3组增加（P0.05）；C组组内比较，无统计学差异(P0.05）。 （4）P2Y2mRNA表达结果与其干预有关。C1组和A1组肺组织P2Y2mRNA表达较其各自ATP干预组降低，与苏拉明干预组比较增高（P0.05）；A1组肺组织P2Y2mRNA表达较C1组增高（P0.05）。 （5）豚鼠α防御素与RL、中性粒细胞及IL-8呈正相关(P0.05)。（二）细胞实验部分结果 （1）哮喘组患者分离培养中性粒细胞中HNP1-3蛋白及mRNA表达较对照组增高，与哮喘ATP干预组比较降低，较哮喘苏拉明干预组增高(P0.05）；对照组较其ATP干预组比较降低，与苏拉明干预组比较增高（P0.05）。 （2）哮喘组分离的中性粒细胞P2Y2mRNA及蛋白、IL-8表达较正常组增高（P0.05）。 （3）P2Y2mRNA及蛋白表达结果与其干预有关，正常组和哮喘组分离的中性粒细胞P2Y2mRNA及蛋白表达较其各自ATP干预组降低，与苏拉明干预组比较增高（P0.05）。 结论： 1. α防御素在支气管哮喘中表达增加。 2. P2Y2受体在支气管哮喘中表达上调诱导中性粒细胞脱颗粒促使α防御 素表达增加从而参与气道炎症反应。
[Abstract]:Background: neutrophilic granulocytes (neutrophils) are widely recognized in bronchial asthma. They are involved in airway inflammation through degranulation and release of inflammatory factors. However, the mechanism of 伪-defensin is still unclear. 伪-defensin is a kind of cationic antimicrobial peptide which mainly exists in neutrophils. It is reported that the P2Y2 receptor acting on the surface of neutrophil membrane promotes the degranulation of neutrophil to participate in inflammatory reaction. Objective: to investigate the expression of 伪-defensin in bronchial asthma and the effect of P2Y2 receptor. Methods: the experiment was divided into two parts. Animal experiment: 72 healthy male guinea pigs were randomly divided into control group (C), asthma group), (A); ovalbumin sensitized guinea pig model, and normal saline as control group. The control group and asthma group were divided into two groups: C1/A1), ATP intervention group (C2/A2) and sulamine intervention group (C3/A3). The samples were obtained after 20 hours of intraperitoneal injection of normal saline, ATP25mg/kg and sulamine 7.5 mg / kg. The total number of (BALF) cells in bronchoalveolar lavage fluid (BALF), eosinophil and neutrophil count, interleukin-8 (IL-8) and 伪 -defensin (RNP) in plasma and the expression of P2Y2 and RNPmRNA in lung tissue were detected. Cell experiment: neutrophils isolated from peripheral blood of patients with acute attack of bronchial asthma and healthy volunteers were cultured after intervention (P2Y2 receptor inducer ATP and antagonist sulamine). The expression of P2Y2 receptor protein and mRNA in supernatant and neutrophil were detected. Results: (1) the number of BALF cells, eosinophils and neutrophils in A1 group was significantly higher than that in C1 group (P0.05). The number of BALF cells and neutrophils in A1 group was higher than that in A3 group, and the expression of RNP protein and mRNA in plasma and lung tissue of A1 group was higher than that of C1 group (P0.05). (2). Compared with A2 group, it was lower than A3 group (P0.05), C1 group was lower than C2 group, higher than C3 group (P0.05). (3), A1 group was higher than C1 group, lower than A2 group, higher than A3 group (P0.05); C group). There was no significant difference (P0.05). (4) P2Y2mRNA expression results were related to its intervention. C1 group and A1 group lung tissue P2Y2mRNA expression was lower than their respective ATP intervention group, higher than Sulamine intervention group (P0.05); The expression of P2Y2mRNA in group A1 was higher than that in group C1 (P0.05). (5). There was a positive correlation between 伪 -defensin and RL, neutrophil and IL-8 (P0.05). (2) the results of cell experiment were as follows: (1) the expression of HNP1-3 protein and mRNA in cultured neutrophils in asthmatic group was higher than that in control group, which was lower than that in asthmatic ATP intervention group and higher than that in asthmatic sulamine intervention group (P0.05); The P2Y2mRNA and protein of neutrophils isolated from asthmatic group were higher than those of control group (P0.05). (2), and the expression of IL-8 was higher than that of normal group (P0.05). (3) the expression of P2Y2mRNA and protein was related to the intervention. The expression of P2Y2mRNA and protein of neutrophils isolated from normal group and asthma group was lower than that of their respective ATP intervention group, and was higher than that of sulamine intervention group (P0.05). Conclusion: 1. 伪-defensin expression increased in bronchial asthma. 2. 2. Upregulation of P2Y2 receptor in bronchial asthma induced neutrophil degranulation and increased 伪 -defensin expression, thus participating in airway inflammation.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R562.25
本文编号：2277415
[Abstract]:Background: neutrophilic granulocytes (neutrophils) are widely recognized in bronchial asthma. They are involved in airway inflammation through degranulation and release of inflammatory factors. However, the mechanism of 伪-defensin is still unclear. 伪-defensin is a kind of cationic antimicrobial peptide which mainly exists in neutrophils. It is reported that the P2Y2 receptor acting on the surface of neutrophil membrane promotes the degranulation of neutrophil to participate in inflammatory reaction. Objective: to investigate the expression of 伪-defensin in bronchial asthma and the effect of P2Y2 receptor. Methods: the experiment was divided into two parts. Animal experiment: 72 healthy male guinea pigs were randomly divided into control group (C), asthma group), (A); ovalbumin sensitized guinea pig model, and normal saline as control group. The control group and asthma group were divided into two groups: C1/A1), ATP intervention group (C2/A2) and sulamine intervention group (C3/A3). The samples were obtained after 20 hours of intraperitoneal injection of normal saline, ATP25mg/kg and sulamine 7.5 mg / kg. The total number of (BALF) cells in bronchoalveolar lavage fluid (BALF), eosinophil and neutrophil count, interleukin-8 (IL-8) and 伪 -defensin (RNP) in plasma and the expression of P2Y2 and RNPmRNA in lung tissue were detected. Cell experiment: neutrophils isolated from peripheral blood of patients with acute attack of bronchial asthma and healthy volunteers were cultured after intervention (P2Y2 receptor inducer ATP and antagonist sulamine). The expression of P2Y2 receptor protein and mRNA in supernatant and neutrophil were detected. Results: (1) the number of BALF cells, eosinophils and neutrophils in A1 group was significantly higher than that in C1 group (P0.05). The number of BALF cells and neutrophils in A1 group was higher than that in A3 group, and the expression of RNP protein and mRNA in plasma and lung tissue of A1 group was higher than that of C1 group (P0.05). (2). Compared with A2 group, it was lower than A3 group (P0.05), C1 group was lower than C2 group, higher than C3 group (P0.05). (3), A1 group was higher than C1 group, lower than A2 group, higher than A3 group (P0.05); C group). There was no significant difference (P0.05). (4) P2Y2mRNA expression results were related to its intervention. C1 group and A1 group lung tissue P2Y2mRNA expression was lower than their respective ATP intervention group, higher than Sulamine intervention group (P0.05); The expression of P2Y2mRNA in group A1 was higher than that in group C1 (P0.05). (5). There was a positive correlation between 伪 -defensin and RL, neutrophil and IL-8 (P0.05). (2) the results of cell experiment were as follows: (1) the expression of HNP1-3 protein and mRNA in cultured neutrophils in asthmatic group was higher than that in control group, which was lower than that in asthmatic ATP intervention group and higher than that in asthmatic sulamine intervention group (P0.05); The P2Y2mRNA and protein of neutrophils isolated from asthmatic group were higher than those of control group (P0.05). (2), and the expression of IL-8 was higher than that of normal group (P0.05). (3) the expression of P2Y2mRNA and protein was related to the intervention. The expression of P2Y2mRNA and protein of neutrophils isolated from normal group and asthma group was lower than that of their respective ATP intervention group, and was higher than that of sulamine intervention group (P0.05). Conclusion: 1. 伪-defensin expression increased in bronchial asthma. 2. 2. Upregulation of P2Y2 receptor in bronchial asthma induced neutrophil degranulation and increased 伪 -defensin expression, thus participating in airway inflammation.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R562.25
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