【摘要】：背景：中性粒細胞在支氣管哮喘中受到廣泛重視，其通過脫顆粒及釋放炎性因子等參與氣道炎癥反應(yīng)，但機制仍不完全清楚。α防御素是主要存在于中性粒細胞內(nèi)的一類陽離子抗菌肽。研究報道，作用于中性粒細胞膜表面P2Y2受體促進中性粒細胞脫顆粒參與炎癥反應(yīng)。 目的：探討α防御素在支氣管哮喘中表達情況及P2Y2受體對其影響。 方法：實驗共分兩部分。 動物實驗部分：72只健康雄性豚鼠隨機分為對照組（C）、哮喘組（A）；卵蛋白致敏復(fù)制哮喘豚鼠模型，用生理鹽水替代卵蛋白作為對照組；再將對照組和哮喘組分成：生理鹽水組（C1/A1）、ATP干預(yù)組（C2/A2）、蘇拉明干預(yù)組（C3/A3），分別腹腔注射生理鹽水、ATP25mg/kg及蘇拉明7.5mg/kg，20小時后獲取標(biāo)本，檢測支氣管肺泡灌洗液（BALF）細胞總數(shù)并對嗜酸性粒細胞及中性粒細胞計數(shù)；檢測豚鼠血漿中白介素-8（IL-8）和α防御素（RNP）及肺組織中P2Y2和RNPmRNA表達水平。 細胞實驗部分：選取支氣管哮喘急性發(fā)作期患者和健康志愿者外周血分離中性粒細胞作為標(biāo)本，，分別進行干預(yù)后培養(yǎng)（P2Y2受體誘導(dǎo)劑ATP和拮抗劑蘇拉明）；檢測細胞上清液中IL-8，HNP1-3以及中性粒細胞中P2Y2受體蛋白及mRNA表達水平。 結(jié)果： （一）動物實驗部分結(jié)果 （1）A1組BALF中細胞總數(shù)、嗜酸性粒細胞及中性粒細胞數(shù)目顯著多于C1組(P0.05)；A1組BALF中細胞總數(shù)及中性粒細胞數(shù)目與A3組比較亦增多，較A2組降低(P0.05)；C組組內(nèi)比較，無統(tǒng)計學(xué)差異(P0.05）。 （2）A1組豚鼠血漿和肺組織中RNP蛋白及mRNA表達較C1組增高，與A2組比較降低，較A3組增高(P0.05）；C1組較C2組比較降低，與C3組比較增高（P0.05）。 （3）A1組豚鼠血漿中IL-8表達較C1組增高，與A2組比較降低，較A3組增加（P0.05）；C組組內(nèi)比較，無統(tǒng)計學(xué)差異(P0.05）。 （4）P2Y2mRNA表達結(jié)果與其干預(yù)有關(guān)。C1組和A1組肺組織P2Y2mRNA表達較其各自ATP干預(yù)組降低，與蘇拉明干預(yù)組比較增高（P0.05）；A1組肺組織P2Y2mRNA表達較C1組增高（P0.05）。 （5）豚鼠α防御素與RL、中性粒細胞及IL-8呈正相關(guān)(P0.05)。（二）細胞實驗部分結(jié)果 （1）哮喘組患者分離培養(yǎng)中性粒細胞中HNP1-3蛋白及mRNA表達較對照組增高，與哮喘ATP干預(yù)組比較降低，較哮喘蘇拉明干預(yù)組增高(P0.05）；對照組較其ATP干預(yù)組比較降低，與蘇拉明干預(yù)組比較增高（P0.05）。 （2）哮喘組分離的中性粒細胞P2Y2mRNA及蛋白、IL-8表達較正常組增高（P0.05）。 （3）P2Y2mRNA及蛋白表達結(jié)果與其干預(yù)有關(guān)，正常組和哮喘組分離的中性粒細胞P2Y2mRNA及蛋白表達較其各自ATP干預(yù)組降低，與蘇拉明干預(yù)組比較增高（P0.05）。 結(jié)論： 1. α防御素在支氣管哮喘中表達增加。 2. P2Y2受體在支氣管哮喘中表達上調(diào)誘導(dǎo)中性粒細胞脫顆粒促使α防御 素表達增加從而參與氣道炎癥反應(yīng)。
[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 偽-defensin is still unclear. 偽-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 偽-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 偽 -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 偽 -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. 偽-defensin expression increased in bronchial asthma. 2. 2. Upregulation of P2Y2 receptor in bronchial asthma induced neutrophil degranulation and increased 偽 -defensin expression, thus participating in airway inflammation.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R562.25

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