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目的研究支气管哮喘(简称哮喘)过敏性刺激诱发气道感觉神经敏化机制。方法成年雄性豚鼠39只,按随机数字表法分为生理盐水致敏/激发对照组(A组,9只)、卵白蛋白(OVA)致敏/生理盐水激发对照组(B组,9只)、OVA致敏/激发实验组(C组,21只)。A组以生理盐水(0.5ml/只)致敏,B、C组以10%OVA(0.5ml/只)致敏,第10天开始雾化吸入生理盐水(A、B组)或1%OVA(C组)进行激发,每天1次,每次30min,根据实验需要又将C组21只豚鼠分为激发1d组(C1组,6只)、连续激发3d组(C2组,6只)、连续激发5d组(C3组,9只)。利用免疫荧光双标技术结合激光共聚焦扫描显微观察与Westernblot技术,研究生长相关蛋白43(GAP43)在气道神经以及结状神经节、颈静脉神经节内分布与水平及与P物质(SP)和胶质源神经生长因子(GDNF)受体c RET表达神经元关系。结果免疫荧光结果显示,C3组豚鼠气道内GAP43免疫反应阳性神经呈网状分布于大、中支气管内,以黏膜下层为主,部分GAP43阳性神经纤维向黏膜层内延伸;在结状神经节和颈静脉神经节内有大量GAP43免疫阳性神经胞体,在结状神经节内主要与SP免疫阳性胞体共存,在颈静脉神经节内主要与c RET免疫阳性胞体共存。Westernblot结果显示,A、B、C1、C2、C3组GAP43蛋白表达水平吸光度(A)值分别为0.38±0.04、0.41±0.03、0.49±0.05、0.79±0.08、0.76±0.04。C1、C2、C3组分别与A、B组比较差异均有统计学意义(P均0.05);C2组GAP43蛋白表达与C1组比较差异有统计学意义(P<0.01),但与C3组GAP43蛋白表达比较差异无统计学意义(P>0.05)。结论哮喘过敏性刺激能诱发气道感觉神经———SP肽能神经、GDNF敏感性神经纤维与胞体表达GAP43蛋白。
Objective To study the sensitization mechanism of airway sensory nerves induced by allergic stimulation of bronchial asthma (asthma). Methods Thirty-nine adult male guinea pigs were randomly divided into normal saline sensitized / challenged control group (A group, n = 9), ovalbumin (OVA) sensitized / saline challenged control group , OVA sensitized / challenge experimental group (C group, 21). Group A was sensitized with saline (0.5ml / mouse), group B and C were sensitized with 10% OVA (0.5ml / mouse), and inhaled saline (group A and B) or OVA (Group C, group C) were stimulated once a day for 30 minutes. According to the experiment, 21 guinea pigs in group C were further divided into 1d group (group C1 and group 6) and 3 groups (group C2 and group 6) Continuous excitation 5d group (C3 group, 9). Immunofluorescence double labeling technique combined with laser confocal scanning microscopy and Western blotting was used to study the distribution and level of GAP43 in the airway nerves, nodose ganglia and jugular ganglia and the relationship between substance P (SP) ) And glial derived neural growth factor (GDNF) receptor c RET expression neurons. Results The results of immunofluorescence showed that the GAP43 immunoreactive nerve in the airway of C3 group was reticularly distributed in the large and middle bronchus, mainly in the submucosa. Some GAP43 positive nerve fibers extended into the mucosal layer. In the nodose ganglia and There are a large number of GAP43 immunopositive neurons in the jugular ganglion, coexisting mainly with SP immunopositive cells in the nodose ganglion, coexisting with c RET immunopositive cells in the jugular ganglia. Western blot results showed that the absorbance (A) values of GAP43 protein expression in A, B, C1, C2 and C3 groups were 0.38 ± 0.04, 0.41 ± 0.03, 0.49 ± 0.05, 0.79 ± 0.08 and 0.76 ± 0.04, respectively. There was significant difference between group C1, C2 and C3 and group A and B (all P <0.05). The expression of GAP43 in group C2 was significantly lower than that of group C1 (P <0.01) There was no significant difference in protein expression (P> 0.05). Conclusion Allergic stimulation of asthma can induce airway sensory nerves - SP peptide nerve, GDNF - sensitive nerve fibers and cell bodies to express GAP43 protein.