甲磺司特对哮喘大鼠气道炎症及IL-5的影响

发布时间：2018-03-19 17:42

本文选题：甲磺司特　切入点：哮喘　出处：《南华大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的在大鼠哮喘模型上观察甲磺司特(Suplatast Tosilate,IPD)干预前后哮喘大鼠气道炎症、肺组织白细胞介素5(Interleukin-5,IL-5)基因表达以及肺泡灌洗液(bronchoalveolar lavage fluids,BALF)中IL-5的含量的变化,探讨甲磺司特在哮喘防治中的可能作用机制。方法 1.取雄性成年SD大鼠(4周龄,体重200±20g)50只,分为五组:对照(Control)组、模型(Model)组、布地奈德(budesonide,BUD)组、IPD早期干预组即IPD(A)组和IPD晚期干预组即IPD(B)组,各组10只。 2.在实验0天(即致敏前)尾静脉采血测定外周血嗜酸性粒细胞(eosinophil,,EOS)总数与百分比。除Control组以外的其余四组大鼠分别于第1天和第8天腹腔注射10㳠OVA混合液1ml(含OVA 100mg、氢氧化铝100mg及灭活百日咳杆菌苗5×109个作为免疫佐剂)致敏。2周后再进行激发,2㳠OVA泵雾化吸入,10分钟/次,每天1次,连续7天,构建大鼠哮喘模型,Control组致敏和激发均以生理盐水代替。BUD组每次激发前BUD泵雾化吸入,IPD(A)组和IPD(B)组分别从致敏和激发阶段开始予IPD(50mg/kg.d)灌胃。 3.末次激发24小时内麻醉动物,再次尾静脉采血测定外周血EOS总数与百分比,气管插管后行气道高反应性(airway hyper responsiveness,AHR)检测;检测后立即腹主动脉放血处死大鼠,取各组动物BALF及肺组织,分别测定BALF中炎性细胞总数和EOS百分比、通过逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR)检测肺组织IL-5mRNA的表达、酶联免疫吸附试验法(Enzyme linked immunosorbent assay, ELISA)测定BALF上清液中IL-5的含量,并分析IL-5与EOS、AHR之间的相关性,结合肺组织病理学检查分析气道炎症状态。结果 1.大鼠哮喘模型的建立:与Control组相比,在激发过程中,Model组大鼠逐渐出现烦躁不安、挠鼻、呼吸急促、点头状呼吸、发绀、活动进食减少或俯卧不动、出现竖毛、毛发失去光泽甚至脱毛、反应迟滞等哮喘样表现。激发7天后,Model组大鼠动物气道高反应性测定显示气道反应性增高,与Control组比较差异有统计学意义(P0.05);肺组织病理显示:气道周围明显炎症改变、支气管管壁增厚、管腔狭窄,气道上皮细胞脱落,粘液分泌明显增加。BALF细胞学显示EOS百分比明显增加(P0.05),提示哮喘大鼠模型复制成功。 2.气道高反应性测定:各组大鼠用盐酸组胺激发,在同一激发浓度,Model组、BUD组、IPD(A)组和IPD(B)组气道阻力明显高于Control组(P0.05) ,差异有统计学意义;与Model组相比,BUD组、IPD(A)组和IPD(B)组气道阻力明显降低(P0.05),差异有统计学意义。而IPD(A)组与BUD组之间相比差异无统计学意义(P0.05)。 3.病理组织学检查:Model组气道周围炎症改变明显、支气管管壁增厚、管腔狭窄,气道上皮细胞脱落,粘液分泌明显增加。与Model组比较,IPD(A)组、IPD (B)和BUD组气道周围炎症明显减轻,气道周围炎症细胞浸润、气道分泌物减少、支气管壁增厚及上皮细胞脱落现象明显改善。 4. BALF细胞学检测:IPD(A)组、IPD(B)组和BUD组炎性细胞总数及EOS百分比明显低于Model组(P0.05);IPD(A)组与BUD组之间炎性细胞总数及EOS百分比则差异无统计学意义(P0.05)。 5.外周血EOS总数与百分比:实验前(即致敏前):5组大鼠外周血EOS的总数与百分比无明显差异(P0.05)。实验后(即激发7天后):IPD(A)组、IPD(B)组和BUD组外周血EOS总数与百分比明显低于Model组(P0.05);IPD(A)组、IPD(B)组和BUD组外周血EOS总数与百分比三组之间差异无统计学意义(P0.05)。 6. BALF上清液中IL-5含量:致敏、激发后Model组BALF上清液中IL-5的含量较Control组明显升高(P0.05);IPD(A)组、IPD (B)组和BUD组IL-5含量明显低于Model组(P0.05);IPD(A)组和BUD组之间相比差异无统计学意义(P0.05)。 7.肺组织IL-5mRNA表达量:致敏、激发后Model组肺组织中IL-5mRNA的表达较Control组明显升高(P0.05);IPD(A)组、IPD (B组)和BUD组IL-5mRNA表达量明显低于Model组(P0.05), IPD(A)组和BUD组之间相比差异无统计学意义(P0.05)。 8. BALF中IL-5含量与气道阻力成正相关(r=0.919,P0.01);肺组织IL-5mRNA的表达量与气道阻力成正相关(r=0.909,P0.01);BALF中IL-5含量与EOS百分比成正相关(r=0.955,P0.01);BALF中EOS百分比与气道阻力成正相关(r=0.865, P0.01);BALF中IL-5含量与BALF中炎性细胞总数成正相关(r=0.955,P0.01);肺组织IL-5mRNA的表达量与BALF中IL-5的含量成正相关(r=0.973,P0.01)。结论 1. IPD能够降低哮喘大鼠气道高反应性,部分抑制气道炎症、抑制EOS的募集。 2. IPD能抑制肺组织IL-5的基因转录,降低BALF中IL-5的含量。 3. IPD早期干预作用与BUD有相似的抗炎效应。IPD晚期干预也有部分抗炎作用。. 4. IPD降低气道高反应性、抑制气道炎症可能是通过降低IL-5的合成实现的,并且在基因转录水平产生影响。
[Abstract]:objective
In the rat model of asthma were suplatasttosilate (Suplatast Tosilate, IPD) before and after the intervention of asthmatic rat airway inflammation, interleukin 5 in lung tissue (Interleukin-5, IL-5) gene expression and bronchoalveolar lavage fluid (bronchoalveolar lavage, fluids, BALF) changes in the content of IL-5 in the discussion of suplatasttosilate in asthma in the prevention and treatment mechanism.
Method
1. male adult SD rats (4 weeks old, weighing 200 + 20g) 50, were divided into five groups: control group (Control), model group (Model), budesonide (budesonide, BUD) group, IPD early intervention group, IPD (A) group and IPD late intervention group, namely, IPD (A) group, 10 in each group.
2. in the 0 day of the experiment (i.e. before sensitization) tail vein blood determination of peripheral blood eosinophils (eosinophil, EOS) and the total percentage. Except the Control group of the other four groups of rats respectively at first days and eighth days by intraperitoneal injection of 10? OVA mixture of 1ml (including OVA 100mg, 100mg aluminum hydroxide pertussis and inactivated vaccine 5 * 109) as adjuvant sensitization after.2 weeks of excitation, 2? OVA pump inhalation, 10 minutes / time, 1 times a day, for 7 consecutive days, to construct the rat asthma model. Control group was sensitized and challenged with saline instead of.BUD group each challenge BUD pump inhalation, IPD (A) group and IPD (B) group were sensitized and challenged from beginning to IPD (50mg/kg.d) by gavage.
At the end of the 3. shot within 24 hours of anesthesia animal, again the tailvein determination of peripheral blood EOS count and percentage of endotracheal intubation, airway hyperresponsiveness (airway hyper, responsiveness, AHR) detection; detection of abdominal aortic blood immediately after the rats were killed, the animal BALF and lung tissue of each group, and the total number of inflammatory cells in BALF the percentage of EOS were measured by reverse transcriptase polymerase chain reaction (Reverse Transcription-Polymerase Chain Reaction, RT-PCR) to detect the expression of IL-5mRNA in lung tissue, enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) for the determination of IL-5 BALF in the supernatant, and analysis of IL-5 and EOS, the correlation between AHR, combined with the analysis of the state of airway inflammation lung pathology examination.
Result
The establishment of 1. asthma model rats: compared with Control group, in the excitation process, the rats in Model group appeared irritability, scratching the nose, shortness of breath, nodded like breathing, cyanosis, eating activity reduced or lying motionless, hair, dull hair and even hair removal, reaction hysteresis and asthma stimulate performance. 7 days later, the rats in group Model animal airway hyperresponsiveness showed airway hyperresponsiveness, have increased significantly compared with the Control group (P0.05); display of lung tissue around the airway: obvious inflammation, bronchial wall thickening, stenosis, airway epithelial cell shedding, mucus secretion increased significantly.BALF cytology show the percentage of EOS increased significantly (P0.05), suggesting that the rat asthma model was established successfully.
Determination of 2. airway hyperresponsiveness in rats: histamine provocation with hydrochloric acid at the same concentration, excitation, Model group, BUD group, IPD group (A) and IPD (B) group airway resistance was significantly higher than Control group (P0.05), the difference was statistically significant; compared with Model group, BUD group, IPD (A) group and IPD (B) group significantly decreased airway resistance (P0.05), the difference was statistically significant. IPD (A) between the group and the BUD group was no significant difference (P0.05).
3. histopathological examination: Model group significantly inflammation around the airway, bronchial wall thickening, stenosis, airway epithelial cell shedding, mucus secretion increased significantly compared with Model group, IPD group (A), IPD (B) and BUD group significantly reduced inflammation around the airway, airway inflammatory cell infiltration around airway secretion, reduce. Bronchial wall thickening and shedding of epithelial cells decreased.
4. BALF cytological examination: the IPD (A) group, the total number of inflammatory cells and the percentage of EOS in IPD (B) group and BUD group were significantly lower than those in Model group (P0.05), and there was no significant difference in the total number of inflammatory cells and percentage of inflammatory cells between IPD (A) group and the group of P0.05.
The peripheral blood EOS 5. number and percentage: before the experiment (i.e. before sensitization): there is no significant difference between the total and the percentage of peripheral blood EOS 5 group rats (P0.05). After the experiment (i.e. excited 7 days): IPD (A) group, IPD (B) in peripheral blood of EOS group and BUD group the total number and the percentage was significantly lower than Model group (P0.05); IPD (A) group, IPD (B) had no statistical significance in peripheral blood in EOS group and BUD group the total number and percentage of differences between the three groups (P0.05).
6. IL-5 content in supernatant of BALF: after sensitization, the IL-5 content in BALF supernatant of Model group was significantly higher than that in Control group (P0.05), IPD (A) group, IPD (B) group and A group were significantly lower than those in group A.
The expression of IL-5mRNA 7. in lung tissue of sensitized, the expression of IL-5mRNA in lung tissue after excitation of Model group obviously increased compared with the Control group (P0.05); IPD (A) group, IPD (B group) and BUD group IL-5mRNA expression was significantly lower than group Model (P0.05), IPD (A) group and BUD group were compared between the difference was statistically significant (P0.05).
8. BALF and the contents of IL-5 in airway resistance was positively correlated (r=0.919, P0.01); the expression of IL-5mRNA in lung tissue and airway resistance was positively correlated (r=0.909, P0.01); BALF and IL-5 levels in the percentage of EOS positive correlation (r=0.955, P0.01); BALF% EOS and airway resistance was positively correlated (r=0.865 P0.01); the total number of inflammatory cells and the content of IL-5 BALF in BALF positive correlation (r=0.955, P0.01); the expression of IL-5 was positively related to the content of BALF and IL-5mRNA in lung tissue in (r=0.973, P0.01).
conclusion
1. IPD can reduce airway hyperresponsiveness in asthmatic rats, partially inhibit airway inflammation and inhibit the recruitment of EOS.
2. IPD can inhibit the gene transcription of IL-5 in lung tissue and reduce the content of IL-5 in BALF.
The early intervention of 3. IPD had similar anti-inflammatory effects with BUD, and the late intervention of.IPD also had some anti-inflammatory effects.
4. IPD reduces airway hyperresponsiveness and inhibits airway inflammation by reducing the synthesis of IL-5 and affects the transcriptional level of the gene.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R562.25

【引证文献】