本文選題：甲磺司特　切入點(diǎn)：哮喘　出處：《南華大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的 在大鼠哮喘模型上觀察甲磺司特(Suplatast Tosilate,IPD)干預(yù)前后哮喘大鼠氣道炎癥、肺組織白細(xì)胞介素5(Interleukin-5,IL-5)基因表達(dá)以及肺泡灌洗液(bronchoalveolar lavage fluids,BALF)中IL-5的含量的變化,探討甲磺司特在哮喘防治中的可能作用機(jī)制。 方法 1.取雄性成年SD大鼠(4周齡,體重200±20g)50只,分為五組:對照(Control)組、模型(Model)組、布地奈德(budesonide,BUD)組、IPD早期干預(yù)組即IPD(A)組和IPD晚期干預(yù)組即IPD(B)組,各組10只。 2.在實(shí)驗(yàn)0天(即致敏前)尾靜脈采血測定外周血嗜酸性粒細(xì)胞(eosinophil,,EOS)總數(shù)與百分比。除Control組以外的其余四組大鼠分別于第1天和第8天腹腔注射10㳠OVA混合液1ml(含OVA 100mg、氫氧化鋁100mg及滅活百日咳桿菌苗5×109個作為免疫佐劑)致敏。2周后再進(jìn)行激發(fā),2㳠OVA泵霧化吸入,10分鐘/次,每天1次,連續(xù)7天,構(gòu)建大鼠哮喘模型,Control組致敏和激發(fā)均以生理鹽水代替。BUD組每次激發(fā)前BUD泵霧化吸入,IPD(A)組和IPD(B)組分別從致敏和激發(fā)階段開始予IPD(50mg/kg.d)灌胃。 3.末次激發(fā)24小時內(nèi)麻醉動物,再次尾靜脈采血測定外周血EOS總數(shù)與百分比,氣管插管后行氣道高反應(yīng)性(airway hyper responsiveness,AHR)檢測;檢測后立即腹主動脈放血處死大鼠,取各組動物BALF及肺組織,分別測定BALF中炎性細(xì)胞總數(shù)和EOS百分比、通過逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(Reverse Transcription-Polymerase Chain Reaction,RT-PCR)檢測肺組織IL-5mRNA的表達(dá)、酶聯(lián)免疫吸附試驗(yàn)法(Enzyme linked immunosorbent assay, ELISA)測定BALF上清液中IL-5的含量,并分析IL-5與EOS、AHR之間的相關(guān)性,結(jié)合肺組織病理學(xué)檢查分析氣道炎癥狀態(tài)。 結(jié)果 1.大鼠哮喘模型的建立:與Control組相比,在激發(fā)過程中,Model組大鼠逐漸出現(xiàn)煩躁不安、撓鼻、呼吸急促、點(diǎn)頭狀呼吸、發(fā)紺、活動進(jìn)食減少或俯臥不動、出現(xiàn)豎毛、毛發(fā)失去光澤甚至脫毛、反應(yīng)遲滯等哮喘樣表現(xiàn)。激發(fā)7天后,Model組大鼠動物氣道高反應(yīng)性測定顯示氣道反應(yīng)性增高,與Control組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05);肺組織病理顯示:氣道周圍明顯炎癥改變、支氣管管壁增厚、管腔狹窄,氣道上皮細(xì)胞脫落,粘液分泌明顯增加。BALF細(xì)胞學(xué)顯示EOS百分比明顯增加(P0.05),提示哮喘大鼠模型復(fù)制成功。 2.氣道高反應(yīng)性測定:各組大鼠用鹽酸組胺激發(fā),在同一激發(fā)濃度,Model組、BUD組、IPD(A)組和IPD(B)組氣道阻力明顯高于Control組(P0.05) ,差異有統(tǒng)計(jì)學(xué)意義;與Model組相比,BUD組、IPD(A)組和IPD(B)組氣道阻力明顯降低(P0.05),差異有統(tǒng)計(jì)學(xué)意義。而IPD(A)組與BUD組之間相比差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 3.病理組織學(xué)檢查:Model組氣道周圍炎癥改變明顯、支氣管管壁增厚、管腔狹窄,氣道上皮細(xì)胞脫落,粘液分泌明顯增加。與Model組比較,IPD(A)組、IPD (B)和BUD組氣道周圍炎癥明顯減輕,氣道周圍炎癥細(xì)胞浸潤、氣道分泌物減少、支氣管壁增厚及上皮細(xì)胞脫落現(xiàn)象明顯改善。 4. BALF細(xì)胞學(xué)檢測:IPD(A)組、IPD(B)組和BUD組炎性細(xì)胞總數(shù)及EOS百分比明顯低于Model組(P0.05);IPD(A)組與BUD組之間炎性細(xì)胞總數(shù)及EOS百分比則差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 5.外周血EOS總數(shù)與百分比:實(shí)驗(yàn)前(即致敏前):5組大鼠外周血EOS的總數(shù)與百分比無明顯差異(P0.05)。實(shí)驗(yàn)后(即激發(fā)7天后):IPD(A)組、IPD(B)組和BUD組外周血EOS總數(shù)與百分比明顯低于Model組(P0.05);IPD(A)組、IPD(B)組和BUD組外周血EOS總數(shù)與百分比三組之間差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 6. BALF上清液中IL-5含量:致敏、激發(fā)后Model組BALF上清液中IL-5的含量較Control組明顯升高(P0.05);IPD(A)組、IPD (B)組和BUD組IL-5含量明顯低于Model組(P0.05);IPD(A)組和BUD組之間相比差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 7.肺組織IL-5mRNA表達(dá)量:致敏、激發(fā)后Model組肺組織中IL-5mRNA的表達(dá)較Control組明顯升高(P0.05);IPD(A)組、IPD (B組)和BUD組IL-5mRNA表達(dá)量明顯低于Model組(P0.05), IPD(A)組和BUD組之間相比差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 8. BALF中IL-5含量與氣道阻力成正相關(guān)(r=0.919,P0.01);肺組織IL-5mRNA的表達(dá)量與氣道阻力成正相關(guān)(r=0.909,P0.01);BALF中IL-5含量與EOS百分比成正相關(guān)(r=0.955,P0.01);BALF中EOS百分比與氣道阻力成正相關(guān)(r=0.865, P0.01);BALF中IL-5含量與BALF中炎性細(xì)胞總數(shù)成正相關(guān)(r=0.955,P0.01);肺組織IL-5mRNA的表達(dá)量與BALF中IL-5的含量成正相關(guān)(r=0.973,P0.01)。 結(jié)論 1. IPD能夠降低哮喘大鼠氣道高反應(yīng)性,部分抑制氣道炎癥、抑制EOS的募集。 2. IPD能抑制肺組織IL-5的基因轉(zhuǎn)錄,降低BALF中IL-5的含量。 3. IPD早期干預(yù)作用與BUD有相似的抗炎效應(yīng)。IPD晚期干預(yù)也有部分抗炎作用。. 4. IPD降低氣道高反應(yīng)性、抑制氣道炎癥可能是通過降低IL-5的合成實(shí)現(xiàn)的,并且在基因轉(zhuǎn)錄水平產(chǎn)生影響。
[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.

【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】：2010
【分類號】：R562.25

【引證文獻(xiàn)】

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1 譚鈺嬪;甲磺司特改善哮喘大鼠氣道炎癥反應(yīng)及對IL-5/GATA-3影響的依賴機(jī)制[D];南華大學(xué);2011年

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本文編號：1635329