吸入性糖皮質(zhì)激素對(duì)哮喘大鼠中TLR-7表達(dá)的影響
發(fā)布時(shí)間：2018-08-04 10:47
【摘要】：目的 通過(guò)吸入糖皮質(zhì)激素（布地奈德）干預(yù)哮喘大鼠，觀察氣道炎癥，測(cè)定氣道阻力、肺組織中TOLL樣受體7（TLR7）的mRNA轉(zhuǎn)錄及蛋白表達(dá)，觀察相關(guān)細(xì)胞因子IFN-γ、IL-12mRNA轉(zhuǎn)錄的變化情況，了解吸入性糖皮質(zhì)激素對(duì)肺組織中TLR7受體的影響及相關(guān)細(xì)胞因子的變化。 方法 1.40只清潔健康，，約1月齡，體重150-170克的雄性SD大鼠，隨機(jī)分配為以下四個(gè)組別：正常對(duì)照組（Control組）、哮喘模型組（Model組）、地塞米松干預(yù)治療組（Dexamethasone，DXM組），布地奈德混懸液干預(yù)治療組（Budesonide，BUD組），每組各10只。 2. Model組、DXM組、BUD組大鼠分別于實(shí)驗(yàn)第1天、第8天腹腔注射10㳠卵清蛋白(Ovalbumin，OVA)溶液1毫升，包含免疫佐劑氫氧化鋁100毫克，實(shí)驗(yàn)第15天，用2㳠卵清蛋白溶液4毫升對(duì)大鼠進(jìn)行霧化吸入激發(fā)，每天霧化1次，每次約為10分鐘，連續(xù)14天。DXM組大鼠在霧化吸入激發(fā)前半小時(shí)，給予腹腔注射地塞米松注射液，劑量為0.5毫克/千克, BUD組給予2毫升（1mg）布地奈德混懸液霧化吸入。在致敏階段與霧化激發(fā)階段，Control組采用0.9%氯化鈉代替卵清蛋白溶液進(jìn)行腹腔注射與霧化吸入。 3.實(shí)驗(yàn)第28天，四組大鼠分別予以水合氯醛腹腔注射麻醉后用肺功能檢測(cè)氣道阻力變化，檢測(cè)完成后用腹主動(dòng)脈放血方法處死大鼠，分別取各組左右兩側(cè)肺組織，其中左肺組織作實(shí)時(shí)熒光定量聚合酶連鎖反應(yīng)法（quantitative real-time PCR，qPCR）來(lái)檢測(cè)肺組織TLR7、IL-12和IFN-γ mRNA的轉(zhuǎn)錄水平，并通過(guò)Western Blot檢測(cè)方法檢測(cè)大鼠肺組織中TLR7的蛋白表達(dá)水平；右側(cè)肺組織通過(guò)病理HE染色方法對(duì)各組大鼠肺組織進(jìn)行形態(tài)學(xué)觀察，分析四組大鼠中氣道炎癥的變化情況。 結(jié)果 1．亞急性哮喘大鼠模型的建立：除Contol組外，其他三組大鼠隨著激發(fā)次數(shù)的增加，開(kāi)始出現(xiàn)煩躁不安、抓耳撓腮、食欲減退、脫毛、呼吸急促、反應(yīng)遲鈍等哮喘樣表現(xiàn)；實(shí)驗(yàn)第28天進(jìn)行氣道反應(yīng)性測(cè)定，顯示Model組大鼠氣道反應(yīng)性有增高，與Control組比較具有統(tǒng)計(jì)學(xué)意義（P＜0.05）；結(jié)合HE染色肺組織切片的形態(tài)學(xué)檢查顯示，Model組氣道周圍大量炎癥細(xì)胞增多，嗜酸性粒細(xì)胞增加，氣道平滑肌增生，管腔內(nèi)粘性分泌物明顯增多，提示大鼠哮喘模型構(gòu)建成功。 2．BUD組病理組織學(xué)與氣道阻力檢測(cè)變化：經(jīng)干預(yù)后的哮喘大鼠肺組織病理學(xué)顯示，與Model組比較，BUD組氣道炎性反應(yīng)減輕，與DXM相類似。大鼠氣道阻力測(cè)定，BUD組氣道阻力在不同濃度鹽酸組胺誘導(dǎo)下，其阻力值與Model組比較減低，差異具有統(tǒng)計(jì)學(xué)意義(P＜0.05)；與Control組比較增高，差異具有統(tǒng)計(jì)學(xué)意義(P＜0.05)；與DXM比較，差異無(wú)統(tǒng)計(jì)學(xué)意義。 3．BUD組肺組織TLR7mRNA與TLR7蛋白表達(dá)量變化：哮喘大鼠Model組TLR7mRNA與蛋白的表達(dá)量均較Control組明顯降低，差異具有統(tǒng)計(jì)學(xué)意義(P＜0.05)；BUD組與DXM組TLR7mRNA及蛋白表達(dá)量同Model組比較均明顯增高，差異具有統(tǒng)計(jì)學(xué)意義(P＜0.05)。BUD組與DXM組TLR7mRNA及蛋白表達(dá)量相比，差異無(wú)統(tǒng)計(jì)學(xué)意義。與Control組比較，明顯低于Control組，差異具有統(tǒng)計(jì)學(xué)意義(P＜0.05)。 4. BUD組肺組織IFN-γ和IL-12的mRNA表達(dá)量變化：Model組IL-12mRNA與IFN-γmRNA表達(dá)量較Control組比較均明顯降低，差異具有統(tǒng)計(jì)學(xué)意義(P＜0.05)； BUD組、DXM組表達(dá)量較Model組比較，IL-12和IFN-γ的mRNA的表達(dá)量均顯著增高，差異具有統(tǒng)計(jì)學(xué)意義(P＜0.05)；但仍低于Control組(P＜0.05)；BUD組與DXM組相比差異無(wú)統(tǒng)計(jì)學(xué)意義。 結(jié)論 1.卵清蛋白致敏與激發(fā)成功構(gòu)建亞急性哮喘大鼠模型。吸入性糖皮質(zhì)激素（布地奈德）能減輕氣道炎癥與降低氣道高反應(yīng)性。 2.布地奈德干預(yù)治療哮喘大鼠后能上調(diào)肺組織TLR7mRNA轉(zhuǎn)錄水平與TLR7蛋白的表達(dá)。 3.布地奈德干預(yù)治療哮喘大鼠后能夠上調(diào)IL-12及IFN-γ mRNA轉(zhuǎn)錄水平。
[Abstract]:objective
The effects of inhaled glucocorticoid (budesonide) on airway inflammation, airway resistance, mRNA transcription and protein expression of TOLL like receptor 7 (TLR7) in the lung tissue were observed and the changes in the related cytokine IFN- gamma and IL-12mRNA transcription were observed. The effects of inhaled glucocorticoids on the TLR7 receptor in lung tissue and related details were investigated. The change of cytokine.
Method
1.40 healthy, 1 month old, and 150-170 g male SD rats were randomly assigned to the following four groups: normal control group (group Control), asthma model group (group Model), dexamethasone intervention group (Dexamethasone, DXM group), budesonide suspension (Budesonide, BUD group), 10 rats in each group.
2. Model, DXM and BUD rats were given first days and 1 ml of 10? Ovalbumin (Ovalbumin, OVA) solution on eighth days, including 100 mg of aluminum hydroxide, an immune adjuvant, fifteenth days, and 2? Ovalbumin solution of 4 ml in rats and 1 times per day for 10 minutes each time for 14 days. Intraperitoneal injection of dexamethasone injection was given to the intraperitoneal injection of 0.5 mg / kg at the dose of 0.5 mg / kg before inhalation and inhalation. 2 ml of budesonide suspension was inhaled in group BUD. In the sensitization stage and atomization stage, 0.9% sodium chloride was used instead of ovalbumin solution for intraperitoneal injection and atomization inhalation.
3. on the twenty-eighth day of the experiment, the four groups of rats were injected with chloral hydrate intraperitoneally to detect the change of airway resistance. After the test was completed, the rats were killed by the abdominal aorta bleeding method, and the left and right lung tissues were taken respectively in each group, and the left lung tissue was made by the real-time fluorescent quantitative polymerase chain reaction (quantitative real-time PCR, qPCR). To detect the transcriptional level of TLR7, IL-12 and IFN- gamma mRNA in lung tissue, and to detect the protein expression level of TLR7 in the lung tissue of rats by Western Blot detection method. The lung tissue of the right lung tissue was observed by pathological HE staining in the right lung tissue, and the changes of airway inflammation in the four groups of rats were analyzed.
Result
The establishment of 1. subacute asthmatic rat models: in addition to the Contol group, the other three groups of rats began to appear agitated, anorexia, anorexia, hairing, shortness of breath, slow reaction and other asthmatic manifestations with the increase of the number of excuses. The airway responsiveness of the three rats in the twenty-eighth days of the experiment showed an increase in airway responsiveness in the group of rats, and the increase of airway responsiveness in the group of rats. The comparison of Control group was statistically significant (P < 0.05). The morphological examination of lung tissue sections stained with HE showed that there were a large number of inflammatory cells around the airway in the Model group, the eosinophil increased, the airway smooth muscle proliferation and the viscous secretions in the lumen increased obviously, suggesting the construction of the rat model of asthma was successful.
Histopathological and airway resistance detection changes in group 2.BUD: the lung histopathology of the asthmatic rats after the dry prognosis showed that the airway inflammatory response of the BUD group was less than that in the Model group. The airway resistance of the rats was similar to that of the DXM. The resistance value of the airway resistance in the BUD group was lower than that of the Model group under the induction of different concentrations of histamine in the group of BUD. There was statistical significance (P < 0.05); compared with Control group, the difference was statistically significant (P < 0.05); compared with DXM, the difference was not statistically significant.
The expression of TLR7mRNA and TLR7 protein in the lung tissue of 3.BUD group: the expression of TLR7mRNA and protein in the Model group of the asthmatic rats was significantly lower than that in the Control group, and the difference was statistically significant (P < 0.05); the TLR7mRNA and protein expressions of BUD and DXM groups were significantly higher than those in Model group, and the difference was statistically significant (P < 0.05) The expression of TLR7 mRNA and protein in M group was significantly lower than that in Control group (P < 0.05).
The expression of mRNA expression of IFN- gamma and IL-12 in the lung tissue of 4. BUD group: the expression of IL-12mRNA and IFN- y mRNA in Model group was significantly lower than that in the Control group, and the difference was statistically significant (P < 0.05). The expression of DXM group was significantly higher than that in the BUD group. But it was still lower than that in group Control (P < 0.05); there was no significant difference between BUD group and DXM group.
conclusion
1. ovalbumin sensitization and stimulation successfully constructed a subacute asthmatic rat model. Inhaled glucocorticoid (budesonide) can reduce airway inflammation and reduce airway hyperresponsiveness.
2. budesonide can increase TLR7mRNA transcription level and TLR7 protein expression in lung tissue after asthma treatment in rats.
3. budesonide can increase the transcription level of IL-12 and IFN- gamma mRNA after treatment in asthmatic rats.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R725.6
本文編號(hào)：2163708
[Abstract]:objective
The effects of inhaled glucocorticoid (budesonide) on airway inflammation, airway resistance, mRNA transcription and protein expression of TOLL like receptor 7 (TLR7) in the lung tissue were observed and the changes in the related cytokine IFN- gamma and IL-12mRNA transcription were observed. The effects of inhaled glucocorticoids on the TLR7 receptor in lung tissue and related details were investigated. The change of cytokine.
Method
1.40 healthy, 1 month old, and 150-170 g male SD rats were randomly assigned to the following four groups: normal control group (group Control), asthma model group (group Model), dexamethasone intervention group (Dexamethasone, DXM group), budesonide suspension (Budesonide, BUD group), 10 rats in each group.
2. Model, DXM and BUD rats were given first days and 1 ml of 10? Ovalbumin (Ovalbumin, OVA) solution on eighth days, including 100 mg of aluminum hydroxide, an immune adjuvant, fifteenth days, and 2? Ovalbumin solution of 4 ml in rats and 1 times per day for 10 minutes each time for 14 days. Intraperitoneal injection of dexamethasone injection was given to the intraperitoneal injection of 0.5 mg / kg at the dose of 0.5 mg / kg before inhalation and inhalation. 2 ml of budesonide suspension was inhaled in group BUD. In the sensitization stage and atomization stage, 0.9% sodium chloride was used instead of ovalbumin solution for intraperitoneal injection and atomization inhalation.
3. on the twenty-eighth day of the experiment, the four groups of rats were injected with chloral hydrate intraperitoneally to detect the change of airway resistance. After the test was completed, the rats were killed by the abdominal aorta bleeding method, and the left and right lung tissues were taken respectively in each group, and the left lung tissue was made by the real-time fluorescent quantitative polymerase chain reaction (quantitative real-time PCR, qPCR). To detect the transcriptional level of TLR7, IL-12 and IFN- gamma mRNA in lung tissue, and to detect the protein expression level of TLR7 in the lung tissue of rats by Western Blot detection method. The lung tissue of the right lung tissue was observed by pathological HE staining in the right lung tissue, and the changes of airway inflammation in the four groups of rats were analyzed.
Result
The establishment of 1. subacute asthmatic rat models: in addition to the Contol group, the other three groups of rats began to appear agitated, anorexia, anorexia, hairing, shortness of breath, slow reaction and other asthmatic manifestations with the increase of the number of excuses. The airway responsiveness of the three rats in the twenty-eighth days of the experiment showed an increase in airway responsiveness in the group of rats, and the increase of airway responsiveness in the group of rats. The comparison of Control group was statistically significant (P < 0.05). The morphological examination of lung tissue sections stained with HE showed that there were a large number of inflammatory cells around the airway in the Model group, the eosinophil increased, the airway smooth muscle proliferation and the viscous secretions in the lumen increased obviously, suggesting the construction of the rat model of asthma was successful.
Histopathological and airway resistance detection changes in group 2.BUD: the lung histopathology of the asthmatic rats after the dry prognosis showed that the airway inflammatory response of the BUD group was less than that in the Model group. The airway resistance of the rats was similar to that of the DXM. The resistance value of the airway resistance in the BUD group was lower than that of the Model group under the induction of different concentrations of histamine in the group of BUD. There was statistical significance (P < 0.05); compared with Control group, the difference was statistically significant (P < 0.05); compared with DXM, the difference was not statistically significant.
The expression of TLR7mRNA and TLR7 protein in the lung tissue of 3.BUD group: the expression of TLR7mRNA and protein in the Model group of the asthmatic rats was significantly lower than that in the Control group, and the difference was statistically significant (P < 0.05); the TLR7mRNA and protein expressions of BUD and DXM groups were significantly higher than those in Model group, and the difference was statistically significant (P < 0.05) The expression of TLR7 mRNA and protein in M group was significantly lower than that in Control group (P < 0.05).
The expression of mRNA expression of IFN- gamma and IL-12 in the lung tissue of 4. BUD group: the expression of IL-12mRNA and IFN- y mRNA in Model group was significantly lower than that in the Control group, and the difference was statistically significant (P < 0.05). The expression of DXM group was significantly higher than that in the BUD group. But it was still lower than that in group Control (P < 0.05); there was no significant difference between BUD group and DXM group.
conclusion
1. ovalbumin sensitization and stimulation successfully constructed a subacute asthmatic rat model. Inhaled glucocorticoid (budesonide) can reduce airway inflammation and reduce airway hyperresponsiveness.
2. budesonide can increase TLR7mRNA transcription level and TLR7 protein expression in lung tissue after asthma treatment in rats.
3. budesonide can increase the transcription level of IL-12 and IFN- gamma mRNA after treatment in asthmatic rats.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R725.6
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