吸入性糖皮质激素对哮喘大鼠中TLR-7表达的影响
发布时间：2018-08-04 10:47
【摘要】：目的 通过吸入糖皮质激素（布地奈德）干预哮喘大鼠，观察气道炎症，测定气道阻力、肺组织中TOLL样受体7（TLR7）的mRNA转录及蛋白表达，观察相关细胞因子IFN-γ、IL-12mRNA转录的变化情况，了解吸入性糖皮质激素对肺组织中TLR7受体的影响及相关细胞因子的变化。 方法 1.40只清洁健康，，约1月龄，体重150-170克的雄性SD大鼠，随机分配为以下四个组别：正常对照组（Control组）、哮喘模型组（Model组）、地塞米松干预治疗组（Dexamethasone，DXM组），布地奈德混悬液干预治疗组（Budesonide，BUD组），每组各10只。 2. Model组、DXM组、BUD组大鼠分别于实验第1天、第8天腹腔注射10㳠卵清蛋白(Ovalbumin，OVA)溶液1毫升，包含免疫佐剂氢氧化铝100毫克，实验第15天，用2㳠卵清蛋白溶液4毫升对大鼠进行雾化吸入激发，每天雾化1次，每次约为10分钟，连续14天。DXM组大鼠在雾化吸入激发前半小时，给予腹腔注射地塞米松注射液，剂量为0.5毫克/千克, BUD组给予2毫升（1mg）布地奈德混悬液雾化吸入。在致敏阶段与雾化激发阶段，Control组采用0.9%氯化钠代替卵清蛋白溶液进行腹腔注射与雾化吸入。 3.实验第28天，四组大鼠分别予以水合氯醛腹腔注射麻醉后用肺功能检测气道阻力变化，检测完成后用腹主动脉放血方法处死大鼠，分别取各组左右两侧肺组织，其中左肺组织作实时荧光定量聚合酶连锁反应法（quantitative real-time PCR，qPCR）来检测肺组织TLR7、IL-12和IFN-γ mRNA的转录水平，并通过Western Blot检测方法检测大鼠肺组织中TLR7的蛋白表达水平；右侧肺组织通过病理HE染色方法对各组大鼠肺组织进行形态学观察，分析四组大鼠中气道炎症的变化情况。 结果 1．亚急性哮喘大鼠模型的建立：除Contol组外，其他三组大鼠随着激发次数的增加，开始出现烦躁不安、抓耳挠腮、食欲减退、脱毛、呼吸急促、反应迟钝等哮喘样表现；实验第28天进行气道反应性测定，显示Model组大鼠气道反应性有增高，与Control组比较具有统计学意义（P＜0.05）；结合HE染色肺组织切片的形态学检查显示，Model组气道周围大量炎症细胞增多，嗜酸性粒细胞增加，气道平滑肌增生，管腔内粘性分泌物明显增多，提示大鼠哮喘模型构建成功。 2．BUD组病理组织学与气道阻力检测变化：经干预后的哮喘大鼠肺组织病理学显示，与Model组比较，BUD组气道炎性反应减轻，与DXM相类似。大鼠气道阻力测定，BUD组气道阻力在不同浓度盐酸组胺诱导下，其阻力值与Model组比较减低，差异具有统计学意义(P＜0.05)；与Control组比较增高，差异具有统计学意义(P＜0.05)；与DXM比较，差异无统计学意义。 3．BUD组肺组织TLR7mRNA与TLR7蛋白表达量变化：哮喘大鼠Model组TLR7mRNA与蛋白的表达量均较Control组明显降低，差异具有统计学意义(P＜0.05)；BUD组与DXM组TLR7mRNA及蛋白表达量同Model组比较均明显增高，差异具有统计学意义(P＜0.05)。BUD组与DXM组TLR7mRNA及蛋白表达量相比，差异无统计学意义。与Control组比较，明显低于Control组，差异具有统计学意义(P＜0.05)。 4. BUD组肺组织IFN-γ和IL-12的mRNA表达量变化：Model组IL-12mRNA与IFN-γmRNA表达量较Control组比较均明显降低，差异具有统计学意义(P＜0.05)； BUD组、DXM组表达量较Model组比较，IL-12和IFN-γ的mRNA的表达量均显著增高，差异具有统计学意义(P＜0.05)；但仍低于Control组(P＜0.05)；BUD组与DXM组相比差异无统计学意义。 结论 1.卵清蛋白致敏与激发成功构建亚急性哮喘大鼠模型。吸入性糖皮质激素（布地奈德）能减轻气道炎症与降低气道高反应性。 2.布地奈德干预治疗哮喘大鼠后能上调肺组织TLR7mRNA转录水平与TLR7蛋白的表达。 3.布地奈德干预治疗哮喘大鼠后能够上调IL-12及IFN-γ mRNA转录水平。
[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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R725.6
本文编号：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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R725.6
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