吸入糖皮质激素对哮喘大鼠肺组织YKL-40表达的影响

发布时间：2018-06-02 10:33

本文选题：吸入性糖皮质激素 + 哮喘　；参考：《南华大学》2014年硕士论文

【摘要】：目的通过对亚急性的哮喘大鼠模型进行的实验研究，了解吸入性糖皮质激素药物使用后，大鼠的气道炎症及气道阻力发生的变化，以及大鼠肺组织的甲壳质酶3样蛋白质1(Chitinase-3-like-1protein，YKL-40)、白细胞介素-8（Interleukin-8，IL-8）表达变化，了解吸入性糖皮质激素对哮喘大鼠肺组织中YKL-40mRNA及蛋白、IL-8mRNA表达水平的影响。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）、地塞米松组（DXM组），布地奈德组（BUD组），每一组分别划分10只SD大鼠。 2. Model组大鼠、DXM组大鼠、BUD组大鼠在实验进展的第1天、第8天给予腹腔注射10㳠的卵清蛋白(OVA)溶液1毫升，稀释有免疫佐剂氢氧化铝0.1克。在实验第15天，用2㳠的卵清蛋白溶液4毫升对大鼠进行雾化吸入以激发哮喘，每1次激发的时间为10分钟，1次/天，连续共两周。其中DXM组大鼠在先给予腹腔注射地塞米松注射液（剂量按0.5毫克/千克计算）后半小时再予以雾化吸入激发OVA，,而布地奈德组先给予布地奈德来泵雾化吸入(按1mg/2ml使用)。相对应正常对照组则采用生理盐水代替卵清蛋白溶液进行腹腔注射与雾化吸入以致敏与激发哮喘产生。 3.在实验第28天，四组大鼠先予以检测气道阻力值，在检测结束后,采用剪断大鼠腹主动脉的方法来处死动物，在获取左右两侧肺组织后，取左肺组织用实时荧光定量PCR方法（qPCR）来检测肺组织中YKL-40与IL-8mRNA的转录水平，并通过WesternBlot检测方法来检测大鼠肺组织中YKL-40的蛋白表达水平；取大鼠右侧肺组织通过HE染色病理检查来分析四组大鼠中气道炎症的变化情况。结果 1.成功建立亚急性哮喘大鼠模型：实验第14天开始予以2㳠卵清蛋白溶液雾化吸入后，随着次数增加逐渐出现以下哮喘发作症状：面部搔抓、呼吸频率加快、点头呼吸、面部紫绀，大小便失禁等，于实验第28天予检测其气道阻力变化，气道阻力结果提示哮喘模型组的气道阻力均有显著增加，与正常对照组比较有显著的差异（P＜0.05）；通过大鼠肺组织的HE染色病理切片进行观察：Model组肺组织中支气管与肺泡周围嗜酸性粒细胞增多，气道内有较多粘性分泌物，支气管管壁稍显增厚。 2.BUD组大鼠肺组织气道炎症的变化:与哮喘大鼠Model组比较，BUD组炎症细胞及粘性分泌物均明显减少。DXM组同BUD组对比，形态学差异不大。control组中未发现明显炎症反应。 3.BUD组大鼠气道阻力变化:经过盐酸组胺雾化吸入后，哮喘大鼠Model组的气道阻力与Control组比较显著增高，差异有统计学意义(P＜0.05)。布地奈德组大鼠气道阻力和哮喘模型大鼠相比较有显著降低，有显著的差异(P＜0.05)，，与正常对照组相比较气道阻力有明显增高，有显著的差异(P＜0.05)，同地塞米松组相比较，发现没有显著差异。 4. BUD大鼠肺组织中YKL-40与IL-8的mRNA变化：哮喘大鼠Model组的肺组织YKL-40mRNA与IL-8的mRNA转录水平与Control组比较显著增高(P＜0.05)。BUD组肺组织的YKL-40mRNA与IL-8的mRNA转录水平与Model组比较显著降低(P＜0.05)，仍显著高于正常对照组(P＜0.05)，同地塞米松组对比没有显著差异。 5. BUD组大鼠肺组织YKL-40蛋白表达量变化情况：哮喘模型组大鼠的肺组织YKL-40的蛋白表达量同正常对照组相比有显著的升高(P＜0.05)，布地奈德组大鼠的肺组织YKL-40的蛋白表达量同哮喘模型组相比较有显著的降低(P＜0.05)，但仍高于正常对照组，同地塞米松组比较没有显著的差异。结论 1.以卵清蛋白做为抗原可成功建立哮喘大鼠亚急性模型。哮喘模型组大鼠的肺组织中YKL-40的蛋白表达量同正常对照组相比有显著的升高，布地奈德组大鼠的肺组织YKL-40的蛋白表达量同哮喘模型组相比有显著的降低，但仍高于正常对照组，同地塞米松组比较没有显著的差异。 2.哮喘大鼠肺组织YKL-40mRNA及蛋白与IL-8mRNA表达水平升高，提示细胞因子YKL-40与IL-8参与了支气管哮喘发病过程。 3.雾化吸入布地奈德能够减轻气道炎症与气道阻力，有效降低大鼠局部肺组织中YKL-40mRNA与IL-8mRNA的转录，减少YKL-40蛋白表达。
[Abstract]:objective
The changes in airway inflammation and airway resistance in rats after inhalation of inhaled glucocorticoids, as well as the changes in the expression of the chitinase 3 like protein 1 (Chitinase-3-like-1protein, YKL-40), and the expression of interleukin -8 (Interleukin-8, IL-8) in rat lung tissue were investigated by an experimental study of the subacute asthmatic rat model. Effects of inhaled corticosteroids on expression of YKL-40mRNA and protein and IL-8mRNA in lung tissue of asthmatic rats.
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 group (group DXM), and budesonide group (group BUD), each group was divided into 10 SD rats.
2. Model rats, group DXM rats, group BUD rats on the first day of the experiment, 1 ml of 10? Ovalbumin solution (1 ml) and 0.1 grams of aluminum hydroxide with an immune adjuvant. On the fifteenth day, 2? Ovalbumin solution was used to stimulate the rats to stimulate asthma, and the time of every 1 excitation was 10 minutes. 1 times per day, for a total of two weeks, group DXM rats were first given intraperitoneal injection of dexamethasone injection (dosage according to 0.5 mg / kg) and then atomized and inhaled to stimulate OVA, while budesonide group was given budesonide first to pump inhalation (according to 1mg/2ml). The protein solution was injected intraperitoneally and nebulized so as to sensitize and stimulate asthma.
3. on the twenty-eighth day of the experiment, the four groups of rats first detected the airway resistance. After the end of the test, the rat abdominal aorta was cut to kill the animals. After obtaining the left and right bilateral lung tissues, the left lung tissue was taken to detect the transcriptional level of YKL-40 and IL-8mRNA in the lung tissue by real time fluorescence quantitative PCR method (qPCR), and through WesternBlot examination. The protein expression level of YKL-40 in the lung tissue of rats was detected and the changes of airway inflammation in the four groups of rats were analyzed by the pathological examination of the right lung tissue by HE staining.
Result
1. a rat model of subacute asthma was successfully established: after fourteenth days of experiment, 2 of the ovalbumin solution was inhaled. With the increase of the number of times, the symptoms of asthma attack gradually appeared following the increasing number of times: the facial scratch, the speed of breathing, the head breathing, the cyanosis of the face, the incontinence, and so on. The airway resistance changes and the airway obstruction were detected on the twenty-eighth day of the experiment. The force results showed that the airway resistance of the asthma model group increased significantly, and there was a significant difference compared with the normal control group (P < 0.05). The HE staining pathological sections of the lung tissue of rats were observed: the increased eosinophils around the bronchi and alveoli, more viscous secretions in the airway and the bronchial tube wall in the lung tissue of the Model group were observed. It's thicker.
The changes of airway inflammation in the lung tissue of group 2.BUD rats: compared with the Model group of the asthmatic rats, the inflammatory cells and the sticky secretions in the group BUD significantly decreased the comparison between the group.DXM and the BUD group, and there was no obvious inflammatory reaction in the group of.Control.
The airway resistance changes in the 3.BUD group: after inhalation of histamine hydrochloride, the airway resistance of the Model group was significantly higher in the group of asthma rats than that in the Control group (P < 0.05). The airway resistance of the budesonide group was significantly lower than that of the asthma model rats. There were significant differences (P < 0.05), with the normal control group. Compared with the dexamethasone group, there was no significant difference in airway resistance between the two groups (P < 0.05).
The mRNA changes of YKL-40 and IL-8 in the lung tissue of 4. BUD rats: the mRNA transcriptional level of YKL-40mRNA and IL-8 in the lung tissue of the group Model of the asthmatic rats was significantly higher than that in the Control group (P < 0.05) the transcriptional level of the YKL-40mRNA in the lung tissue of the.BUD group was significantly lower than that in the control group (0.05), still significantly higher than that in the normal control group (< 0.05). There was no significant difference in the comparison with the dexamethasone group.
The changes in the expression of YKL-40 protein in the lung tissue of 5. BUD rats: the protein expression of YKL-40 in the lung tissue of the asthmatic model group was significantly higher than that in the normal control group (P < 0.05). The protein expression of YKL-40 in the lung tissue of the budesonide group was significantly lower than that in the asthma model group (P < 0.05), but still higher than that in the model group (P < 0.05). There was no significant difference between the control group and the dexamethasone group.
conclusion
1. the subacute model of asthmatic rats was successfully established with ovalbumin as antigen. The protein expression of YKL-40 in the lung tissue of the asthmatic model group was significantly higher than that in the normal control group. The protein expression of YKL-40 in the lung tissue of the budesonide group was significantly lower than that of the asthma model group, but it was still higher than the normal control group. There was no significant difference in the group, compared with the dexamethasone group.
2. the expression level of YKL-40mRNA and protein and IL-8mRNA in lung tissue of asthmatic rats increased, suggesting that cytokines YKL-40 and IL-8 are involved in the pathogenesis of bronchial asthma.
3. inhalation of budesonide can reduce airway inflammation and airway resistance, effectively reduce the transcription of YKL-40mRNA and IL-8mRNA in the local lung tissue of rats and reduce the expression of YKL-40 protein.
【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

【参考文献】