哮喘大鼠基因的动态变化及三氧化二砷的干预研究

发布时间：2018-05-03 14:00

本文选题：哮喘 + 基因芯片　；参考：《南华大学》2013年硕士论文

【摘要】：目的利用全基因表达谱芯片技术观察哮喘大鼠和应用三氧化二砷干预后不同时间点的基因差异表达的变化，阐明哮喘的发病机制和三氧化二砷治疗哮喘的分子机制。方法健康雄性清洁级SD大鼠70只，，按随机表随机分成3组：正常对照组A组10只，哮喘模型组B组30只(分别为二周B1组、四周B2组、八周B3组，每组各10只），三氧化二砷干预组C组30只(分别为二周C1组、四周C2组、八周C3组，每组各10只）。以腹腔内注射联合雾化吸入卵蛋白建立哮喘模型，末次激发后24h处死大鼠检测支气管肺泡灌洗液（BALF）中的白细胞数及嗜酸性粒细胞数；左肺行染色、病理图像分析；再切取右肺提取肺组织总RNA行全基因组表达谱芯片检测，以基因表达倍数值2.0和基因表达倍数值－2.0为阈值来确定差异表达基因，然后用GO及Pathway分析对差异表达基因进行功能分类分析，并对基因芯片结果进行实时定量PCR验证。结果 1、哮喘模型组BALF中白细胞总数、嗜酸性粒细胞（Eos）比例较正常组均有明显增高，三氧化二砷组较哮喘模型组明显降低，差异有显著性(P 0.05)。 2、哮喘模型组大鼠气道支气管及血管周围存在大量炎症细胞浸润，以嗜酸性粒细胞、淋巴细胞为主，气道平滑肌增厚，基底膜增厚，管腔狭窄，粘膜下细胞外基质沉积，且随着时间的延长，以上变化逐渐加重；三氧化二砷组较哮喘模型组上述改变减轻，正常组无上述改变。 3、在24358个基因表达谱的筛选中，哮喘模型组对比正常组共有438个差异基因表达，其中348个上调，90个下调；三氧化二砷组对比哮喘模型组共有236个基因表达差异，其中96个上调，140个下调。其中GZMA、MAL基因在哮喘模型组中表达下调，三氧化二砷干预组后表达上调，通过实时定量PCR验证后与基因芯片筛选结果表达方向一致。结论 1、GZMA、MAL等基因可能参与了哮喘的发生发展过程。 2、三氧化二砷治疗哮喘的机理可能与上调GZMA、MAL基因在肺组织中的表达有关。
[Abstract]:Purpose In order to elucidate the pathogenesis of asthma and the molecular mechanism of arsenic trioxide treatment, the changes of gene differential expression at different time points after arsenic trioxide intervention in asthmatic rats were observed by whole gene expression microarray technique. Method Seventy healthy male SD rats of clean grade were randomly divided into three groups: normal control group (n = 10), asthma model group (B group) (n = 30) (group B _ 1 for two weeks, group B _ 2 for four weeks, group B _ 3 for eight weeks). There were 10 rats in each group, and 30 rats in group C in arsenic trioxide intervention group (10 rats in each group) (C1 group for two weeks, C 2 group for four weeks and C 3 group for eight weeks). The asthmatic model was established by intraperitoneal injection and atomization inhalation of ovalbumin. The white blood cells and eosinophils in bronchoalveolar lavage fluid (BALF) were measured 24 hours after the last stimulation, and the left lung was stained and analyzed by pathological images. The total RNA of lung tissue was extracted from the right lung and detected by whole genome expression microarray. The differentially expressed genes were determined by the threshold of gene expression doubling value 2.0 and gene expression doubling value -2.0. Then go and Pathway analysis were used to analyze the function of differentially expressed genes, and real-time quantitative PCR was used to verify the results of gene chip. Result 1. The percentage of total white blood cells and eosophils in BALF of asthmatic model group was significantly higher than that of normal group, and that of arsenic trioxide group was significantly lower than that of asthmatic model group (P 0.05). 2. In the asthma model group, there were a large number of inflammatory cells infiltrating around the airway bronchus and blood vessels, mainly eosinophils and lymphocytes, airway smooth muscle thickening, basement membrane thickening, lumen stenosis, and extracellular matrix deposition. The above changes were gradually aggravated with the prolongation of time, and the above changes in arsenic trioxide group were lighter than those in asthma model group, but no such changes were found in normal group. 3. In the screening of 24358 gene expression profiles, there were 438 differentially expressed genes in asthmatic model group compared with normal group, 348 of which were up-regulated and 90 were down-regulated, and 236 genes were differentially expressed in arsenic trioxide group compared with asthmatic model group. Of these, 96 were up-regulated and 140 downgraded. The expression of GZMA-MAL gene was down-regulated in asthmatic model group and up-regulated in arsenic trioxide intervention group. The expression direction of GZMA-MAL gene was consistent with the result of gene chip screening by real-time quantitative PCR. Conclusion Genes such as GZMA-MAL may be involved in the occurrence and development of asthma. The mechanism of arsenic trioxide in the treatment of asthma may be related to the up-regulation of GZMA-MAL gene expression in lung tissue.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R562.25

【共引文献】