Expression data analysis in lungs from mice induced for pulmonary arterial hypertension (PAH) by inhalation of Stachybotrys chartarum spores. Mus musculus

It has been reported that repeated intra-tracheal instillation of S. chartarum spores induced significant pulmonary arterial remodeling in mice, which resulted in pathological changes like human pulmonary arterial hypertension (PAH) and elevation right ventricle systolic pressure. Then, we used microarrays to know the complex molecular mechanisms that underlie pathogenesis of PAH. Overall design: Isolates of Stachybotrys chartarum were used. Ddy mice were anesthetized and the spore suspension was intratracheally injected 12 times, i.e. 1×104 spores into each mouse at 4-5 day intervals for 8 weeks. Mice were sacrificed one week after the final injection and then examined. Lung tissue specimens from mice model of PAH (n=3) and normal controls (n=3) were obtained. RNA targets preparation were performed according to the manufacturer's protocol using GeneChip(R) 3' IVT Express Kit (Affymetrix). One hundred nanograms of total RNA was converted into double-stranded cDNA template for transcription. In vitro transcription synthesized amplified RNA (aRNA) and incorporated a biotin-conjugated nucleotide. After purification and fragmentation of aRNA, 12.5 ug of them was hybridized to GeneChip(R) Mouse Genome 430 2.0 Array (Affymetrix).The Probe Array was scanned using a GeneChip(R) Scanner 3000 7G.

已有研究表明，反复经气管内滴注烟色葡萄穗霉（S. chartarum, Stachybotrys chartarum）孢子可在小鼠体内诱发显著的肺动脉重构，进而引发与人类肺动脉高压（PAH, pulmonary arterial hypertension）相似的病理改变，并导致右心室收缩压升高。本研究借助微阵列（microarrays）技术，阐明肺动脉高压发病机制背后的复杂分子机制。整体实验设计：本研究采用烟色葡萄穗霉分离株。将Ddy小鼠麻醉后，每隔4~5天经气管内注射孢子悬液，共给药12次，每只小鼠每次注射1×10^4个孢子，造模周期持续8周。末次注射一周后处死小鼠并开展相关检测。分别收集肺动脉高压模型组小鼠（n=3）与正常对照组小鼠（n=3）的肺组织标本。依照GeneChip® 3' IVT Express试剂盒（Affymetrix）的制造商操作规程进行RNA靶标制备：取100 ng总RNA反转录为双链cDNA模板，用于转录反应。体外转录过程合成扩增RNA（aRNA, amplified RNA）并掺入生物素标记核苷酸。对扩增RNA进行纯化与片段化后，取12.5 μg片段化产物与GeneChip® Mouse Genome 430 2.0 芯片（Affymetrix）进行杂交。使用GeneChip® Scanner 3000 7G扫描仪对探针阵列进行扫描。