微小RNA表达谱在血管钙化动物模型中的差异性表达

MicroRNA differential expression in animal model of vascular calcification

目的通过维生素D3结合尼古丁法构建血管钙化动物模型，观察血管钙化过程中的微小RNA（miRNA，miR）谱变化，并通过生物信息学预测分析miRNA靶基因，采用Affemitrix miRNA芯片检测血管钙化模型组和对照组主动脉组织中miRNA表达谱变化（设2倍改变为阈值），筛选差异表达的miRNAs。方法7周龄雄性SD大鼠30只，随机分为两组，每组15只，采用维生素D3（300 kIU/kg）肌肉注射和尼古丁（5 mg/kg）灌胃建立大鼠血管钙化动物模型。对照组给予等体积的生理盐水处理。诱导4周后处死大鼠并分离主动脉组织。提取组织RNA，纯化获得miRNAs，采用Affemitrix miRNA芯片检测血管钙化模型组和对照组主动脉组织中miRNA表达谱变化（设2倍改变为阈值），筛选差异表达的miRNAs。结果诱导第20天模型组大鼠血钙水平显著升高（P＝0.000）。碱性磷酸酶（ALP）的检测结果显示，与对照组比较，模型组大鼠ALP活性明显升高，第20天时，模型组大鼠ALP活性增高了85.7%。miRNA芯片结果表明在模型组中，mmu-miR-297a的表达量明显下调。结论成功构建血管钙化动物模型，通过筛选差异表达的miRNAs，可见mmu-miR-297a在血管钙化模型中表达下调。

ObjectiveTo build animal model of vascular calcification using vitamin D3＋ nicotine method, observe spectral changes of microRNA （miRNA, miR） in the process of vascular calcification, predict miRNA target genes through the analysis of the bioinformatics, to detect miRNA expression changes （set 2 times the change of threshold） in the aorta tissues of vascular calcification model group and control group using Affemitrixmicrornas chip, and screen differentially expressed miRNAs.MethodsThe rat vascular calcification model was established using vitamin D3 plus nicotine, and the miRNA expression profile was analyzed by miRNA chip assay. Potential target of one selected miRNA with sharpest variation in expression was predicted by both PicTar and TargetScan.ResultsAt 20th day of induction, the blood calcium in the model group was significantly increased （P=0.000）. Alkaline phosphatase （ALP） activity was significantly increased in the model group as compared with that in the control group. At 20th day, ALP activity in the model group was increased by 85.7%. Micrornas chip results showed that the expression of mmu-miR-297a was significantly down-regulated in the model group.ConclusionThis study successfully constructed vascular calcification animal model. Through the screening of differentially expressed miRNAs, we found that miR-297a expression was significantly down-regulated in the vascular calcification model.