小鼠肾脏缺血-再灌注损伤miRNAs差异表达谱研究

摘要

To screen the differentially-expressed microRNAs (miRNAs) in mouse models with renal ischemia-reperfusion injury (IRI),aiming to offer foundation for unraveling the molecular mechanism of the incidence and progression of IRI.Methods The mouse models with acute IRI were established by renal artery clamping.Fifteen mice were divided into the IRI group and sham surgery group (E group).The animals in the IRI group were subdivided into the A group (45 min ischemia followed by 24 h reperfusion),B group (25 min ischemia followed by 24 h reperfusion),C group (45 min ischemia followed by 4 h reperfusion) and D group (25 min ischemia followed by 4 h reperfusion) (n=3 for each group).The severity ofIRI was evaluated by histological changes and renal function.The differentially-expressed miRNAs in the IRI mouse models at different ischemia time (25 and 45 min) and reperfusion time (4 and 24 h) were screened by using cluster analysis of miRNAs microarray data.The differential expression of miR-695 and miR-145 was validated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).Results Both histological changes and renal function confirmed that the IRI mouse models were successfully established.Compared with the sham surgery group,71 differentially-expressed miRNAs were detected in the IRI group including 30 down-regulated miRNAs and 40 up-regulated miRNAs.The results of qRT-PCR demonstrated that if the standardized expression level of miRNAs in the E group was 1,the relative expression levels of miR-695 and miR-145 were 11.82 and 0.31 in the IRI group (both P＜0.05),which were consistent with the chip results.Conclusions After renal IRI,different changes occur in the gene expression profile of miRNAs.These differentially-expressed miRNAs act as molecular biomarkers for renal IRI with potential clinical and scientific research values.%目的 筛选小鼠肾脏缺血-再灌注损伤(IRI)差异性表达的微小核糖核酸(miRNAs),为进一步深入阐明肾脏IRI发生、发展的分子机制奠定基础.方法 应用微型动脉夹夹闭小鼠双侧肾动脉制备急性肾缺血损伤模型,将15只小鼠分为IRI组和假手术组(E组),IRI组再分为A组(缺血时间45 min,再灌注时间24 h)、B组(缺血时间25 min,再灌注时间24 h)、C组(缺血时间45 min,再灌注时间4h)、D组(缺血时间25 min,再灌注时间4 h),每组3只.采用肾脏组织形态学改变和肾功能评估结果来鉴定各组小鼠的IRI程度;采用miRNAs芯片聚类分析对小鼠IRI特定缺血时间(25、45 min)和再灌注时间(4、24 h)下肾脏差异性表达差异表达的miRNAs进行筛选鉴定;采用实时定量逆转录聚合酶链反应(qRT-PCR)对小鼠IRI后差异表达miRNAs芯片miR-695与miR-145进行验证.结果 肾脏组织形态学改变和肾功能评估结果显示成功建立IRI模型.与假手术组相比,肾脏IRI组共检出71种差异性表达显著的miRNAs,其中30种下调表达,41种上调表达.qRT-PCR法检测结果表明,以E组特定miRNAs的表达量标准化为1,miR-695及miR-145在肾脏IRI组的相对表达量分别为11.82和0.31(均为 P＜0.05),与芯片结果基本一致.结论 肾脏IRI后,miRNAs表达谱发生差异性改变,这些差异性表达的miRNAs将可作为肾脏IRI的分子标志物而具有潜在的临床及科研应用价值.