microRNA在缺血再灌注致急性肾损伤中的作用及机制研究

Immune-inflammatory responses is involved in the progression of ischemic-reperfusion(I/R) kidney injury, which can be departed into three phases: initiation, development, repair. On the basis of our previous study of gene-chip analysis on microRNA, we hypothesized that a number of microRNA were involved in the process of AKI, and our aim was to screen the degree/stage-related microRNA through correlation analysis with inflammation. In order to make the optimal choice of therapeutic target, we will take the form of gene modification to intervene in all the degree/stage-related microRNA and evaluate the degree of inflammation,changes of cell phenotype, prognosis of animal etc.so as to obtain innovative therapeutic targets. considering the exploration of pathogenesis in I/R induced AKI model was of great importance, not only would we analyze the target gene(mRNA) by expression level of mRNA, luciferase, we also intend to make mRNA-related pathway analysis, which could provide strategic reference for prevention and treatment of AKI.

缺血再灌注致急性肾损伤（AKI）是一个涉及到免疫炎症反应的病理过程，并可根据病程的变化分为始动、发展、修复阶段。我们前期通过对microRNA芯片分析认为在AKI进程中有一组microRNA参与各阶段并发挥作用。因此本项目拟用microRNA显著性水平及炎症程度阶段相关与否，筛查参与AKI病程的microRNA。采用mimic/inhibitor基因转染技术对其表达进行干预，观察炎症程度、细胞表型、动物预后等损伤及修复相关指标变化，确定何种阶段相关的microRNA可能成为潜在的治疗靶标。通过mRNA显著性、Luciferase技术、Pathway analysis等技术鉴定microRNA的靶基因并分析相关信号通路，进一步探究缺血性AKI的病理机制，为其防治提供新的策略。

本研究首先成功构建缺血-再灌注和顺铂致急性肾损伤两种大鼠动物模型。再利用成功构建动物模型，收集大鼠不同时间点尿液、肾皮质标本，分别对尿液和肾皮质进行miRNA的高通量分析，随后进行定量PCR验证芯片结果的可靠性。在尿液和肾脏标本中分析出差异表达的miRNA。同时在心脏外科手术术后并发AKI患者尿液中筛选出差异表达的miRNA，如microRNA-30c-5p、microRNA-192-5p。在体外利用肾小管上皮细胞构建低氧－复氧细胞模型，采用mimics和inhibitor转染，外源性调节microRNA－30c－5p的表达，观察该miRNA在低氧－复氧所致的凋亡过程中的功能及作用。结果显示，上调microRNA－30c－5p的表达后，细胞凋亡水平显著降低，同时可以稳定低氧诱导因子－1a（HIF－1a）的表达。进一步的实验证实，该miRNA通过作用于靶基因SOCS3，减轻其对HIF1a的抑制作用，进而对肾脏缺血再灌注起保护作用。