MicroRNAs在肝星状细胞活化过程中表达差异谱的研究

MicroRNAs expression profiles in activation of hepatic stellate cells

目的应用MicroRNAs基因芯片技术筛选静止和活化肝星状细胞差异表达的MicroRNAs。方法用链酶蛋白酶和胶原酶原位灌流,Nycodenz密度梯度离心分离大鼠HSCs,并进行体外培养,借助荧光倒置显微镜观察细胞形态,MicroRNAs基因芯片技术和荧光定量PCR检验MicroRNAs在HSCs静止期(2天)和活化期(14天)的表达。结果HSCs的miRNAs表达谱中差异表达miRNAs共21个,其中上调12个,下调9个(P<0.01);荧光定量RT-PCR证实其表达水平在静止和活化期肝星状细胞中均有差异(P<0.01)。结论MicroRNAs基因芯片筛选差异表达miRNAs可望为肝纤维化的基因治疗提供全新的靶标和策略,为肝纤维化的发病机制和诊断治疗研究提供了新的思路。

Objective To determine the miRNAs expression profile of quiescent and culture activated rat hepatic stellate cells. Methods HSCs were isolated from the livers of adult Sprague-Dawley rats（weighing 400～500 g） by in situ perfusion and purified by single-step density gradient centrifugation with Nycodenz,became highly activated after 14 days＇ cultivation.Cell morphological characteristics were identified by means of phase-contrast microscopy,fluorescence microscopy. The difference in miRNAs expression between quiescent hepatic stellate cells at 2 days and their corresponding activated cells that underwent activation in vitro over a period of 2 weeks was detected by both microarrays and quantitative PCR. Results Twelve miRNAs were expressed above the threshold levels in activated hepatic stellate cells,with a progressively lower numbers when activated（P0.05）.Only 9 miRNAs were expressed above the threshold levels in quiescent hepatic stellate cells.The expression of selected miRNAs was further validated and found their differential expression in cell-specific manners（P0.05）. Conclusion The expression of a number of miRNAs in quiescent and activated hepatic stellate cells may suggest the potential roles of these miRNAs and applicability as diagnostic and prognostic signatures in liver fibrosis.