摘要
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.
出处
《胃肠病学和肝病学杂志》
CAS
2010年第1期20-24,共5页
Chinese Journal of Gastroenterology and Hepatology
基金
上海市科委基础研究重点项目基金资助(07JC14044)
