Department of Gastroenterology, Shanghai East Hospital Affiliated to Tongji University, Shanghai 200120, China.
World J Gastroenterol. 2012 Apr 21;18(15):1745-52. doi: 10.3748/wjg.v18.i15.1745.
To identify differentially expressed genes in quiescent and activated hepatic stellate cells (HSCs) and explore their functions.
HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation. Total RNA and mRNA of quiescent HSCs, and culture-activated HSCs were extracted, quantified and reversely transcripted into cDNA. The global gene expression profile was analyzed by microarray with Affymetrix rat genechip. Differentially expressed genes were annotated with Gene Ontology (GO) and analyzed with Kyoto encyclopedia of genes and genomes (KEGG) pathway using the Database for Annotation, Visualization and Integrated Discovery. Microarray data were validated by quantitative real-time polymerase chain reaction (qRT-PCR). The function of Wnt5a on human HSCs line LX-2 was assessed with lentivirus-mediated Wnt5a RNAi. The expression of Wnt5a in fibrotic liver of a carbon tetrachloride (CCl(4))-induced fibrosis rat model was also analyzed with Western blotting.
Of the 28 700 genes represented on this chip, 2566 genes displayed at least a 2-fold increase or decrease in expression at a P < 0.01 level with a false discovery rate. Of these, 1396 genes were upregulated, while 1170 genes were downregulated in culture-activated HSCs. These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms. The most enriched GO terms included response to wounding, wound healing, regulation of cell growth, vasculature development and actin cytoskeleton organization. KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs. Wnt5a was significantly increased in culture-activated HSCs as compared with quiescent HSCs. qRT-PCR validated the microarray data. Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation, downregulated expressions of type I collagen and transforming growth factor-β1. Wnt5a was upregulated in the fibrotic liver of a CCl(4)-induced fibrosis rat model.
Wnt5a is involved in the activation of HSCs, and it may serve as a novel therapeutic target in the treatment of liver fibrosis.
鉴定静息和激活的肝星状细胞（HSCs）中的差异表达基因，并探讨其功能。
通过胶原酶和蛋白酶原位灌注和密度梯度尼科丹兹离心从正常 Sprague Dawley 大鼠中分离 HSCs。提取、定量静息 HSCs 和培养激活 HSCs 的总 RNA 和 mRNA，并逆转录为 cDNA。使用 Affymetrix 大鼠基因芯片通过微阵列分析全基因表达谱。使用京都基因与基因组百科全书（KEGG）途径的基因本体论（GO）注释差异表达基因，并进行分析。使用数据库进行注释、可视化和综合发现对微阵列数据进行验证。通过定量实时聚合酶链反应（qRT-PCR）验证微阵列数据。使用慢病毒介导的 Wnt5a RNAi 评估 Wnt5a 对人 HSCs 系 LX-2 的作用。还使用 Western blot 分析四氯化碳（CCl4）诱导的纤维化大鼠模型肝纤维化中 Wnt5a 的表达。
该芯片上代表的 28700 个基因中，有 2566 个基因的表达水平至少增加或减少了 2 倍，P<0.01，假发现率为 0.01。其中，1396 个基因上调，而 1170 个基因下调在培养的激活的 HSCs 中。这些差异表达的转录物根据生物过程 GO 术语分为 545 个 GO。最富集的 GO 术语包括对创伤的反应、伤口愈合、细胞生长的调节、脉管系统发育和肌动蛋白细胞骨架组织。KEGG 途径分析表明，Wnt5a 信号通路参与了 HSCs 的激活。与静息 HSCs 相比，培养的激活的 HSCs 中 Wnt5a 的表达明显增加。qRT-PCR 验证了微阵列数据。慢病毒介导的 Wnt5a 表达抑制在激活的 LX-2 中导致增殖显著受损，下调 I 型胶原和转化生长因子-β1 的表达。在四氯化碳（CCl4）诱导的纤维化大鼠模型的纤维化肝中，Wnt5a 上调。
Wnt5a 参与 HSCs 的激活，可能成为治疗肝纤维化的新治疗靶点。
