Cellular and Molecular Biology of the Hepatic Stem Cell

本研究建立了一种诱导分化的方法，即先用去甲基化剂5-氮杂-2 '-脱氧胞苷（5-aza-2'-deoxycytidine）处理，然后用FGF+OSM+HGF联合诱导，再用合成的糖皮质激素地塞米松（Dex）处理。这种处理导致细胞大小和细胞器复杂性显著增大，如通过FACS和共聚焦显微镜所示。RLE的形态成熟被细胞增殖的减少所阻碍。更重要的是，RT-PCR分析揭示了肝细胞特异性标志物AFP、TTR、达特和连接蛋白32的诱导。此外，编码肝脏富集转录因子HNF 1a/B、HNF 3a/B、HNF 4和C/EBPb以及Hex和加塔4/5的mRNA沿着肝细胞分化而显着诱导。RT-PCR结果得到了cDNA微阵列分析的支持。处理方案后基因表达谱的比较反映了肝细胞分化程序的激活，如通过分化相关基因集的表达增加和细胞周期调节基因集的表达减少所判断的。与肝特化/分化相关的表达基因中包括BMP 4、BMP 6、Enothelin 1以及参与血红素和异生物质代谢的基因。此外，观察到对细胞外基质重塑和细胞骨架组织至关重要的结构基因表达的改变，包括MMP 1、TIMP-1、胶原、层粘连蛋白、α-辅肌动蛋白。重要的是，b-连环蛋白、Hdac 1以及c-myc的表达随着肝细胞分化而沿着增加。相反，在体外胚胎肝干细胞分化系统中，Wnt/β-catenin通路被报道下调。因此，不同的调控途径可能负责控制成体干细胞与胚胎干细胞的分化。此外，我们发现诱导涉及胆管上皮以及胰腺分化的基因，反映了RLE细胞的广泛发育能力。正在进行由单个分化因子及其组合诱导的基因表达谱的分析，其中基因通常且优先在纯化的肝细胞、卵圆细胞或胆管上皮细胞中表达，以鉴定成体肝干细胞分化途径的特征性基因表达模块。 表征伴随干细胞室的扩增和分化的分子和细胞事件对于理解肝干细胞的生物学和促进其临床应用是至关重要的。TNFa是一种诱导肝细胞增殖和凋亡的炎性细胞因子，参与了不同类型的肝损伤。在这项工作中，我们研究了大鼠肝上皮细胞系（RLE 13）的分化是否促进了对TNF α介导的凋亡的敏感性的获得。为了诱导向肝细胞谱系的分化，我们用5-氮杂-2 '-脱氧胞苷+地塞米松+制瘤素M的组合处理RLE细胞。而对照RLE对TNF α具有抗性，在分化的RLE中观察到大量凋亡。为了阐明TNF α诱导的细胞凋亡的机制，研究了caspase激活和线粒体功能的级联反应。在对照和分化的RLE中，TNF α诱导了细胞凋亡所需的所有早期事件，包括caspase 8的活化、线粒体膜电位的丧失、细胞色素c的释放和caspase 9的裂解。然而，只有在分化的RLE中，TNFa处理才导致效应物半胱天冬酶3的活化。IkBa的磷酸化和降解的Western印迹分析以及NF κ B DNA结合活性的凝胶位移分析显示，在对照和分化的RLE中，该途径的激活相似。因此，NFkB存活途径的失调被排除作为TNF α在两种细胞类型中引起的差异应答的潜在解释。有趣的是，肝细胞分化与凋亡抑制蛋白家族（IAP）成员XIAP表达模式的变化相关。而控制RLE显示丰富的XIAP存在于可溶性胞质级分，这种蛋白质是很难检测到分化的RLE。由于在全细胞裂解物中的蛋白质印迹分析显示XIAP在两种细胞类型中的可比较的全局表达，因此我们正在测试以下假设：涉及该蛋白质的细胞内分布、构象或结合复合物的变化可以解释XIAP不能抑制分化的RLE中的细胞凋亡。

A differentiation protocol consisting of a sequential treatment with a demethylating agent 5-aza-2'-deoxycytidine followed by a combination of FGF+OSM+HGF in the continuos presence of a synthetic glucocorticoid dexamethasone (Dex) was developed and applied to stem-like rat liver epithelial (RLE) cell lines derived from adult rat. This treatment resulted in a remarkable enlargement in cell size and organelle complexity as shown by FACS and confocal microscopy. Morphological maturation of RLE was paralleled by a decrease in cell proliferation. More significantly, RT-PCR analysis revealed an induction of hepatocyte specific markers AFP, TTR, TAT and connexin 32. In addition, mRNAs encoding liver enriched transcription factors HNF 1a/b, HNF 3a/b, HNF 4, and C/EBPb as well as Hex and GATA 4/5 were markedly induced along with hepatocytic differentiation. The RT-PCR results were supported by a cDNA microarray analysis. Comparison of the gene expression profiles following the treatment protocol reflected an activation of the hepatocytic differentiation program as judged by increased expression of a differentiation-associated gene set and decreased expression of a cell cycle regulated gene set. Among the expressed genes related to hepatic specification/differentiation were BMP4, BMP6, Enothelin 1, as well as genes involved in heme and xenobiotic metabolism. In addition, alteration in expression of structural genes critical for extracellular matrix remodeling and organization of cytoskeleton including MMP1, TIMP-1, collagens, laminin, a-actinin was observed. Of importance, expression of b-catenin, Hdac1 as well as c-myc was increased along with hepatocytic differentiation. In contrast, down-regulation of Wnt/b-catenin pathway was reported in vitro system of fetal hepatic stem cell differentiation. Hence, it is possible that different regulatory pathways are responsible for the control of adult vs. embryonic stem cells differentiation. Furthermore, we found induction of genes implicated in biliary epithelial as well as pancreatic differentiation reflecting a broad developmental capacity of the RLE cells. Analysis of gene expression profiles induced by individual differentiating factors and their combinations, with genes commonly and preferentially expressed in purified hepatocytes, oval cells or biliary epithelial cells is underway to identify genes expression modules characteristic for the differentiation pathways of adult hepatic stem cells. Characterization of molecular and cellular events accompanying the expansion and differentiation of the stem cell compartment is of fundamental importance for understanding the biology of the liver stem cells and facilitating its clinical application. TNFa, an inflammatory cytokine that induces both hepatocyte proliferation and apoptosis, has been involved in different types of liver damage. In this work, we examined whether the differentiation of a rat liver epithelial cell line (RLE13) promoted the acquisition of sensitivity to TNFa-mediated apoptosis. To induce differentiation towards the hepatocyte lineage we treated RLE cells with a combination of 5-Aza-2'-deoxycytidine+Dexamethasone+Oncostatin M. While control RLE were resistant to TNFa, a massive apoptosis was observed in differentiated RLE. To clarify the mechanism of apoptosis induced by TNFa, cascade of caspase activation and mitochondrial functions were investigated. In both control and differentiated RLE, TNFa induced all early events required for apoptosis, including activation of caspase 8, loss of mitochondria membrane potential, release of cytochrome c and cleavage of caspases 9. However, only in differentiated RLE did TNFa treatment culminated in activation of the effector caspase 3. Western blot analysis of the phosphorylation and degradation of IkBa as well as gel shift analysis of NFkB DNA-binding activity showed a similar activation of this pathway in both control and differentiated RLE. Therefore, a deregulation of the NFkB survival pathway was excluded as a potential explanation for the differential response elicited by TNFa in both cell types. Interestingly, hepatocytic differentiation was associated with changes in the expression pattern of XIAP, a member of the inhibitor of apoptosis protein family (IAPs). While control RLE showed abundant presence of XIAP in soluble cytosolic fractions, this protein was hardly detectable in differentiated RLE. Since western blot analysis in whole cell lysates showed a comparable global expression of XIAP in both cell types, we are testing the hypothesis that changes in intracellular distribution, conformation or binding complexes involving this protein could account for the inability of XIAP to inhibit apoptosis in differentiated RLE.