Cellular and Molecular Biology of the Hepatic Stem Cell

肝干细胞的细胞和分子生物学

• 批准号: 6761549
• 负责人: SNORRI S THORGEIRSSON
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6761549
• 关键词: biliary tract; cell growth regulation; cell population study; cell proliferation; cytokine receptors; dexamethasone; flow cytometry; gene expression; interleukin 6; liver cells; liver regeneration; molecular biology; nuclear factor kappa beta; stem cell factor; stem cells; tissue /cell culture; transcription factor; tumor necrosis factor alpha; western blottings

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.

研究人员开发了一种分化方案，包括用去甲基化剂5-氮杂-2'-脱氧胞苷进行顺序处理，然后在持续存在合成糖皮质激素地塞米松（Dex）的情况下，结合FGF+OSM+HGF，并将其应用于成年大鼠干细胞样大鼠肝上皮（RLE）细胞系。这种处理导致细胞大小和细胞器复杂性显著增大，如流式细胞仪和共聚焦显微镜所示。RLE的形态成熟与细胞增殖的减少是平行的。更重要的是，RT-PCR分析显示肝细胞特异性标志物AFP、TTR、TAT和connexin 32的诱导作用。此外，编码肝脏富集转录因子HNF 1a/b、HNF 3a/b、HNF 4、C/EBPb以及Hex和GATA 4/5的mrna在肝细胞分化过程中被显著诱导。RT-PCR结果得到了cDNA芯片分析的支持。治疗方案后基因表达谱的比较反映了肝细胞分化程序的激活，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来判断。表达的与肝脏分化相关的基因包括BMP4、BMP6、Enothelin 1以及血红素和外源代谢相关的基因。此外，观察到细胞外基质重塑和细胞骨架组织的关键结构基因MMP1、TIMP-1、胶原、层粘连蛋白、a-actin的表达变化。重要的是，b-catenin、Hdac1和c-myc的表达随着肝细胞分化而增加。相比之下，Wnt/b-catenin通路在胎儿肝干细胞分化的体外系统中被下调。因此，可能有不同的调控途径负责控制成体干细胞与胚胎干细胞的分化。此外，我们发现与胆道上皮和胰腺分化相关的基因诱导反映了RLE细胞的广泛发育能力。通过分析单个分化因子及其组合诱导的基因表达谱，以及纯化肝细胞、卵圆细胞或胆道上皮细胞中常见和优先表达的基因，确定成体肝干细胞分化途径的特征基因表达模块。