STUDIES ON DIFFERENTIATION AND REGENERATION OF FETAL LIVER STEM CELLS AND THEIR APPLICATION FOR HEPATIC FAILURE

肝祖细胞的生长速度快于造血细胞。培养10d后，位于聚集体中心的细胞呈白蛋白染色，周边细胞呈CK19染色。腺病毒介导的HNF-4基因转移导致HNF-4、ApoA1、APOC3、PXR、TAT基因表达增加。经hnf-4基因转染组的小鼠存活率显著高于对照组(P=0.032)。经HNF-4转基因的小鼠血浆白蛋白、总胆固醇和血糖水平高于对照组小鼠。利用基因芯片分析，我们发现在基底膜基质Engelbreth-Holm-Sarm(EHS)凝胶上培养的肝细胞中，磷脂酰肌醇4，5-二磷酸(PI(4，5)P2)磷酸酶基因表达上调，从而导致EHS凝胶上肝细胞PI(4，5)P2水平下降。EHS凝胶上肝细胞的这些变化伴随着肌动蛋白解聚和di…的促进。肝细胞表型分化更明显。PI(4，5)P2或磷脂酶C抑制剂U73122可降低肝细胞白蛋白和肝细胞核因子4(HNF-4)的mRNA表达。相反，肌动蛋白干扰剂明胶增加了白蛋白和HNF-4的mRNA表达。这些结果表明，肌动蛋白细胞骨架的组织通过PI(4，5)P2参与了ECM对肝细胞分化的调控。为了阐明这种关系，我们使用了小干扰RNA(SiRNA)，它可以强烈而特异地抑制细胞培养中的基因表达。SiHNF-4处理可降低原代大鼠肝细胞HNF-4mRNA的表达水平。在siHNF-4存在的情况下，在EHS凝胶上培养的肝细胞中上调的白蛋白的mRNA表达被下调。此外，siHNF-4不影响肝细胞的形态和肌动蛋白组装。这些结果表明，HNF-4直接调节肝脏特异性基因的表达，并可能位于细胞骨架组织的下游，其机制是EHS凝胶调节肝细胞的分化表型。较少

Hepatic progenitor cells grew more rapidly than hematopoietic cells. After 10days culture, cells located in the center of aggregates for were stained for albumin, but peripheral cells for CK19. Adenovirus-mediated HNF-4 gene transfer resulted in increases in the expressions of HNF-4, ApoA1 ApoC3, PXR, TAT mRNA. Mice treated with HNF-4-transfected progenitor cells significantly survived than control mice (P=0.032). Plasma levels of albumin, total cholesterol, and glucose were higher in mice treated with cells transtected by HNF-4 than in control mice.Using a cDNA microarray analysis, we identified that phosphatidylinositol 4,5-bisphosphate (PI (4,5)P_2) phosphatase mRNA was up-regulated in hepatocytes cultured on a basement membrane matrix, Engelbreth-Holm-Swarm (EHS) gel, which led to the finding that there was a decrease in the PI(4,5)P_2 levels of hepatocytes on EHS gel. These changes in hepatocytes on the EHS gel were accompanied by the promotion of the actin depolymerization and di … More fferentiated phenotypes of hepatocytes. Treatment with PI(4,5)P_2 or a phospholipase C inhibitor, U73122, resulted in a decrease in the mRNA expressions of albumin and hepatocyte nuclear factor 4 (HNF-4) in hepatocytes. In contrast, the actin-disrupting agent gelsolin increased the mRNA expressions of albumin and HNF-4. These findings indicated that the organization of the actin cytoskeleton via PI(4,5)P_2 is involved in the regulation of hepatocyte differentiation by the ECM.To elucidate the relationship, we used small interfering RNA (siRNA), which obtains strong and specific knockdown of gene expression in cell culture. Treatment with siHNF-4 declined the expression levels for HNF-4mRNA in primary rat hepatocytes. The mRNA expression of albumin that was up-regulated in hepatocytes cultured on EHS gel, was decreased in the presence of siHNF-4. Moreover, siHNF-4 did not affect the morphology and actin assembly of hepatocytes. These findings demonstrated that HNF-4 directly regulates liver-specific gene expression and might be downstream of cytoskeletal organization in the mechanism by which the differentiated phenotype of hepatocytes is regulated by EHS gel. Less