Cloning and characterization of newly described proteins from a fat-storing cell library : their significance in the activation of the cells.

脂肪储存细胞(FSC)转化为肌成纤维细胞，在肝纤维化形成中起重要作用。我们从大鼠储脂细胞文库中分离到两个cDNAC72和C81，其中C72包含一个开放阅读框架，编码一个468个氨基酸残基的蛋白质。C72基因的核苷酸序列与I型前胶原C-蛋白酶增强子蛋白(PCPE)基因同源。经Northern印迹分析，从新鲜分离和早期传代的FSC和成纤维细胞中提取的总RNA中约有1.7kb的C72c DNA。与新鲜分离的FSC相比，早期传代的FSC中C72mRNA的表达增加3倍，而在大鼠全肝、新鲜分离的肝细胞、内皮细胞和Kupffer细胞中均未检测到C72mRNA的表达。而在CCl_4-肝硬变模型中，C72mRNA的表达被诱导。另一方面，我们发现该基因的C末端含有与RNA结合的共同序列所特有的基序，这表明PCPE具有除增强子活性以外的其他功能。反义寡核苷酸下调PCPE的表达，抑制FSC.C81cDNA非胶原蛋白的合成和胶原的合成，编码563个氨基酸残基的开放阅读框。其推导的氨基酸序列与以前报道的蛋白质没有明显的同源性。C81cDNA与新鲜分离的FSC中的2.1kb的mRNA杂交，早期传代的FSC杂交条带消失，成纤维细胞中未检测到信号，这些cDNA可作为追踪HSC向肌成纤维细胞转化的有用工具。

Fat-storing cells (FSC) transform into myofibroblasts and play an important role in liver fibrogenesis. We isolated two cDNAs, C72 and C81, from a rat fat-storing cell library.C72 cDNA contained an open reading frame coding for a protein of 468 amino acid residues. C72 cDNA had a nucleotide sequence homologous to that of the gene for the type I procollagen C-proteinase enhancer protein (PCPE). C72 cDNA recognized by Northern blot a 1.7 kb mRNA in total RNA extracted from freshly isolated and early passaged FSC and fibroblasts. The expression of C72 mRNA was increased 3-fold in early passaged FSC as compared to freshly isolated FSC.C72 mRNA was not detected in total rat liver, freshly isolated hepatocytes, endothelial or Kupffer cells. However, the expression of C72 mRNA was induced in CC1_4-cirrhotic livers. On the other hand, we revealed that the derived C-terminus of the cDNA contains motifs specific to RNA-binding consensus sequence, suggesting that PCPE has a function other than the enhancer activity. The down-regulation of PCPE with an antisense oligonucleotide inhibited non-collagenous protein production as well as collagen production in FSC.C81 cDNA contained an open reading frame which codes 563 amino acid residues. Its deduced amino acid sequence showed no significant homology to previously reported proteins. C81 cDNA hybridized with a 2.1 kb mRNA presented in freshly isolated FSC.The hybridization band disappeared in early passaged FSC,and no signals were detected in fibroblasts.From these results, these cDNAs can be useful tools to follow the transformation of HSC to myofibroblasts during development of hepatic fibrosis.