The Mechanisms of Hepatic Stellate Cell Activation : Possible Contribution of Type I Procollagen C-Proteinase Enhancer Protein.

此前，我们从肝星状细胞(HSC)文库中克隆了I型前胶原C-蛋白酶增强子(PCPE)基因。PCPE基因编码的氨基酸序列的C末端含有RNA结合蛋白所特有的基序。在“激活”过程中，HSC中PCPE蛋白的表达逐渐增加，在这个过程中，细胞转化为肌成纤维细胞样细胞。在HSC中加入PCPE mRNA的反义寡核苷酸(AS)后，HSC中PCPE蛋白的表达最低。在这些细胞中，与加入无意义寡核苷酸(NS)的细胞相比，非胶原蛋白和胶原的合成都受到了抑制。因此，PCPE蛋白可能通过与RNA结合而参与HSC的蛋白质合成。首先，我们研究了生长因子对HSC功能的影响。在活化的HSCs的培养液中加入PDGF。24小时后，BrdU和Smo…双重免疫染色在活化的HSC中进行更多的OTH肌肉α肌动蛋白的表达。我们发现，处于细胞周期S期的肝星状细胞经培养激活后，不表达或很少表达平滑肌α肌动蛋白。接下来，我们发现静止的HSCs同时表达Flt-1和KDR/Flk-1。当HSCs被激活时，Flt-1的表达较静止的HSCs增加。相反，KDR/Flk-1在细胞中的表达减少。在静止和激活的HSC中，血管内皮生长因子对DNA和胶原的合成没有影响。将血管内皮生长因子加入活化的肝星状细胞培养上清液中，可抑制活化的肝星状细胞的收缩，抑制平滑肌α肌动蛋白的表达。将AS或NS加入活化的HSCs培养液中，连续提取细胞总RNA至36h，细胞总RNA含量在AS和NS处理的细胞中无明显差异。在HSCs中加入放线菌素D后，AS组细胞总RNA含量下降的速度明显快于NS组。结论：PCPE蛋白可能参与了HSCs中RNA的稳定，从而促进了HSCs的蛋白质合成。PCPE与多种生长因子的相互作用是今后HSCs研究的重点。较少

Previously, we isolated type I procollagen C-proteinase enhancer (PCPE) cDNA from the library of hepatic stellate cells (HSC). The C-terminus of the deduced amino acid sequence of PCPE cDNA was shown to contain motifs specific for RNA-binding proteins. PCPE protein expression increased progressively in HSCs under an "activation" process, in which the cells are transformed to myofibroblast-like cells. PCPE protein expression was minimal in HSC following incubation with addition of an antisense oligonucleotide (AS) for PCPE mRNA to the culture medium. In these cells, the syntheses of non-collagenous protein as well as collagen were attenuated in comparison with the cells with the nonsense oligonucleotide (NS) addition. Thus, PCPE protein may contribute to the protein production in HSC through binding to RNAs.First, we studied the effect of growth factors on HSC function. PDGF was added to the culture medium of activated HSCs. Twenty-four hours later, double immunostaining of BrdU and smo … More oth muscle α actin in activated HSCs were performed. We found that HSC at the S-phase of the cell cycle after activation by culture express none or less smooth muscle α actin. Next, we found that quiescent HSCs expressed both flt-1 and KDR/flk-1. When HSCs were activated, flt-1 expression were increased compared to that in the quiescent HSCs. In contrast, KDR/flk-1 expression in the cells diminished. VEGF produced no changes in DNA and collagen syntheses by the quiescent and activated HSCs. When VEGF was added to the culture medium of activated HSCs, the expression of smooth muscle α actin was attenuated and contraction of activated HSCs was inhibited.Finally, we studied the role of PCPE protein in the metabolism of RNA in HSCs. AS or NS were added to the culture medium of activated HSCs, and total RNA was extracted from the cells serially up to 36 h. Cellular contents of total RNA were not different between the cells treated with AS and NS. When actinomycin D was added to HSCs, the amout of total RNA was decreased more rapidly in cells treated with AS than that with NS.In conclusion, PCPE protein may be involved in the stabilization of RNAs leading to contribution of protein production in HSCs. The interaction between PCPE and various growth factors should be investigated in HSCs in future. Less