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

肝星状细胞激活的机制：I 型原胶原 C-蛋白酶增强蛋白的可能贡献。

基本信息

批准号：
09670570
负责人：
MATSUI Atsushi
金额：
$ 0.45万
依托单位：
Saitama Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670570/
关键词：
Hepatic Stellate Cell PCPE RNA-binding Protein PDGF VEGF Smooth Muscle α Actin コラゲン bFGF TGFβ

项目摘要

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

此前，我们从肝星状细胞 (HSC) 文库中分离出 I 型前胶原 C 蛋白酶增强子 (PCPE) cDNA。 PCPE cDNA 推导的氨基酸序列的 C 末端显示含有 RNA 结合蛋白特异性的基序。在“激活”过程中，HSC 中的 PCPE 蛋白表达逐渐增加，其中细胞转化为肌成纤维细胞样细胞。在向培养基中添加 PCPE mRNA 的反义寡核苷酸 (AS) 进行孵育后，HSC 中的 PCPE 蛋白表达量最小。在这些细胞中，与添加无义寡核苷酸（NS）的细胞相比，非胶原蛋白以及胶原蛋白的合成减弱。因此，PCPE蛋白可能通过与RNA结合来促进HSC的蛋白质生产。首先，我们研究了生长因子对HSC功能的影响。将PDGF添加到活化的HSC的培养基中。 24 小时后，对活化的 HSC 中的 BrdU 和其他肌肉 α 肌动蛋白进行双重免疫染色。我们发现，培养激活后处于细胞周期S期的HSC不表达或表达较少的平滑肌α肌动蛋白。接下来，我们发现静止的 HSC 同时表达 flt-1 和 KDR/flk-1。当 HSC 被激活时，与静止状态的 HSC 相比，flt-1 表达增加。相反，细胞中的 KDR/flk-1 表达减少。 VEGF 不会对静止和激活的 HSC 的 DNA 和胶原蛋白合成产生任何变化。当活化的HSCs培养基中添加VEGF时,平滑肌α肌动蛋白的表达减弱,活化的HSCs的收缩受到抑制。最后,我们研究了PCPE蛋白在HSCs RNA代谢中的作用。将AS或NS添加到活化的HSC的培养基中，并连续提取细胞总RNA长达36小时。 AS和NS处理的细胞之间总RNA的细胞含量没有差异。当向HSC中添加放线菌素D时，AS处理的细胞中总RNA的量比NS处理的细胞减少得更快。总之，PCPE蛋白可能参与RNA的稳定，从而导致HSC中蛋白质的产生。未来应在 HSC 中研究 PCPE 与各种生长因子之间的相互作用。较少的