Arkadia在肝纤维化SnoN蛋白降解中的作用及分子机制研究

Smad transcriptional co-repressor SnoN acts as an antagonist that tightly controls the trans-activation of TGF-β1/Smad target,and the loss of SnoN is a critical,pathogenic event that may lead to hepatic fibrosis.Our previous study found that SnoN protein is reduced progressively in the fibrotic liver from different liver fibrosis animal model,and in liver tissues from patients with hepatitis B virus.Our study also found SnoN is downregulated in fibrotic liver by a mechanism independent of gene transcription stability.Arkadia, an E3 ubiquitin ligase that is implicated in the degradation of SnoN, was expressed in the liver tissus. However, the role and mechanism underlying Arkadia degradation SnoN in the fibrotic liver remains unknown. In this study,we will first use the carbon tetrachloride(CCL4) induced liver fibrosis and regression rat model, expression levels of SnoN mRNA and protein were observed by qRT-PCR and Western blots assay.The ubiquition activity and degredation of SnoN in the fibrotic liver were also assessed by an in vitro assay.This study initially explored the regulation and mechanism of SnoN expression in fibrotic liver through detecting liver Arkadia expression and relationship between Arkadia and SnoN by immunohistochemial staining and immunoprecipitation analysis.The rat was also received a mesenteric vein injection of overexpression of Arkadia lentivirus or downexpression of Arkadia lentirus.Then the role of Arkadia in SnoN degradation was further checked in vivo with Western blot analysis and immunohistochenminal.Second,we will use rat primary hepatic stellate cell as an in vitro system,this study explored the molecular basis on the change of SnoN expression by TGF-β1 and explored the effect and mechanism of TGF-β1/Smad signaling pathway on Arkadia degradation SnoN protein by siRNA strategy, Western blot, qRT-PCR and immunofluorescence staining. Our study is of great significance on illustration the mechanism of liver fibrosis.The study findings will help to understand the role and mechnism of Arkadia in regulating SnoN protein expression in liver fibrosis.

SnoN蛋白能阻断TGF-β1/Smad信号，抑制肝纤维化。我们前期研究发现，不同肝纤维化动物模型及病人纤维化肝组织中,SnoN蛋白减少，而SnoN基因转录水平未改变,可能与泛素化降解SnoN蛋白的关键酶Arkadia有关，但其作用和机制尚不清楚。本项目：1.动态观察四氯化碳诱导大鼠肝纤维化形成及逆转中Arkadia与SnoN蛋白的降解关系；并通过慢病毒转染技术，分别敲低、过表达肝组织Arkadia，观察对纤维化肝组织SnoN蛋白降解率、泛素化活性和表达量的影响，明确Arkadia在SnoN蛋白降解中的作用。2.利用原代大鼠肝星状细胞，免疫共沉淀测Arkadia与SnoN及其降解的主要信号分子Smad2、Smad3的相互作用，并分别增加、阻断Smad2、Smad3激活观察其对Arkadia降解SnoN的影响，探讨Arkadia降解SnoN的分子机制。本研究将有利于深入阐明肝纤维化发病机制。

本项目立题宗旨是探讨Arkadia在肝纤维化大鼠SnoN蛋白降解中的作用及分子机制。对于肝纤维化的研究，稳定、可靠的肝纤维化动物模型和体外培养肝星状细胞无疑是强有力研究对象，在此已毋庸赘言。本课题立题之初主要考虑应用四氯化碳诱导肝纤维化大鼠模型和大鼠原代肝星状细胞为对象进行研究。而在研究进展开后，为了更加深入地探讨，我们并不局限于申请书所述的动物模型。研究中使用了胆管结扎诱导肝纤维化大鼠、四氯化碳诱导肝纤维化大鼠、不同分期肝纤维化病人肝穿刺标本、以及大鼠和人的肝星状细胞，对Arkadia在纤维化肝组织SnoN蛋白降解中的作用及分子机制进行了深入探讨。.研究主要从以下几个方面展开：1.分别以胆管结扎诱导肝纤维化大鼠、四氯化碳诱导肝纤维化大鼠、不同分期肝纤维化病人的肝穿刺标本为主要研究对象，秉承从个体到整体的思路，从基因、蛋白表达定位和定量入手，逐个检测不同纤维化模型以及人纤维化肝组织中Arkadia和SnoN的表达情况，全面、详细研究了在体肝组织中Arkadia和SnoN表达变化的关系及其共定位情况；2.分别以大鼠和人肝星状细胞为研究对象，应用慢病毒敲低和过表达技术干预肝星状细胞Arkadia的表达，建立了Arkadia稳定过表达以及敲低的HSC-T6和LX-6细胞；3.应用“2”建立的细胞系，采用免疫细胞化学、QRT-PCR、Western blotting、流式细胞技术、免疫共沉淀等技术，对Arkadia在SnoN蛋白降解中的作用及机制进行探讨，发现肝星状细胞中Arkadia和SnoN蛋白存在共定位表达，敲低Arkadia可以显著抑制FN、Collagen I、 Collagen III、α-SMA及TIMP-1的生成，增强Arkadia的表达可以显著促进FN、α-SMA的表达，对Collagen I、 TIMP-1的促进作用不明显，但Arkadia的促纤维化作用并非通过降解SnoN分子实现的，而是通过增强TGF-β1信号通路而实现的。围绕这些研究内容，目前共发表受本课题资助的论文6篇，另2篇SCI论文投寄中；协助培养博士生1名，硕士生1名。项目的执行取得了预期的成果，超额完成了考核指标，并为以后的研究打下了基础。.在本项目的基础上，相关后续工作设想如下： 1. 肝纤维化中SnoN蛋白降解的分子机制研究;2. 对Arkadia在肝纤维化中的作用进行深入研究。

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