【作者】 肖静；

【导师】 蔡国平；

【作者基本信息】 清华大学 ， 生物学， 2010， 博士

【摘要】 干扰素诱导蛋白p204是干扰素诱导蛋白p200家族的成员之一。近年来的研究表明,p204参与了多种中胚层来源组织的发生及发育过程。其中p204参与调控的中胚层组织包括:骨,肌肉以及软骨等。然而,至今尚未有关于p204参与调控脂肪组织生成的相关报道。我们的预实验结果显示,p204在3T3-L1前脂肪细胞中表达,并且其表达量随着脂肪分化的进程而升高。据此,我们推测p204可能参与了脂肪分化的调控。本课题的主要目的就是探究p204在脂肪分化过程中的功能及相关机制。本研究结果显示,在脂肪分化早期p204表达短时增高而后恢复。并且p204蛋白从细胞质转移到了细胞核中。为进一步研究p204在脂肪分化过程中的功能,我们分别应用RNA干扰技术构建p204低表达模型以及通过转染p204重组质粒构建p204过表达模型。研究结果显示,抑制p204的表达可显著的抑制脂肪生成,而过表达p204可以促进脂肪生成。与此同时,一些脂肪分化标记分子,包括PPARγ(过氧化物酶体增生物激活受体γ), C/EBPα(CCAAT/增强子结合蛋白α),LPL(脂蛋白脂肪酶)及adipsin(降脂蛋白)的基因表达量在p204低表达模型中减低而在p204过表达模型中升高。应用免疫共沉淀技术与免疫荧光技术,我们发现p204与C/EBPδ存在蛋白质相互作用并且两者共定位于细胞核中。进一步研究表明,应用RNA干扰技术抑制p204在3T3-L1细胞系中的表达,可导致p204与C/EBPδ形成的蛋白质复合体遭到破坏。而这可能是导致C/EBPδ蛋白与PPARγ基因启动子元件形成的蛋白质基因复合物发生解离从而抑制PPARγ基因转录活性的原因之一。综上所述,我们的研究结果表明p204在脂肪分化早期的短时上调在脂肪的发生中是不可或缺的。因为如果人为干扰了p204在脂肪分化早期的表达模式将会导致脂肪生成的不可逆损伤。更多还原

【Abstract】 A member of the interferon-inducible p200 family of proteins, p204, has recently been reported to function in the development of many mesoderm-derived tissues, such as bone, muscle, and cartilage. However, no published study has yet investigated the role of p204 in adipogenesis. Our preliminary experiments showed that p204 can be found in 3T3-L1 preadipocytes and its expression was up-regulated in a differentiation-dependent manner. As such, we hypothesized that p204 is associated with adipogenesis and focused on the influence of p204 on adipogenesis.In the present study we investigated the transient elevated expression and cytoplasm-to-nucleus translocation of p204 in the early stage of adipogenesis. To determine the effect of p204 on adipogenesis, p204-siRNA and expression vector were produced for p204 suppression and overexpression, respectively. The knockdown of p204 resulted in a significantly depressed adipocyte differentiation, whereas p204 overexpression promoted adipocyte differentiation. The mRNA expression of adipogenic markers, such as peroxisome-proliferator-activated receptor (PPAR)γ, CCAAT/enhancer-binding-protein (C/EBP)α, lipoprotein lipase (LPL), and adipsin, was decreased by p204 suppression and increased by p204 overexpression. A co-immunoprecipitation assay coupled with an indirect immunofluorescence assay also indicated that p204 interacted and co-localized with C/EBPδin the nucleus. Furthermore, the knockdown of p204 disrupted the interaction between p204 and C/EBPδand partially suppressed the PPARγtranscriptional activity by dissociating C/EBPδwith the PPARγpromoter element.Collectively, our data indicate that the transient expression of p204 in the early stage is indispensable for adipocyte differentiation. Disruption of p204 expression patterns at this stage leads to irreversible damage in fat formation.更多还原