Communication between Kupffer cells and stellate cells

描述（由申请方提供）：星状细胞的活化是肝纤维化发病机制中的关键问题，由受损肝细胞和活化枯否细胞释放的因子介导。了解枯否细胞调节瘢痕组织组分的形成和分泌的机制与潜在的治疗干预非常相关，这构成了本申请的主要目标。我们假设枯否细胞衍生因子/反应性物质在星状细胞纤维化反应中起关键作用。在本申请中，我们提出：1）使用原代大鼠Kupffer细胞和原代星状细胞的体外共培养模型来探索Kupffer细胞对星状细胞胶原I产生的影响。我们将通过进行核体外转录运行试验、启动子研究、mRNA稳定性和北方印迹分析来分析对I型胶原表达的影响是否涉及转录调控。为了研究蛋白质合成是否受到影响，将计算I型胶原合成和降解的速率; 2）为了确定枯否细胞衍生的反应物质是否是星状细胞中I型胶原上调的介体，将测量促氧化剂和亚硝化物质的浓度，并研究与抗氧化剂和抑制剂一起孵育共培养物的效果。鉴定共培养物中反应性物质（例如NADPH氧化酶、黄嘌呤氧化酶、线粒体、细胞色素P450 2 E1和诱导型一氧化氮合酶）来源的研究将涉及添加抑制剂或化学诱导剂。使用来自Kupffer细胞的条件培养基加入到星状细胞中，并用siRNA转染以选择性地沉默一种细胞类型或另一种细胞类型中的反应性物质的来源，将有助于剖析每种细胞类型对在共培养物中观察到的效应的贡献; 3）表征作为代表性多不饱和脂肪酸的花生四烯酸的贡献，对共培养物中胶原I表达的影响，并将其与其他脂肪酸的表达进行比较。机制方法将包括评估脂质过氧化反应和花生四烯酸代谢途径的贡献;和4）评估慢性乙醇喂养对与枯否细胞共培养的星状细胞的胶原蛋白I表达的贡献。将对照星状细胞中I型胶原的基础表达与乙醇处理大鼠的星状细胞进行比较，并评估反应性物质的贡献。机制研究将遵循，以了解如何库普弗细胞调节乙醇处理大鼠的星状细胞的纤维化反应。我们希望这种共培养模型将有助于我们确定枯否细胞对星状细胞纤维化反应的影响，并剖析预防肝脏疾病的潜在治疗靶点。

DESCRIPTION (provided by applicant): Activation of stellate cells, a key issue in the pathogenesis of hepatic fibrosis, is mediated by factors released from damaged hepatocytes and activated Kupffer cells. Understanding the mechanisms by which Kupffer cells modulate the formation and secretion of the components of the scar tissue is of great relevance for potential therapeutic intervention, and this constitutes the major goal of this application. We hypothesize that Kupffer cell-derived factors/reactive species play a critical role in the stellate cell fibrogenic response. In this application we propose: 1) to explore the impact of Kupffer cells on stellate cell collagen I production using an in vitro co-culture model of primary rat Kupffer cells and primary stellate cells. We will analyze whether the effect on collagen I expression involves transcriptional regulation by carrying out nuclear in vitro transcription run-on assays, promoter studies, mRNA stability, and Northern blot analysis. To study if protein synthesis is affected, the rate of collagen I synthesis and degradation will be calculated; 2) to determine if Kupffer cell-derived reactive species are the mediators for collagen I up-regulation in stellate cells, concentration of prooxidant and nitrosative species will be measured and the effect of incubating the co-cultures with antioxidants and inhibitors will be studied. Studies to identify the source of reactive species in the co-culture (e.g. NADPH oxidase, xanthine oxidase, mitochondria, cytochrome P450 2E1, and inducible nitric oxide synthase) will involve addition of inhibitors or chemical inducers. The use of conditioned medium from Kupffer cells added to stellate cells, and transfection with siRNA to selectively silence the sources of reactive species in one cell type or the other will help to dissect the contribution of each cell type to the effects observed in the co-culture; 3) to characterize the contribution of arachidonic acid, as a representative polyunsaturated fatty acid, on collagen I expression in the co-cultures and to compare it to that of other fatty acids. Mechanistic approaches will include evaluating the contribution of lipid peroxidation reactions and the arachidonic acid metabolic pathways; and 4) to assess the contribution of chronic ethanol feeding to collagen I expression by stellate cells in co-culture with Kupffer cells. The basal expression of collagen I in control stellate cells will be compared to that of stellate cells from ethanol-treated rats, and the contribution of reactive species will be assessed. Mechanistic studies will follow to understand how Kupffer cells modulate the fibrogenic response in stellate cells from ethanol-treated rats. We hope that this co-culture model will help us to define the effects of Kupffer cells on the stellate cell fibrogenic response and to dissect potential therapeutic targets for preventing liver disease.