LPS刺激调控U937细胞表达B7-H1机制的实验研究

发布时间：2018-07-10 09:46

本文选题：转录调控 + 启动子　；参考：《第三军医大学》2005年博士论文

【摘要】：革兰氏阴性菌外膜的主要成分LPS是导致脓毒性休克的首要诱因。一旦细菌被裂解,其胞壁成分LPS进入体液,单核-巨噬细胞系统就成了最先接触LPS的第一道屏障,并在LPS导致的急性炎症及后续免疫应答中扮演重要角色。LPS要激活单核-巨噬细胞,必须先与血浆中的LPS结合蛋白(LBP)及可溶性CD14结合形成复合物,进而结合单核-巨噬细胞膜上的TLR4/MD2,再通过MyD88、IRAK、TRAF6/ECSIT和NIK/IKK一系列的信号事件引发以NF-κB为主的转录因子的核转位,从而诱导或抑制一系列的基因转录表达,导致炎症反应。U937细胞是目前运用较多的用以研究单核-巨噬细胞分化及功能的细胞模型,而LPS是诱导其产生炎症介质的最有效刺激物。U937细胞上负性共刺激分子B7-H1的表达受到LPS刺激的调控,我们用1μg/ml终浓度的LPS刺激U937细胞24h后,进行荧光半定量Real-Time PCR和FCS检测,发现LPS刺激在mRNA和蛋白两个环节上调U937上B7-H1基因的组成性表达。对巨噬细胞系U937细胞而言,既然LPS刺激可以上调其B7-H1的表达,而转录调控事件最终要通过转录因子的介导利用增强子和协同激活因子对该基因启动子区的结合而达到增强转录的目的,并且LPS的下游信号事件主要是通过转录因子NF-κB的核转位而调节基因表达的,那么B7-H1基因的启动子区序列有何特征,哪些区域(即最小核心启动子)是基本转录所必需,而哪些区域又参与活化转录,它的转录起始位点(TSS)又定位在哪里,B7-H1基因的启动子区有无NF-κB的结合元件,对B7-H1基因的活化转录是否很重要,如果不是很重要,那么其启动子区的哪些转录因子结合元件对LPS刺激后该基因在单核-巨噬细胞的转录增强效应至关重要?上述这些问题一直尚未见文献报告阐述。为了探索B7-H1的转录调控机制尤其是受到LPS刺激后的转录调控机制,我们主要进行了如下三个方面的工作: 1.以U937细胞为模型,分析了LPS刺激影响U937细胞上B7-H1表达的调控模式,通过荧光半定量Real-Time PCR和FCS检测发现U937细胞组成性表达B7-H1,LPS刺激后在mRNA和蛋白水平两个层次增强其表达,说明U937细胞是研究LPS刺激后B7-H1转录调控机制的合适细胞模型。
[Abstract]:LPS, the main component of the outer membrane of Gram-negative bacteria, is the primary cause of septic shock. Once the bacteria are lysed, the cell wall component LPS enters the body fluid, and the monocyte-macrophage system becomes the first barrier against LPS. In order to activate mononuclear macrophages, LPS must bind to LPS-binding protein (LBP) and soluble CD14 in plasma to form a complex. And then combined with TLR4 / MD2 on the monocyte-macrophage cell membrane, and then through a series of signal events, MyD88-IRAKN TRAF6 / ECSIT and Nike / IKK, trigger the nuclear translocation of NF- 魏 B-dominated transcription factors, thus inducing or inhibiting a series of transcriptional expressions of genes. Inflammatory response. U937 cells are the most widely used cell models to study the differentiation and function of mononuclear macrophages. The expression of B7-H1, a negative costimulatory molecule on U937 cells, was regulated by LPS. We stimulated U937 cells with 1 渭 g/ml final concentration of LPS-induced U937 cells for 24 hours, and then detected the expression of B7-H1 in U937 cells by fluorescence semi-quantitative Real-Time PCR and FCS detection. It was found that LPS stimulated U937 up-regulated the constitutive expression of B7-H1 gene in both mRNA and protein. In the case of macrophage cell line U937, since LPS could up-regulate the expression of B7-H1, However, the transcriptional regulation events are ultimately enhanced by the binding of enhancers and co-activators to the promoter region of the gene, which is mediated by transcription factors. The downstream signal events of LPS regulate gene expression mainly through nuclear translocation of transcription factor NF- 魏 B. so what are the characteristics of the promoter region of B7-H1 gene and which regions (that is, the smallest core promoter) are essential for basic transcription. Which regions are involved in activated transcription, and where its transcription initiation site (TSS) is located with or without NF- 魏 B binding elements in the promoter region of the B7-H1 gene, is important to the activation of B7-H1 gene transcription, if not very important. Which transcription factor binding elements in its promoter region are critical to the transcriptional enhancement of the gene in monocyte-macrophages after LPS stimulation? These problems have not been described in the literature report. In order to explore the transcriptional regulation mechanism of B7-H1, especially the transcriptional regulation mechanism stimulated by LPS, we mainly carried out the following three aspects: 1. The regulation of B7-H1 expression in U937 cells stimulated by LPS was analyzed. By fluorescence semi-quantitative Real-Time PCR and FCS analysis, it was found that the expression of B7-H1 in U937 cells was enhanced at both mRNA and protein levels after LPS stimulation. These results suggest that U937 cells are suitable for studying the transcriptional regulation mechanism of B7-H1 stimulated by LPS.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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