本文選題：轉(zhuǎn)錄調(diào)控 + 啟動子�。� 參考：《第三軍醫(yī)大學》2005年博士論文

【摘要】：革蘭氏陰性菌外膜的主要成分LPS是導致膿毒性休克的首要誘因。一旦細菌被裂解,其胞壁成分LPS進入體液,單核-巨噬細胞系統(tǒng)就成了最先接觸LPS的第一道屏障,并在LPS導致的急性炎癥及后續(xù)免疫應答中扮演重要角色。LPS要激活單核-巨噬細胞,必須先與血漿中的LPS結(jié)合蛋白(LBP)及可溶性CD14結(jié)合形成復合物,進而結(jié)合單核-巨噬細胞膜上的TLR4/MD2,再通過MyD88、IRAK、TRAF6/ECSIT和NIK/IKK一系列的信號事件引發(fā)以NF-κB為主的轉(zhuǎn)錄因子的核轉(zhuǎn)位,從而誘導或抑制一系列的基因轉(zhuǎn)錄表達,導致炎癥反應。U937細胞是目前運用較多的用以研究單核-巨噬細胞分化及功能的細胞模型,而LPS是誘導其產(chǎn)生炎癥介質(zhì)的最有效刺激物。U937細胞上負性共刺激分子B7-H1的表達受到LPS刺激的調(diào)控,我們用1μg/ml終濃度的LPS刺激U937細胞24h后,進行熒光半定量Real-Time PCR和FCS檢測,發(fā)現(xiàn)LPS刺激在mRNA和蛋白兩個環(huán)節(jié)上調(diào)U937上B7-H1基因的組成性表達。 對巨噬細胞系U937細胞而言,既然LPS刺激可以上調(diào)其B7-H1的表達,而轉(zhuǎn)錄調(diào)控事件最終要通過轉(zhuǎn)錄因子的介導利用增強子和協(xié)同激活因子對該基因啟動子區(qū)的結(jié)合而達到增強轉(zhuǎn)錄的目的,并且LPS的下游信號事件主要是通過轉(zhuǎn)錄因子NF-κB的核轉(zhuǎn)位而調(diào)節(jié)基因表達的,那么B7-H1基因的啟動子區(qū)序列有何特征,哪些區(qū)域(即最小核心啟動子)是基本轉(zhuǎn)錄所必需,而哪些區(qū)域又參與活化轉(zhuǎn)錄,它的轉(zhuǎn)錄起始位點(TSS)又定位在哪里,B7-H1基因的啟動子區(qū)有無NF-κB的結(jié)合元件,對B7-H1基因的活化轉(zhuǎn)錄是否很重要,如果不是很重要,那么其啟動子區(qū)的哪些轉(zhuǎn)錄因子結(jié)合元件對LPS刺激后該基因在單核-巨噬細胞的轉(zhuǎn)錄增強效應至關(guān)重要?上述這些問題一直尚未見文獻報告闡述。 為了探索B7-H1的轉(zhuǎn)錄調(diào)控機制尤其是受到LPS刺激后的轉(zhuǎn)錄調(diào)控機制,我們主要進行了如下三個方面的工作: 1.以U937細胞為模型,分析了LPS刺激影響U937細胞上B7-H1表達的調(diào)控模式,通過熒光半定量Real-Time PCR和FCS檢測發(fā)現(xiàn)U937細胞組成性表達B7-H1,LPS刺激后在mRNA和蛋白水平兩個層次增強其表達,說明U937細胞是研究LPS刺激后B7-H1轉(zhuǎn)錄調(diào)控機制的合適細胞模型。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R392

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本文編號：2112989