LPS诱导GCH1基因表达上调的机制研究

发布时间：2018-04-30 20:41

本文选题：GCH1 + 内含子　；参考：《清华大学》2010年硕士论文

【摘要】：GCH1基因编码的GTP环化水解酶是体内合成5,6,7,8-四羟基生物蝶呤(tetrahydrobiopterin,BH4)的限速酶。BH4是芳香族氨基酸羟化酶和一氧化氮合酶的必需辅酶,BH4缺陷导致儿茶酚胺合成不足、苯丙氨酸代谢障碍以及一氧化氮合酶去偶联,与多种病理现象密切相关,如血管功能紊乱、运动功能障碍等。通过诱导GCH1基因的表达,可以促使BH4的合成增加。因此,研究GCH1基因的转录调控机制具有重要的意义。GCH1在多种组织和细胞中表达,可以被多种刺激因子诱导。本研究在小鼠巨噬细胞RAW264.7中,用脂多糖(LPS)诱导GCH1表达上调。将插入人类GCH1基因启动子的报告质粒转入RAW264.7细胞,用双荧光素酶报告基因的检测方法,发现内含子1对于LPS激活GCH1启动子的转录是必要的。通过不同物种间GCH1基因内含子1部分的序列比对,发现有332 bp的高度保守序列。用报告基因的检测方法,发现这332 bp区域作为增强子,负责响应LPS的刺激。LPS刺激RAW264.7细胞激活GCH1转录的机制可能涉及MEK1/2、NF-κB信号通路,而与PI3K、MAPK、JNK无关。此外,在人脐带内皮细胞HUVEC细胞中,TNFα/IFNγ可诱导GCH1的转录激活,并且此332 bp增强子同样为响应刺激的关键元件,此过程也涉及到NF-κB信号通路。总之,本研究发现在GCH1基因的内含子1中,有332 bp的增强子元件,负责在LPS或细胞因子刺激下诱导GCH1的转录激活,对于进一步研究GCH1基因的转录调控机制提供了新的线索,并为治疗BH4缺陷相关的疾病提供了理论依据。
[Abstract]:The GTP cyclase encoded by GCH1 gene is the rate-limiting enzyme. BH4 is an essential coenzyme of aromatic amino acid hydroxylase and nitric oxide synthase, which leads to the deficiency of catecholamine synthesis. Phenylalanine metabolic disorder and nitric oxide synthase decoupling are closely related to many pathological phenomena, such as vascular dysfunction, motor dysfunction and so on. By inducing the expression of GCH1 gene, the synthesis of BH4 can be increased. Therefore, it is important to study the transcriptional regulation mechanism of GCH1 gene. GCH1 is expressed in various tissues and cells and can be induced by multiple stimulators. In this study, lipopolysaccharide (LPS) was used to induce up-regulation of GCH1 expression in mouse macrophages (RAW264.7). The reporter plasmid inserted into the promoter of human GCH1 gene was transferred into RAW264.7 cells and detected by double luciferase. It was found that intron 1 was necessary for LPS to activate the transcription of GCH1 promoter. According to the sequence alignment of intron 1 of GCH1 gene among different species, a highly conserved 332bp sequence was found. Using reporter gene detection method, we found that the 332bp region acted as an enhancer, which was responsible for the activation of GCH1 transcription by RAW264.7 cells in response to LPS stimulation. The mechanism may be involved in MEK1 / 2 NF- 魏 B signaling pathway, but not in PI3KmMAPK- JNK. In addition, TNF- 伪 / IFN- 纬 can induce transcriptional activation of GCH1 in human umbilical cord endothelial cell (HUVEC) cells, and this 332bp enhancer is also a key element in response to stimulation, which also involves NF- 魏 B signaling pathway. In conclusion, we found that there are 332 BP enhancer elements in intron 1 of GCH1 gene, which is responsible for inducing the transcriptional activation of GCH1 under the stimulation of LPS or cytokines, which provides a new clue for the further study of the transcriptional regulation mechanism of GCH1 gene. It provides a theoretical basis for the treatment of BH4 defect related diseases.
【学位授予单位】：清华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

【共引文献】