兰州大学机构库 >生命科学学院
CRP -286位SNP影响表达的机制研究
Alternative TitleThe effect of -286 site SNP on the expression of CRP
刘洋
Thesis Advisor吉尚戎
2017-05-01
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
KeywordC-反应蛋白 DNA甲基化 DNA去甲基化 -286位点
Abstract

C-反应蛋白(CRP)是一种典型的急性期蛋白,主要在肝脏中表达。在机体炎症反应或组织受损时,CRP的含量会在短时间内迅速升高。已有的工作证实,CRP急性期转录水平的升高和组织特异性表达是通过启动子近端转录因子调控来实现的。然而有趣的是,CRP基因启动子区在人和小鼠间是高度保守的,但只有人类CRP具有典型的的急性期。这种现象不能单纯通过转录因子的调控来解释,暗示存在其他的途径调控CRP急性期的转录水平。DNA甲基化是基因转录的重要调控机制之一,我们推测CRP的组织特异性和急性期表达可能受DNA甲基化调控。因此,通过检测兔不同组织和人肝源细胞Hep3B CRP基因的甲基化水平,我们发现存在明显差异,且Hep3B 细胞中基线期至急性期存在快速去甲基化的现象。同时,利用荧光定量PCR检测,我们发现Hep3B细胞急性期时CRP转录水平显著升高,而兔肝脏组织中的转录水平也较其他组织有显著升高。由此我们认为DNA甲基化与CRP的转录负相关,即当甲基化程度低时,CRP转录水平高。为了探究这种快速的甲基化变化是如何实现的,我们对去甲基化水平进行检测,看是否存在5-羟甲基胞嘧啶(5hmC)的快速修饰过程,来实现这一变化。结果显示在Hep3B细胞启动子区,CRP基因急性期几乎检测不到5hmC,在基线期可以检测到一定量的5hmC。而在CRP基因区,急性期和基线期几乎都检测不到5hmC的存在。我们认为,5hmC处于一种预激发状态,一旦刺激产生应激反应,5hmC会全部快速去甲基化,所以急性期几乎不存在5hmC。而基线期的5hmC可以迅速响应去甲基化过程,这有利于甲基化的快速变化。根据我们的检测及文献报道,CRP基因启动子区-286位点可以显著影响血浆中CRP的水平,荧光素酶报告基因的实验也印证了这一现象。然而通过软件分析,发现该位点并不存在转录因子的结合,所以这一现象很难通过转录因子解释,而该位点恰好包含一个CpG。故可能是DNA甲基化的状态,调节了CRP的表达。

Other Abstract

As typical acute phase protein, C-reaction protein (CRP) was mainly expressed in hepatocytes. The level of CRP exaggerated very soon when body was under inflammation or tissue injury. Previous work indicated that the tissue specific and acute phase expression of CRP were regulated by transcription factors within proximal promoter. Interestingly, the promoter of CRP was highly conserved between human and rodent, while it presented typical acute phase only in human. Transcription factor regulation could not explain that solely, implicating there existing other modulation pathways. Being one of the most pivotal mechanisms regulating transcription, DNA methylation might play an important role in the tissue specific and acute phase expression. The level of methylation and demethylation were determined in cell lines Hep3B and rabbit hepatocytes. Results revealed that the DNA methylation within CRP promoter was lower either in Hep3B or rabbit hepatocyte consistent with CRP mRNA expression. Moreover, CRP expression during acute phase may depend on rapid demethylation. To discover the mechanisms of rapid demethylation, the level of DNA 5-hydroxymethyl cytosine (5hmC) was examined. In Hep3B cells, rarely any 5hmC in promoter during acute phase was detected while it presented very low level on baseline. In the gene body, 5hmC was detected neither during acute phase nor baseline. Assuming that 5hmC was under pre-excitation state, it underwent rapid demethylation thoroughly when stimulation occurred. Our previous work and other researches proved that -286 position rs3091244 on the promoter of CRP gene had huge impact on CRP level in plasma, which was also supported by luciferase report gene test. To explore the mechanisms whereby rs3091244 regulated CRP expression, we analyzed the transcription factors binding to this site. It indicated that there is no binding site whether this site was occupied by A or C. It implicated that this site was regulated by DNA methylation.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222027
Collection生命科学学院
Recommended Citation
GB/T 7714
刘洋. CRP -286位SNP影响表达的机制研究[D]. 兰州. 兰州大学,2017.
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