Egr-1启动子放射诱导调控Bax基因表达的研究

发布时间：2018-03-04 03:00

本文选题：Egr-1启动子　切入点：Bax　出处：《天津医科大学》2005年硕士论文　论文类型：学位论文

【摘要】：当细胞受到辐射时,某些编码转录因子的短暂早期基因被激活。这些被激活的基因包括早期生长反应因子基因家族。这些基因可感受X射线产生的氧自由基的诱导而引起细胞的反应。以前的研究证明Egr-1被辐射诱导是由Egr-1启动子区所含的6个CC(A+Trich)_6GG(CArG)模体介导。我们可以利用这个放射诱导启动子来控制肿瘤组织内的Bax的表达。在Bcl-2家族中,Bax是促凋亡蛋白。Bax以无活性状态存在于细胞浆内。在被不同刺激诱导后,Bax的构象发生改变并转移到线粒体上。在线粒体膜上,它形成低聚物并使线粒体膜形成孔,使细胞色素c和其它细胞毒性因子可以排出。因此,Bax的活性调控就成为细胞是否死亡的关键因素。在Bax与其它Bcl-2成员之间的反应成为了此调控的关键。当Bcl-2过表达时,Bax与Bcl-2形成异源二聚体,细胞继续生存。当Bax过表达时,Bax本身形成同源二聚体,细胞凋亡。我构建一个由Egr-1启动子上游连接Bax cDNA的质粒,它可以在受到辐射后被激活而引起Bax表达,从而提高放射诱导凋亡。第一部分 Egr-1启动子和Bax α cDNA的制备为了构建放射诱导基因表达系统,我从BALB/c小鼠的基因组中提取Egr-1启动子的基因序列。通过RT-PCR,我从乳腺癌细胞中提取Bax α基因序列。第二部分 pEgr-Bax表达载体的构建和鉴定为了研究放射诱导Bax基因表达的可能性,我构建了一个pEgr-Bax表达载体。当它受到辐射时可提高Bax基因的表达。我从pIRES-EGFP质粒中剔除CMV,代之为Egr-1启动子。然后将Bax cDNA连接到pIRES-EGFP质粒的多克隆位点上。通过PCR测定Egr-1启动子和Bax cDNA的序列长度分别为476bp和593bp。通过测序证明碱基序列正确。这个载体构建成功可以为肿瘤治疗提供一种方法。
[Abstract]:When cells are exposed to radiation, Some of the transcriptional factor transcriptional genes are activated. These activated genes include the early growth response factor gene family. These genes can sense the induction of oxygen free radicals produced by X-rays and induce cell response. Previous studies have shown that the radiation-induced Egr-1 is mediated by six CC(A TrichGG CArGG motifs in the Egr-1 promoter region. We can use this radiation-induced promoter to control the expression of Bax in tumor tissues. In the Bcl-2 family, the apoptosis-promoting protein. Bax exists in the cytoplasm in an inactive state. The conformation of Bax is changed and transferred to the mitochondria after being induced by different stimuli. On the mitochondrial membrane, it forms an oligomer and makes the mitochondrial membrane form a pore. So that cytochrome c and other cytotoxic factors can be excreted. Therefore, the activity regulation of Bax becomes the key factor of cell death. The reaction between Bax and other Bcl-2 members becomes the key to this regulation. Bax and Bcl-2 form heterodimer. When Bax was overexpressed, homologous dimer and apoptosis were formed. I constructed a plasmid connected with Bax cDNA upstream by Egr-1 promoter, which can activate Bax expression after irradiation, thus enhancing radiation-induced apoptosis. Part one. Preparation of Egr-1 Promoter and Bax 伪 cDNA. In order to construct a radiation-induced gene expression system, I extracted the gene sequence of Egr-1 promoter from the genome of BALB/c mice and extracted the sequence of Bax 伪 gene from breast cancer cells by RT-PCR. Part two. Construction and Identification of pEgr-Bax expression Vector. In order to study the possibility of radiation-induced Bax gene expression, I constructed a pEgr-Bax expression vector. When it was exposed to radiation, I could improve the expression of Bax gene. I removed CMV from the pIRES-EGFP plasmid and replaced it with Egr-1 promoter. Then I linked Bax cDNA to the polyclonal site of pIRES-EGFP plasmid. The sequence length of Egr-1 promoter and Bax cDNA were 476bp and 593bprespectively. The sequence of Egr-1 promoter and Bax cDNA were proved to be correct by sequencing. The successful construction of the vector could provide a method for tumor therapy.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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