论文标题:Egr-1启动子放射诱导调控Bax基因表达的研究
A Study on Regulating Bax Expression by Radiation-inducible Egr-1 Promoter
论文作者 张春智
论文导师 王平；牛瑞芳,论文学位 硕士,论文专业 肿瘤学
论文单位 天津医科大学,点击次数 146,论文页数 49页File Size3867k
2005-05-01论文网 http://www.lw23.com/lunwen_2361237/ Egr-1启动子;Bax;放射治疗;凋亡;基因治疗;表达载体
Egr-1 Promoter ;Bax ;radiotherapy ;apoptosis ;gene therapy; expression vector
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。
The cellar exposure to ionizing radiation is associated with transcriptional activation of certain immediate early genes that encode transcription factors. These genes include members of the early growth response gene families. The induction of these genes following X-irradiation may represent cellular responses to oxidative stress. Previous studies have demonstrated that induction of Egr-1 gene transcription is mediated by activation of CC(A+T rich)_6 GG (CArG) motifs in the Egr-1 Promoter(5,6). We took advantage of a radiation-inducible promoter in order to control Bax gene expression within the tumor mass.The Bcl-2 family member Bax is an apoptosis-promoting protein that normally resides in an inactive state within the cytoplasm of healthy cells. Upon induction of apoptosis by diverse stimuli, Bax undergoes a conformational change and translocates to mitochondria, where it oligomerizes and forms pores that allow the release of cytochrome c and other cytotoxic foctors. Regulation of Bax activity is thus a critical determinant of cell fate. Protein-protein interaction between Bax and other Bcl-2 family member are key to this regulation. When Bax heterodimerizes with Bcl-2 overexpressed , the cell continue living. When Bax homodimerizas with itself overexpressed, the cell will apoptosis.I construct a containing a Egr-1 promoter.upstream to a cDNA encoding a Bax protein is transcriptionally activated within the irradiated field to enhance radiation-inducible apoptosis.Part ⅠPrepare Egr-1 Promoter and Bax α cDNAIn order to construct a radiation-inducible gene expression system, the promoter sequence of radiation-inducible Egr-1 gene was amplified from genomic DNA of BALB/c mouse with PCR method. By RT-PCR, Bax α was amplified from breast-cancer cell.Part ⅡThe Construction and Identification of pEgr-Bax Expression Vector For the purpose of studying the feasibility of radiation-inducible Bax expression, I construct a pEgr-Bax which enhance Bax expression by ionizing radiation. I delete CMV from pIRES-EGFP .I substitute Egr-1 promoter for CMV. Bax cDNA was inserted into polyclonia site of pIRES-EGFP . The identification of pEgr-Bax by PCR yielded the results of 476bp and 593bp segments respectively. DNA sequencing demonstrated Egr-1 promoter and Bax cDNA were inserted into pIRES-EGFP and the sequence was proved correct. The success of construction of pEgr-Bax make it possible to therapy cancer with pEgr-Bax which induce Bax expression by ionizing radiation.
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