本文選題：Egr-1啟動子　切入點：Bax　出處：《天津醫(yī)科大學》2005年碩士論文　論文類型：學位論文

【摘要】：當細胞受到輻射時,某些編碼轉錄因子的短暫早期基因被激活。這些被激活的基因包括早期生長反應因子基因家族。這些基因可感受X射線產(chǎn)生的氧自由基的誘導而引起細胞的反應。以前的研究證明Egr-1被輻射誘導是由Egr-1啟動子區(qū)所含的6個CC(A+Trich)_6GG(CArG)模體介導。我們可以利用這個放射誘導啟動子來控制腫瘤組織內的Bax的表達。 在Bcl-2家族中,Bax是促凋亡蛋白。Bax以無活性狀態(tài)存在于細胞漿內。在被不同刺激誘導后,Bax的構象發(fā)生改變并轉移到線粒體上。在線粒體膜上,它形成低聚物并使線粒體膜形成孔,使細胞色素c和其它細胞毒性因子可以排出。因此,Bax的活性調控就成為細胞是否死亡的關鍵因素。在Bax與其它Bcl-2成員之間的反應成為了此調控的關鍵。當Bcl-2過表達時,Bax與Bcl-2形成異源二聚體,細胞繼續(xù)生存。當Bax過表達時,Bax本身形成同源二聚體,細胞凋亡。 我構建一個由Egr-1啟動子上游連接Bax cDNA的質粒,它可以在受到輻射后被激活而引起B(yǎng)ax表達,從而提高放射誘導凋亡。 第一部分 Egr-1啟動子和Bax α cDNA的制備 為了構建放射誘導基因表達系統(tǒng),我從BALB/c小鼠的基因組中提取Egr-1啟動子的基因序列。通過RT-PCR,我從乳腺癌細胞中提取Bax α基因序列。 第二部分 pEgr-Bax表達載體的構建和鑒定 為了研究放射誘導Bax基因表達的可能性,我構建了一個pEgr-Bax表達載體。當它受到輻射時可提高Bax基因的表達。我從pIRES-EGFP質粒中剔除CMV,代之為Egr-1啟動子。然后將Bax cDNA連接到pIRES-EGFP質粒的多克隆位點上。通過PCR測定Egr-1啟動子和Bax cDNA的序列長度分別為476bp和593bp。通過測序證明堿基序列正確。這個載體構建成功可以為腫瘤治療提供一種方法。
[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 偽 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 偽 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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346

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本文編號：1563815