發(fā)布時間：2018-03-09 00:05

  本文選題：HPD　切入點：肺癌　出處：《安徽醫(yī)科大學》2015年碩士論文　論文類型：學位論文

【摘要】：4-羥苯丙酮酸二加氧酶(4-Hydroxyphenylpyruvate Dioxygenase, HPD),是一種參與酪氨酸代謝途徑的代謝酶,主要在肝臟和腎臟表達,在其他組織呈現(xiàn)極低水平表達或不表達。前期研究表明HPD的表達異常及突變與可導(dǎo)致酪氨酸血癥,此外在肝損傷過程中也發(fā)現(xiàn)HPD表達上調(diào),提示HPD可能是肝損傷的重要標志物。我們實驗室前期利用組織芯片、qRT-PCR和Western-Blotting等技術(shù)意外發(fā)現(xiàn)HPD在肺癌組織及細胞中高表達,而在癌旁組織中表達水平較低。但關(guān)于HPD在肺癌中異常表達的分子機制及其對肺癌細胞行為影響的研究,目前尚無任何報道。為進一步確證HPD與肺癌的關(guān)系,本課題進行了以下兩方面的研究：第一部分：檢測敲低HPD表達對化療藥物誘導(dǎo)的肺癌細胞系A(chǔ)549凋亡的影響。首先將前期設(shè)計的HPD RNA干涉片段克隆到慢病毒表達載體上,然后通過瞬轉(zhuǎn)篩選得到干涉效果較明顯的干涉載體,并包裝病毒顆粒,感染肺癌細胞系,經(jīng)嘌呤篩選得到敲低HPD的A549穩(wěn)定細胞株及對照穩(wěn)定株,用抗腫瘤藥物Gefitinib處理以上各種穩(wěn)定株,檢測其凋亡情況。第二部分：構(gòu)建了HPD基因啟動子熒光素酶報告基因載體并進行了轉(zhuǎn)錄活性分析,初步探索了HPD的轉(zhuǎn)錄調(diào)控特點。首先利用UCSC在線數(shù)據(jù)庫分析人HPD基因組結(jié)構(gòu),并獲得其5'端上游啟動子區(qū)域-2000 bp-+39bp的DNA)序列。以人基因組DNA為模板,克隆到pGL3-Basic載體上,設(shè)計不同引物,進一步構(gòu)建系列缺失體,最后通過熒光素酶報告基因?qū)嶒灧治銎滢D(zhuǎn)錄活性。第三部分：通過生物信息學數(shù)據(jù)庫預(yù)測HPD基因啟動子序列上潛在的轉(zhuǎn)錄因子結(jié)合位點,并結(jié)合文獻報道初步探索了HNF1α (Hepatocyte nuclear factor1α),HNF4a (Hepatocyte nuclear factor la)和Nrf2 (Nuclear factor, erythroid 2-like 2)等轉(zhuǎn)錄因子對HPD基因的轉(zhuǎn)錄調(diào)控作用。本論文發(fā)現(xiàn)敲低HPD表達增強了肺癌細胞A549對化療藥物Gefitinib的敏感性,構(gòu)建了HPD啟動子報告基因載體,明確了其在肺癌中特異表達的關(guān)鍵結(jié)構(gòu)域,并發(fā)現(xiàn)HNF1α和Nrf2對HPD基因表達的調(diào)控作用。本論文初步揭示了HPD在肺癌中異常高表達的調(diào)控機制,為深入研究HPD的基因表達調(diào)控奠定了基礎(chǔ)。
[Abstract]:4-Hydroxyphenylpyruvate Dioxygenase (HPDD), a metabolic enzyme involved in tyrosine metabolism, is expressed mainly in liver and kidney. Previous studies have shown that abnormal expression and mutation of HPD may lead to tyrosinemia, and that the expression of HPD is up-regulated during liver injury. The results suggest that HPD may be an important marker of liver injury. In our laboratory, we found that HPD was overexpressed in lung cancer tissues and cells by using tissue microarray qRT-PCR and Western-Blotting techniques. However, there is no report on the molecular mechanism of abnormal expression of HPD in lung cancer and its effect on the behavior of lung cancer cells. In order to further confirm the relationship between HPD and lung cancer, In this paper, the following two aspects were studied: the first part: the effect of low HPD expression on apoptosis of lung cancer cell line A549 induced by chemotherapeutic drugs was detected. Firstly, the previously designed HPD RNA interference fragment was cloned into lentivirus expression vector. Then the interference vector with obvious interference effect was obtained by transient screening, and the virus particles were packaged and infected with lung cancer cell line. The stable cell line A549 with low HPD knockout and the control stable cell line were obtained by purine screening. The stable strains were treated with anti-tumor drug Gefitinib and their apoptosis was detected. Part two: the luciferase reporter gene vector of HPD gene promoter was constructed and the transcriptional activity was analyzed. The transcriptional regulatory characteristics of HPD were preliminarily explored. Firstly, the genomic structure of human HPD was analyzed by UCSC online database, and the 5'end upstream promoter region -2000bp-39bp DNA sequence was obtained. Using human genomic DNA as template, it was cloned into pGL3-Basic vector. Different primers were designed to construct a series of deletions. Finally, the transcriptional activity of luciferase reporter gene was analyzed by experiments. Part three: the potential transcription factor binding sites on the promoter sequence of HPD gene were predicted by bioinformatics database. The transcriptional regulation of HPD gene by transcription factors such as HNF1 偽 nuclear nuclear factor1 偽 HNF4a Hepatocyte nuclear factor la. and Nrf2 nuclear factor, erythroid 2-like 2) was preliminarily explored. It was found that the low expression of HPD enhanced the sensitivity of lung cancer cell A549 to the chemotherapeutic drug Gefitinib. The HPD promoter reporter gene vector was constructed, the key domain of its specific expression in lung cancer was identified, and the regulatory effect of HNF1 偽 and Nrf2 on the expression of HPD gene was found. In this paper, the regulatory mechanism of abnormal overexpression of HPD in lung cancer was revealed. It lays a foundation for further study on the regulation of HPD gene expression.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R734.2

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本文編號：1586117