應用RDPCR檢測職業(yè)砷暴露工人P53基因的損傷
發(fā)布時間：2018-11-15 14:32
【摘要】：依賴隨機化末端連結物PCR (Randomized terminal linker-dependent PCR, RDPCR)是以連接介導PCR (Ligation-mediated PCR, LMPCR)為基礎建立起來的,可以定位檢測任何類型DNA損傷的一種分子生物學方法。目前該法已成功應用于體外細胞染毒實驗和體內動物實驗,來檢測p53基因、ras基因的損傷,但仍未有應用于人群流行病學研究的報道。Southern雜交技術能將PCR過程中產生的特異性片段檢測出來,具有快速、靈敏、準確等優(yōu)點,被廣泛的應用于鑒別DNA分子中的特異堿基序列等各項研究中。 無機砷進入機體會導致細胞DNA損傷,這些損傷部位也包括p53基因,而p53基因的損傷可能是砷遺傳毒性和致癌作用分子機制的關鍵所在。檢測職業(yè)砷暴露人群p53基因的特定損傷位點,對于研究砷的致癌效應和遺傳毒性具有重要意義。 目的：1)將RDPCR方法結合Southern blot雜交技術應用于人群流行病學研究,為RDPCR的推廣和應用積累資料；2)定性檢測p53基因第五外顯子損傷,驗證前期研究結果,并為進一步精確定位損傷位點奠定基礎。 方法：1)離心柱法提取外周血液樣本基因組DNA,雙引物擴增p53基因第五外顯子雙鏈并純化；2)制作單鏈探針,并檢測其可靠性；3)以純化的p53基因第五外顯子為模版進行RDPCR擴增,終產物凝膠電泳后進行Southern blot雜交,鑒定基因損傷。 結果：1)經檢測,根據瓊脂糖凝膠電泳及與雙鏈外顯子雜交結果證實探針制備成功并可靠；2)完成檢測的68例暴露樣本和7例對照樣本中,30例暴露樣本明確檢測到目標基因的損傷,對照樣本均未測到損傷,暴露組損傷率為44.1%,對照組損傷率為0,暴露組損傷率顯著高于對照組(P0.05)；3)各損傷條帶的凝膠電泳和雜交條帶出現(xiàn)位置大致相同,略滯后于外顯子雙鏈位置。 結論：本研究從定性角度進一步證實了前期采用實時熒光定量方法檢測的兩組樣本p53基因第五外顯子損傷情況；各損傷樣本經檢測所得的雜交條帶位置基本一致,初步判斷砷致p53基因第五外顯子損傷具有特定位點,進一步可采用克隆測序等技術確認具體損傷位點；本研究成功將該技術應用于人群分子流行病學研究,進一步擴展了該技術的應用范圍。
[Abstract]:The random-dependent terminal junction (PCR (Randomized terminal linker-dependent PCR, RDPCR) is based on ligation-mediated PCR (Ligation-mediated PCR, LMPCR) and can be used as a molecular biological method for detecting any type of DNA damage. At present, this method has been successfully used in the cell toxicity test in vitro and in vivo animal experiments to detect the damage of p53 gene and ras gene. However, it has not been reported that Southern hybridization can detect the specific fragments produced in the process of PCR, which has the advantages of fast, sensitive, accurate and so on. It has been widely used in the identification of specific base sequences in DNA molecules. Inorganic arsenic can cause DNA damage in cells, including p53 gene. The damage of p53 gene may be the key to the molecular mechanism of arsenic genotoxicity and carcinogenesis. The detection of specific damage sites of p53 gene in occupational arsenic exposed population is of great significance in studying the carcinogenic effect and genetic toxicity of arsenic. Objective: 1) to apply RDPCR method combined with Southern blot hybridization technique in population epidemiological study, and to accumulate data for the popularization and application of RDPCR. 2) qualitative detection of p53 gene exon 5 damage, verification of previous research results, and lay a foundation for further accurate location of damage sites. Methods: 1) the double strand of p53 gene exon 5 was amplified and purified by double primer amplification of genomic DNA, from peripheral blood samples extracted by centrifugal column method, 2) the single strand probe was made and its reliability was tested. 3) the purified p53 gene exon 5 was used as template for RDPCR amplification, and the final product was analyzed by Southern blot hybridization after gel electrophoresis to identify the gene damage. Results: 1) according to the results of agarose gel electrophoresis and double strand exon hybridization, the probe was successfully prepared and reliable. 2) among the 68 exposed samples and 7 control samples, 30 exposed samples detected the damage of the target gene. The damage rate of the exposure group was 44.1%, and the damage rate of the control group was 0. The injury rate of the exposed group was significantly higher than that of the control group (P0.05). 3) the positions of gel electrophoresis and hybridization bands of the damaged bands were approximately the same, which lagged behind the position of exon double strand. Conclusion: this study further confirmed the damage of p53 gene exon 5 in two groups of samples detected by real-time fluorescence quantitative method from qualitative point of view. The location of the hybridization bands was basically the same among the damage samples. It was preliminarily determined that arsenic induced p53 gene exon 5 damage had a specific site, and further specific damage sites could be identified by cloning and sequencing techniques. This study successfully applied this technique to the study of population molecular epidemiology, and further expanded the scope of application of this technique.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R135.1
本文編號：2333572
[Abstract]:The random-dependent terminal junction (PCR (Randomized terminal linker-dependent PCR, RDPCR) is based on ligation-mediated PCR (Ligation-mediated PCR, LMPCR) and can be used as a molecular biological method for detecting any type of DNA damage. At present, this method has been successfully used in the cell toxicity test in vitro and in vivo animal experiments to detect the damage of p53 gene and ras gene. However, it has not been reported that Southern hybridization can detect the specific fragments produced in the process of PCR, which has the advantages of fast, sensitive, accurate and so on. It has been widely used in the identification of specific base sequences in DNA molecules. Inorganic arsenic can cause DNA damage in cells, including p53 gene. The damage of p53 gene may be the key to the molecular mechanism of arsenic genotoxicity and carcinogenesis. The detection of specific damage sites of p53 gene in occupational arsenic exposed population is of great significance in studying the carcinogenic effect and genetic toxicity of arsenic. Objective: 1) to apply RDPCR method combined with Southern blot hybridization technique in population epidemiological study, and to accumulate data for the popularization and application of RDPCR. 2) qualitative detection of p53 gene exon 5 damage, verification of previous research results, and lay a foundation for further accurate location of damage sites. Methods: 1) the double strand of p53 gene exon 5 was amplified and purified by double primer amplification of genomic DNA, from peripheral blood samples extracted by centrifugal column method, 2) the single strand probe was made and its reliability was tested. 3) the purified p53 gene exon 5 was used as template for RDPCR amplification, and the final product was analyzed by Southern blot hybridization after gel electrophoresis to identify the gene damage. Results: 1) according to the results of agarose gel electrophoresis and double strand exon hybridization, the probe was successfully prepared and reliable. 2) among the 68 exposed samples and 7 control samples, 30 exposed samples detected the damage of the target gene. The damage rate of the exposure group was 44.1%, and the damage rate of the control group was 0. The injury rate of the exposed group was significantly higher than that of the control group (P0.05). 3) the positions of gel electrophoresis and hybridization bands of the damaged bands were approximately the same, which lagged behind the position of exon double strand. Conclusion: this study further confirmed the damage of p53 gene exon 5 in two groups of samples detected by real-time fluorescence quantitative method from qualitative point of view. The location of the hybridization bands was basically the same among the damage samples. It was preliminarily determined that arsenic induced p53 gene exon 5 damage had a specific site, and further specific damage sites could be identified by cloning and sequencing techniques. This study successfully applied this technique to the study of population molecular epidemiology, and further expanded the scope of application of this technique.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R135.1
【參考文獻】
相關期刊論文 前10條
1 楊琳;羅建民;溫樹鵬;劉小軍;杜行嚴;李燕;;三氧化二砷對淋巴瘤T2細胞株SHP-1基因的去甲基化作用[J];癌癥;2009年03期
2 龔小衛(wèi);魏潔;李煜生;程蔚蔚;王旭;鄧鵬;姜勇;;p53基因敲除對細胞遷移的影響[J];南方醫(yī)科大學學報;2007年08期
3 趙煥英;包金風;;實時熒光定量PCR技術的原理及其應用研究進展[J];中國組織化學與細胞化學雜志;2007年04期
4 王茜;砷的致癌性與p53基因[J];國外醫(yī)學(衛(wèi)生學分冊);2002年04期
5 康玲,姚華,肖碧玉,王國荃;新疆地方性砷中毒患者外周血淋巴細胞微核率及染色體畸變研究[J];環(huán)境與健康雜志;2001年04期
6 王惠惠;徐苑苑;李冰;李昕;薛鵬;賀淼;王毅;孫貴范;;亞砷酸鈉致Chang liver細胞氧化應激作用的研究[J];環(huán)境與健康雜志;2008年07期
7 吳青;衡正昌;;聯(lián)苯胺對大鼠P53基因損傷作用及器官特異性研究[J];四川大學學報(醫(yī)學版);2006年01期
8 文衛(wèi)華;成會榮;衡正昌;黃玉云;董西明;史忠文;徐華;;實時熒光定量PCR檢測職業(yè)砷暴露人群p53基因損傷[J];四川大學學報(醫(yī)學版);2007年04期
9 潘雪莉,張愛華,黃曉欣,蔣憲瑤;PCR-SSCP及克隆測序法研究燃煤型砷中毒患者p53基因突變與皮膚癌的關系[J];環(huán)境與職業(yè)醫(yī)學;2004年05期
10 高緒芳;帥培強;饒朝龍;文衛(wèi)華;衡正昌;;聯(lián)苯胺對k-ras基因DNA損傷位點研究[J];復旦學報(醫(yī)學版);2007年01期
，本文編號：2333572
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