【摘要】：依賴(lài)隨機(jī)化末端連結(jié)物PCR (Randomized terminal linker-dependent PCR, RDPCR)是以連接介導(dǎo)PCR (Ligation-mediated PCR, LMPCR)為基礎(chǔ)建立起來(lái)的,可以定位檢測(cè)任何類(lèi)型DNA損傷的一種分子生物學(xué)方法。目前該法已成功應(yīng)用于體外細(xì)胞染毒實(shí)驗(yàn)和體內(nèi)動(dòng)物實(shí)驗(yàn),來(lái)檢測(cè)p53基因、ras基因的損傷,但仍未有應(yīng)用于人群流行病學(xué)研究的報(bào)道。Southern雜交技術(shù)能將PCR過(guò)程中產(chǎn)生的特異性片段檢測(cè)出來(lái),具有快速、靈敏、準(zhǔn)確等優(yōu)點(diǎn),被廣泛的應(yīng)用于鑒別DNA分子中的特異堿基序列等各項(xiàng)研究中。 無(wú)機(jī)砷進(jìn)入機(jī)體會(huì)導(dǎo)致細(xì)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(jī)制的關(guān)鍵所在。檢測(cè)職業(yè)砷暴露人群p53基因的特定損傷位點(diǎn),對(duì)于研究砷的致癌效應(yīng)和遺傳毒性具有重要意義。 目的：1)將RDPCR方法結(jié)合Southern blot雜交技術(shù)應(yīng)用于人群流行病學(xué)研究,為RDPCR的推廣和應(yīng)用積累資料；2)定性檢測(cè)p53基因第五外顯子損傷,驗(yàn)證前期研究結(jié)果,并為進(jìn)一步精確定位損傷位點(diǎn)奠定基礎(chǔ)。 方法：1)離心柱法提取外周血液樣本基因組DNA,雙引物擴(kuò)增p53基因第五外顯子雙鏈并純化；2)制作單鏈探針,并檢測(cè)其可靠性；3)以純化的p53基因第五外顯子為模版進(jìn)行RDPCR擴(kuò)增,終產(chǎn)物凝膠電泳后進(jìn)行Southern blot雜交,鑒定基因損傷。 結(jié)果：1)經(jīng)檢測(cè),根據(jù)瓊脂糖凝膠電泳及與雙鏈外顯子雜交結(jié)果證實(shí)探針制備成功并可靠；2)完成檢測(cè)的68例暴露樣本和7例對(duì)照樣本中,30例暴露樣本明確檢測(cè)到目標(biāo)基因的損傷,對(duì)照樣本均未測(cè)到損傷,暴露組損傷率為44.1%,對(duì)照組損傷率為0,暴露組損傷率顯著高于對(duì)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(jié)論：本研究從定性角度進(jìn)一步證實(shí)了前期采用實(shí)時(shí)熒光定量方法檢測(cè)的兩組樣本p53基因第五外顯子損傷情況；各損傷樣本經(jīng)檢測(cè)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(diǎn),進(jìn)一步可采用克隆測(cè)序等技術(shù)確認(rèn)具體損傷位點(diǎn)；本研究成功將該技術(shù)應(yīng)用于人群分子流行病學(xué)研究,進(jìn)一步擴(kuò)展了該技術(shù)的應(yīng)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(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R135.1

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