应用RDPCR检测职业砷暴露工人P53基因的损伤

发布时间：2018-11-15 14:32

【摘要】：依赖随机化末端连结物PCR (Randomized terminal linker-dependent PCR, RDPCR)是以连接介导PCR (Ligation-mediated PCR, LMPCR)为基础建立起来的,可以定位检测任何类型DNA损伤的一种分子生物学方法。目前该法已成功应用于体外细胞染毒实验和体内动物实验,来检测p53基因、ras基因的损伤,但仍未有应用于人群流行病学研究的报道。Southern杂交技术能将PCR过程中产生的特异性片段检测出来,具有快速、灵敏、准确等优点,被广泛的应用于鉴别DNA分子中的特异碱基序列等各项研究中。无机砷进入机体会导致细胞DNA损伤,这些损伤部位也包括p53基因,而p53基因的损伤可能是砷遗传毒性和致癌作用分子机制的关键所在。检测职业砷暴露人群p53基因的特定损伤位点,对于研究砷的致癌效应和遗传毒性具有重要意义。目的：1)将RDPCR方法结合Southern blot杂交技术应用于人群流行病学研究,为RDPCR的推广和应用积累资料；2)定性检测p53基因第五外显子损伤,验证前期研究结果,并为进一步精确定位损伤位点奠定基础。方法：1)离心柱法提取外周血液样本基因组DNA,双引物扩增p53基因第五外显子双链并纯化；2)制作单链探针,并检测其可靠性；3)以纯化的p53基因第五外显子为模版进行RDPCR扩增,终产物凝胶电泳后进行Southern blot杂交,鉴定基因损伤。结果：1)经检测,根据琼脂糖凝胶电泳及与双链外显子杂交结果证实探针制备成功并可靠；2)完成检测的68例暴露样本和7例对照样本中,30例暴露样本明确检测到目标基因的损伤,对照样本均未测到损伤,暴露组损伤率为44.1%,对照组损伤率为0,暴露组损伤率显著高于对照组(P0.05)；3)各损伤条带的凝胶电泳和杂交条带出现位置大致相同,略滞后于外显子双链位置。结论：本研究从定性角度进一步证实了前期采用实时荧光定量方法检测的两组样本p53基因第五外显子损伤情况；各损伤样本经检测所得的杂交条带位置基本一致,初步判断砷致p53基因第五外显子损伤具有特定位点,进一步可采用克隆测序等技术确认具体损伤位点；本研究成功将该技术应用于人群分子流行病学研究,进一步扩展了该技术的应用范围。
[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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R135.1

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