从人口腔细胞获取基因组DNA作基因多态性分析的可行性  

Feasibility of Genetic Polymorphisms Analysis Using Genomic DNA Obtained from Human Buccal Cells

作　　者：程家蓉[1] 关赛芳[1] 王学励[1] 韩丽华[1] 高玉堂[1] 

机构地区：[1]上海交通大学肿瘤研究所上海市肿瘤研究所流行病学研究室,上海200032

出　　处：《癌症》2005年第7期893-897,共5页Chinese Journal of Cancer

摘　　要：背景与目的口腔洗液上皮细胞应用于检测基因多态性已有多篇文献报道,但口腔洗液中含有少量食物残渣是否给基因多态性检测带来干扰已备受关注。本研究比较口腔上皮细胞和血样品DNA中某些基因的多态性,同时以同样的方法检测食物中动植物组织的DNA,以排除食物残渣干扰实验结果的可能性,并探讨改良口腔洗液上皮细胞收集方法。方法收集62例人口腔洗液,每例收集40ml,加10ml异丙醇固定。其中30例进行细胞涂片检查上皮细胞含量,同时采集外周血5ml。另采集人们常食用的米饭、青菜、毛豆、苹果、猪肉、牛肉、鸡肉、鸭肉。所有样品分别经蛋白酶K消化,酚氯仿抽提,乙醇沉淀提取基因组DNA。结合PCR-RFLP技术进行口腔细胞和血细胞基因多态性分析比较。并用Alu片段测定人口腔洗液、外周血及上述食物DNA。结果62例口腔洗液DNA含量为(135.15±64.30)μg(22.36～330.70μg);其中30例经镜检含有人上皮细胞者有75%～95%,DNA含量为(143.44±61.64)μg(51.01～283.58μg);30例外周血中DNA含量为(91.19±38.01)μg(30.83～178.63μg)。琼脂糖凝胶电泳结果显示62例口腔上皮细胞和30例血样品均具有高分子量DNA,其中61例口腔细胞和30例血样品均能扩增茁-globin、Alu以及NAT2、GSTM1、GSTT1、CYP1A1和CYP2E1基因片段,但所有的食物DNA全部未能扩增。30例口腔洗液与相应血液的基因多态性结果一致。结论口腔洗液DNA大部分来源于人,即使含有少量食物残渣也不会给基因多态性分析带来干扰,其基因多态性检测结果与血样品DNA分析结果完全一致。BACKGROUND & OBJECTIVE: Using DNA samples obtained from buccal cells for genetic polymorphism analysis in molecular epidemiological studies has been repeatedly reported, but whether DNA from food remnants in mouth influences the result is still concerned. This study was to compare genetic polymorphisms of buccal cell DNA with those of buffy coat DNA, and with plant and animal DNA from foods to rule out the possibility of interference from food remnants, to improve technique of buccal cell collection and elevate DNA yield. METHODS: Buccal cells were collected from mouthwash (40 ml/case) of 62 subjects, and fixed with isopropyl alcohol; buffy coats of peripheral blood were collected from 30 of these subjects. Common foods (rice, greengrocery, soybean, apple, pork, beef, chicken, and duck) were also collected. DNA of all samples was extracted by chloroform-phenol method. NAT2, GSTM1, GSTT1, CYP1A1, and CYP2E1 genetic polymorphisms were assayed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Alu (human mutual DNA sequence) was also tested. RESULTS: DNA yield of 62 individual mouthwash samples was (135.15±64.30) μg (22.36-330.70 μg); 30 individual mouthwash samples contained 75%-95% oral epithelial cells with DNA yield of (143.44±61.64) μg (51.01-283.58 μg). DNA yield of 30 buffy coat samples was (91.19±38.01) μg (30.83-178.63 μg). Electrophoresis showed that all 62 buccal cell samples and 30 buffy coat samples contained DNA fragments in high molecular weight; ?茁-globin, Alu, NAT2, GSTM1, GSTT1, CYP1A1, and CYP2E1 gene fragments were successfully amplified from 61 buccal cells samples and 30 buffy coat samples, which showed no difference between the 2 kinds of samples from individual collections; these gene fragments were not amplified from all food DNA samples. CONCLUSIONS: The majority of DNA from mouthwash is human-origin. A little amount of food remnants would not influence the measurements of genetic polymorphisms. The genetic polymorphisms show no difference

关 键 词：口腔细胞 基因组DNA 基因多态性 上皮细胞 

分 类 号：R181.2[医药卫生—流行病学]