一种新型大量粪便肠黏膜提取方法在结直肠癌筛查中的应用

Application of a novel method to collect large amount of fecal mucosa in screening colorectal cancer

目的探讨一种新型的大量粪便肠黏膜提取方法在结直肠癌筛查中的应用。方法收集2014年3—4月间，于南京中医药大学第三附属医院拟行手术治疗的10例结直肠癌患者术前1次完整粪便标本和手术切除癌组织标本。同时收集6例肠镜检查未见异常的6例同期住院患者的肠黏膜组织及1次完整粪便标本。使用自制的肠黏膜收集器（该肠黏膜收集器由上、中、下3个体积为1000ml容器组成，3层底部均分别有层滤网，孔径分别为100、200及300目，逐层过滤后收集液体）肠黏膜脱落细胞，采用粪便DNA试剂盒提取脱落细胞DNA，检测DNA浓度及纯度（A260/A280），并对提取的粪液以及肠黏膜组织进行甲基化测序。结果DNA甲基化测序发现，FBN1、SPG20及SNCA基因在lO例结直肠癌患者的癌组织和粪便提取液标本中CpG岛均出现甲基化。而在6例对照患者的活检肠黏膜和粪便提取液标本中均未发现甲基化。粪便重量在100g以下时，粪液收集率在60％～80％之间；粪便重量超过100g后，粪液收集率不稳定；粪便重量在50～100g之间时，DNA的A260/A280值在1．6～1．8之间，浓度在5．0～56．1μg／ml之间。结论本研究采用自行设计的大量粪便肠黏膜收集器一次性处理50～100g粪便时，粪液收集率较为稳定，可获取纯度和浓度较高的DNA，达到检测结直肠肿瘤甲基化状态的需要，便于结直肠癌的筛查。

Objective To explore the application of a novel device of collecting large amount of fecal mucosa for detecting the DNA methylation and screening colorectal cancer. Methods Preoperative complete fecal sample and surgical specimen of 10 patients with colorectal cancer, and complete fecal sample and normal bowel mucosal samples confirmed by colonoscopy of 6 hospitalization cases at The Third Affiliated Hospital, Nanjing University of TCM from March to April 2014 were collected. A self-made bowel mucosa collector （consisting of upper, middle, lower three containers of 1 000 ml volume, with filter screen in each bottom whose pore diameter is 100, 200 and 300 mesh.） was used to collect mucosal exfoliation cells. Fecal DNA kit was applied to extract DNA of exfoliation cells and the concentration and purity of DNA were measured by UV spectrophotometer （A250/A280）, meanwhile DNA methylation of fecal fluid and mucosal tissues was detected by bisulfite sequencing pCR （BSP）. Results DNA methylation sequencing showed that FBN1, SPG20, and SNCA genes presented methylation in CpG island in fecal fluid and cancer tissues from 10 colorectal cancer patients, but did not presented methylation in fecal fluid and mucosa from 6 control cases. When fecal amount was below 100 g, collection rate of fecal fluid was 60% to 80%; when fecal amount was over 100 g, collection rate of fecal fluid was unstable. When fecal amount was 50 to 100 g, DNA A260/A280 value was 1.6 to 1.8, and DNA concentration was 5.0 to 56.1 ng/L. Conclusion Collection rate of fecal fluid with this self-made fecal mucosa collector is quite stable when managing fecal amount of 50 to 100 g once, and can obtain higher purity and concentration of DNA, meeting the demand of methylation detection for screening colorectal cancer.