结直肠癌患者外周血cf-DNA检测及其应用价值初探

发布时间：2018-04-22 19:06

本文选题：结直肠癌 + 外周血游离DNA　；参考：《北京协和医学院》2016年博士论文

【摘要】：研究背景结直肠癌是世界上最常见的癌症之一,随着我国生活习惯和饮食结构的改变以及人口老龄化进程的加快,在我国结直肠癌发病率近几年呈上升趋势。提高结直肠癌的早期诊断率对于及时发现、治疗及改善患者预后非常重要,然而结直肠癌的早期诊断仍然有许多困难。血清肿瘤标志物检测是常用的结直肠癌早期筛查方法之一,然而其敏感度及特异度却不尽如人意。近年来,外周血cf-DNA作为一种新的肿瘤标志物在结直肠癌的早期诊断方面起到重要作用。本研究通过提取患者外周血cf-DNA及肿瘤组织DNA,进行外周血cf-DNA浓度测定、高通量外显子测序及生物信息学分析,试图找出对结直肠癌早期诊断有意义的相关位点；并通过免疫组化染色方法,初步了解PTEN蛋白、NOK蛋白在结直肠癌及正常结肠黏膜组织中的表达情况,了解其表达与肿瘤分化程度是否存在相关性可能。研究方法1、选取2015.9-2016.5期间于北京协和医院初诊为溃疡性结肠炎、结直肠腺瘤、早癌、进展期癌的患者,通过肠镜或手术方式收集腺瘤及肿瘤原发灶标本和癌旁正常组织(同一例患者的癌组织与癌旁组织成对采集),同时收集患者外周血标本。通过DNA提取、片段化、扩增、捕获等步骤,检测外周血cf-DNA浓度,并对样本进行高通量外显子测序。2、选取2015.1-2016.6于北京协和医院接受手术治疗的结肠癌患者肿瘤蜡块,取距癌肿边缘8cm以上邻近无增生的正常结肠黏膜作对正常组织行自身对照,经HE染色再次确认原始组织学诊断后行PTEN及NOK免疫组化检测。研究结果1、共获得来自6个患者的外周血、原发灶及癌旁正常组织共19份样本,经外周血cf-DNA浓度测定,炎症性肠病及结直肠癌病人外周血cf-DNA浓度均较结直肠腺瘤病人为高。2、初步结果显示PTEN在正常结肠黏膜组织中的表达高于结肠癌组织,而NOK在肿瘤组织中表达则高于正常结肠黏膜组织,且NOK在中、高分化结肠癌组织中趋于胞膜分布。结论外周血cf-DNA浓度检测结合高通量外显子测序可能用于结直肠癌的辅助诊断,但还需要扩大样本量及进一步实验研究。PTEN的缺失、突变可能与结肠癌发生相关,NOK可能在结肠癌的发生、发展过程中有一定作用,值得后续进一步实验研究其分子机理及临床应用前景。
[Abstract]:Background Colorectal cancer is one of the most common cancers in the world. With the changes of living habits and diet structure and the acceleration of population aging, the incidence of colorectal cancer in China has been increasing in recent years. It is very important to improve the early diagnosis rate of colorectal cancer for timely detection, treatment and improvement of prognosis. However, there are still many difficulties in early diagnosis of colorectal cancer. Detection of serum tumor markers is one of the commonly used early screening methods for colorectal cancer, but its sensitivity and specificity are not satisfactory. In recent years, peripheral blood cf-DNA, as a new tumor marker, plays an important role in the early diagnosis of colorectal cancer. In this study, we extracted cf-DNA from patients' peripheral blood and tumor DNA, determined the concentration of cf-DNA in peripheral blood, sequenced high throughput exons and analyzed bioinformatics to find the relevant sites for early diagnosis of colorectal cancer. The expression of PTEN protein in colorectal cancer and normal colonic mucosa was studied by immunohistochemical staining, and the correlation between the expression and the degree of tumor differentiation was investigated. Methods 1. The patients who were first diagnosed as ulcerative colitis, colorectal adenoma, early cancer, advanced cancer in Peking Union Hospital from May to June 2015.were selected. The specimens of adenoma and primary tumor and adjacent normal tissue were collected by endoscopy or surgery. The cancer tissue and adjacent tissue of the same patient were collected in pairs and the peripheral blood samples were collected at the same time. The concentration of cf-DNA in peripheral blood was detected by DNA extraction, fragmentation, amplification and capture, and the samples were sequenced by high-throughput exon .2The wax pieces of colon cancer patients who underwent surgical treatment in Peking Union Hospital from January to June 2016 were selected. The normal colon mucosa above the edge of 8cm was used as a self control group. The original histologic diagnosis was confirmed by HE staining and PTEN and NOK were detected by immunohistochemistry. Results 1. A total of 19 samples of peripheral blood, primary focus and adjacent normal tissues from 6 patients were obtained. The cf-DNA concentration in peripheral blood was measured. The concentration of cf-DNA in peripheral blood of patients with inflammatory bowel disease and colorectal cancer was higher than that of patients with colorectal adenoma. The preliminary results showed that the expression of PTEN in normal colonic mucosa was higher than that in colon carcinoma. The expression of NOK in tumor tissues was higher than that in normal colonic mucosa, and NOK tended to be cellular membrane distribution in well-differentiated colon cancer tissues. Conclusion Peripheral blood cf-DNA concentration detection combined with high-throughput exon sequencing may be used in the auxiliary diagnosis of colorectal cancer, but it is necessary to expand the sample size and further study the deletion of .PTEN. The mutation may be related to the carcinogenesis of colon cancer. NOK may play a certain role in the development of colon cancer. It is worthy of further experimental study on its molecular mechanism and clinical application prospect.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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