肺腺癌单细胞基因突变检测的细胞回收液的影响研究  

Effect of recycled liquid of lung adenocarcinoma mutation detection on single cell level

作　　者：冯琪文 周慧 刘彦辰 徐涛 

机构地区：[1]锦州医科大学有机化学教研室,辽宁锦州121001 [2]香港城市大学生物医药健康中心,广东深圳518000

出　　处：《生物医学工程与临床》2018年第1期87-93,共7页Biomedical Engineering and Clinical Medicine

基　　金：2015年辽宁省大学生创新创业训练计划项目(XZJJ2015D.13);2015年校长基金-奥鸿博泽大学生科技创新基金(201510160000005);辽宁省高等学校优秀人才支持计划(LJQ2015066);深圳市科技计划基础研究项目(JCYJ20150601102053070);国家自然科学基金青年基金项目(81201689)

摘　　要：目的研究不同的单细胞回收液对肺癌单细胞突变检测的影响,为后续的肿瘤基因异质性检测和研究奠定基础。方法以肺癌细胞系A549细胞为研究对象,采用微流控芯片技术,将肺癌A549细胞进行分离;将A549单细胞分别回收于达氏修正依氏培养液(DMEM)、磷酸盐缓冲溶液(PBS)和0.9%氯化钠溶液(生理盐水)中,每组30个单细胞;分别加入裂解液进行单细胞裂解,用实时荧光定量聚合酶链反应(PCR)对单细胞进行KRAS(G12S)基因突变检测,比较3种溶液体系对测定结果的影响。结果采用微流控芯片进行单细胞分离和回收,只要有单个细胞越过阀门,就能够被成功分离回收,回收效率能够达到100%。采用0.9%氯化钠溶液筛分出的单细胞KRAS(G12S)突变检测检出率最高,能够达到90%,平均Ct值最低;采用DMEM筛分出的单细胞KRAS(G12S)突变检测检出率最低,平均Ct值最高。结论采用0.9%氯化钠溶液进行单细胞分离、回收,实时荧光定量PCR扩增效率最高,检测效果最好,最适合用于癌症单细胞水平突变检测。Objective To investigate the impact of single cell isolation on mutation detection of lung adenocarcinoma single cell, and lay the foundation for further detection and research of tumor heterogeneity. Methods Single A549 cells were isolated by microfluidic chips technique, and recycled with Dulbecco ＇s modified Eagle＇s medium（DMEM）, phosphate buffered saline（PBS） and 0.9 % sodium chloride solution（physiological solution）, each group of 30 single cells. Lysate was added to lysis single cell to detect KRAS（G12S） gene mutation of single cell by real-time fluorescence quantitative polymerase chain reac-tion（PCR）, and the influence of three solution systems was compared by measurement results. Results For single cell isolation and recovery by microfluidic chip, the single cell was successfully isolated and recycled when it crossed the valve, and recovery rate was 100 %. The mutation detection rate of single cell KRAS（G12S） by 0.9 % sodium chloride solution reached 90 %, and the mean Ct value was the lowest. Mutation detection rate of single cell KRAS（G12S） by DMEM was the lowest and the mean Ct value was the highest. Conclusion For isolation and recovery of single cell by 0.9 % sodium chloride solution, the amplification efficiency of PCR is the highest and detection result is the best, and suitable for cancer single cell mutation detection.

关 键 词：肺腺癌 微流控芯片 单细胞 突变检测 实时荧光定量 聚合酶链反应(PCR) 

分 类 号：R446.9[医药卫生—诊断学]