实时动态活细胞成像系统在单克隆筛选和克隆形成能力检测中的应用  

Real-time live-cell analysis system for screening single tumor cell clones and analyzing their colony-forming ability

作　　者：刘长政[1] 焦晓磊[2] 高敦芹 邢龙彬 刘辉[2] 骆莹[2] 高英堂[2] 

机构地区：[1]天津医科大学三中心临床学院,天津市300170 [2]卫生部人工细胞工程技术研究中心天津市人工细胞重点实验室天津市肝胆疾病研究所天津市第三中心医院,天津市300170

出　　处：《世界华人消化杂志》2017年第10期881-890,共10页World Chinese Journal of Digestology

基　　金：天津市卫计委重点攻关基金资助项目;Nos.15KG113;16KG150;天津市应用基础与前沿技术研究计划青年基金资助项目;No.15JCQNJC115000~~

摘　　要：目的利用IncuCyte ZOOM系统筛选肿瘤细胞单克隆和克隆形成能力检测.方法采用差速消化与差速贴壁分离培养原代肿瘤细胞;在有限稀释法基础上,采用IncuCyte ZOOM系统的实时动态追踪和全孔成像技术对肿瘤细胞进行单克隆筛选及克隆形成能力分析.结果从30例肿瘤组织中培养6株原代肿瘤细胞(TJ3ZX-02至07),进一步从5株原代肿瘤细胞(TJ3ZX-03至07)中成功筛选出89个持续增殖的单克隆,其中67个克隆扩增培养并冻存;并且通过时序图准确有效地排除了18个不能持续增殖的单克隆和28个由2个及以上细胞组成的多克隆.2例单克隆株(TJ3ZX-06-B11,TJ3ZX-07-H11)克隆形成能力分析表明,14 d时平皿方法的克隆形成率(35.17%,13.17%)高于IncuCyte ZOOM 96孔全孔成像(23.13%,5.51%),且差异有统计学意义(P<0.05);延长至21 d时全孔成像的克隆形成率为(35.63%,13.22%),与平皿方法比较无统计学差异.结论 IncuCyte ZOOM系统能够简便、准确、省时省力地实现单克隆细胞筛选和克隆形成率检测.AIM To screen single tumor cell clones and evaluating their colony-forming ability by IncuCyte ZOOM.METHODS Primary tumor cells were isolated by differential digestion and differential adherence method.On the basis of limited dilution,dynamic realtime tracking technology and full aperture imaging technology were used to track single cell clones and evaluate their colony-formation ability.RESULTS Six lines of primary tumor cells（TJ3ZX-02 to 07） were isolated from 30 tumor tissues,and 89 persistently proliferative tumor cell clones were screened from five primary tumor cell lines（TJ3ZX-03 to 07）,of which 67 were expanded and cryopreserved.Eighteen monoclonal cell lines were excluded due to the lack of expansion ability,and 28 polyclonal cell lines were excluded because of consisting of two or more cell types as revealed by the Sequence Diagram.The analysis of cloneforming ability of two monoclonal cell strains（TJ3ZX-06-B11,TJ3ZX-07-H11） showed that the clone-forming rates for the plate method（35.17%,13.17%） were significantly higher than those for IncuCyte ZOOM（23.13%,5.51%） at 14 d（P〈0.05）,although there was no significant difference at 21 d（35.63%and13.22%for IncuCyte ZOOM）.CONCLUSION IncuCyte ZOOM is simple,accurate and time-saving for screening single clones and measuring their colony-forming ability.

关 键 词：原代肿瘤细胞 单克隆细胞 克隆形成率 

分 类 号：R730.3[医药卫生—肿瘤]