遗传 ›› 2017, Vol. 39 ›› Issue (8): 753-762.doi: 10.16288/j.yczz.17-057

• 技术与方法 • 上一篇    下一篇

组织单腺体内单细胞的基因变异分析方法

周彦1(),王超杰2(),朱纯超2,陈江荣3,程酩3,邓宇亮1,郭妍3()   

  1. 1. 上海交通大学系统生物医学研究院,上海 200240
    2. 上海交通大学医学院附属仁济医院,上海 200240
    3. 上海交通大学生物医学工程学院,上海 200240
  • 收稿日期:2017-02-21 修回日期:2017-06-15 出版日期:2017-08-20 发布日期:2017-12-25
  • 作者简介:周彦,硕士研究生,专业方向：单细胞的基因变异分析方法。E-mail: systemguy@sjtu.edu.cn|王超杰,博士,住院医师,研究方向：胃癌。E-mail: wangcja1@sina.com.cn|郭妍,副研究员,研究方向：胃癌早期病变的机理研究。E-mail: yanguo@sjtu.edu.cn
  • 基金资助:
    国家自然科学重大研究计划项目(91529302)

Single-cell gene variation analysis method for single gland

Yan Zhou1(),Chaojie Wang2(),Chunchao Zhu2,Jiangrong Chen3,Ming Cheng3,Yuliang Deng1,Yan Guo3(),   

  1. 1. Shanghai Center for Systems Biomedicine, Shanghai 200240, China
    2. Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
    3. School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China
  • Received:2017-02-21 Revised:2017-06-15 Online:2017-08-20 Published:2017-12-25
  • Supported by:
    the National Natural Science Foundation of China(91529302)

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摘要：

从单细胞尺度进行细胞异质性的分析是深度理解细胞群体关系的关键。组织中的单细胞由于细胞类型不同,尺寸往往相差很大,但是目前常用的基于微孔板和Fluidigm公司的微流控的单细胞组学研究方法,需要入口的单细胞大小相近。本研究以胃组织为例,建立了一种组织单细胞的基因变异分析方法,实现了尺寸差异较大的单细胞的基因变异分析。在该方法中,先将胃组织裂解获得单个腺体,再将单个腺体酶解得到不同大小的腺体内单细胞,然后把这些单细胞铺在聚乙烯萘膜载玻片上,进行激光显微切割分选、全基因组放大,最后测其微卫星的长度。利用该方法,成功在肠上皮化生腺体内部检测到微卫星长度的变化,并灵活地对尺寸差异大的组织细胞以及肠化生腺体细胞进行了精细分析。此外,这种单细胞分析方法还可以对带有不同标记的细胞进行低通量和高通量的基因组分析,为单细胞尺度上的组织异质性研究提供了一种高度灵活的分析方法。

关键词: 单细胞分析方法, 全基因组放大, 激光显微切割, 胃组织, 微卫星

Abstract:

Single-cell analysis of heterogeneity has become the cutting-edge technology for profound understandings of relationships between cell populations. At present, common methods used in single cellular genomic research are mainly microfluidic technologies (Fluidigm) or based on microwells, both requiring a uniform size of cells at the entrance. However, the size of cells in specific tissues can vary from type to type. To address this issue, we need to establish a method to identify genomic features of individual cells of different sizes. In this paper, we developed a robust method in the analysis of single cellular genomic mutations among gastric tissues. Briefly, the single gastric gland was isolated from the whole tissue, and further enzymatically digested into single cells of various sizes by trypsin. These single cells were then spread on the polyethylene naphthalene slides and selected by the laser microdissection method. Whole genome amplification (WGA) and capillary electrophoresis were performed subsequently to detect single cell microsatellite. This method enabled us to detect the existence of microsatellite instability (MSI) of each single cell within the intestinal metaplasia, and to carry out a flexible and fine analysis of single cells with different sizes in tissues and glands. This reliable and practical method is well performed in both low and high-throughput genome analysis when combined with cell labeling methods, thus providing a novel and highly flexible way to study tissue heterogeneity on the single cell scale.

Key words: method to analysis single cell, whole genome amplification, laser capture microdissection, gastric tissue, microsatellite