遗传 ›› 2016, Vol. 38 ›› Issue (7): 666-673.doi: 10.16288/j.yczz.16-085

• 技术与方法 • 上一篇    下一篇

线粒体遗传疾病细胞模型的构建:永生淋巴细胞系和转线粒体细胞系

孙吉吉, 赵晓旭, 乔丽华, 梅霜, 聂志鹏, 张青海, 冀延春, 蒋萍萍, 管敏鑫   

  1. 浙江大学遗传学研究所,浙江大学医学院医学遗传学系,杭州 310058
  • 收稿日期:2016-03-11 出版日期:2016-07-20 发布日期:2016-07-20
  • 作者简介:孙吉吉,硕士,实验师,研究方向:分子与细胞生物学。E-mail: jijisun@zju.edu.cn
  • 基金资助:
    国家自然科学基金项目(编号:31371270)资助[Supported by the National Natural Science Foundation of China (No; 31371270)]

Cellular models for mitochondrial DNA-based diseases: lymphoblastoid cell lines and transmitochondrial cybrids

Jiji Sun, Xiaoxu Zhao, Lihua Qiao, Shuang Mei, Zhipeng Nie, Qinghai Zhang, Yanchun Ji, Pingping Jiang, Min-Xin Guan   

  1. Institute of Genetics, Zhejiang University and Department of Genetics, Zhejiang University School of Medicine, Hangzhou 310058, China
  • Received:2016-03-11 Online:2016-07-20 Published:2016-07-20

摘要: 线粒体基因组突变会引发多种线粒体遗传疾病。永生淋巴细胞系和转线粒体细胞系是线粒体遗传疾病研究的重要工具之一。永生淋巴细胞系使特殊遗传信息在细胞水平得以永久保存。转线粒体细胞系技术的发展为线粒体遗传疾病的分子机制研究提供了体外细胞平台。早期转线粒体细胞系的胞质供体直接来源于未进行永生化的患者各组织细胞或血小板。本文结合永生化手段,以线粒体4401A>G为例,详细介绍了从冻存全血构建永生化淋巴细胞,然后再与ρ0 206一起构建转线粒体细胞系的原理、方法和技术路线,并将两种细胞模型的构建归纳为4个步骤:(1)永生淋巴细胞构建;(2)转化;(3)筛选;(4)鉴定。为真实反应线粒体突变的功能,本文对构建的永生淋巴系和转线粒体细胞系的线粒体突变位点、拷贝数以及转线粒体细胞系的细胞核型进行了分析与鉴定,选取了各参数一致的细胞系用于贮存与分析,以减少实验误差对后续突变位点功能研究的影响。两种细胞模型在细胞水平上为诠释线粒体遗传疾病的分子机制发挥了重要作用,但在具体应用中需注意它们的局限性,尤其是组织特异性的线粒体疾病。

关键词: 线粒体遗传疾病, 永生淋巴细胞系, 转线粒体细胞系, 转化, 融合

Abstract: Mitochondrial DNA (mtDNA) mutations cause a variety of mitochondrial DNA-based diseases which have been studied using Lymphoblastoid cell lines (LCLs) and transmitochondrial cybrids. Individual genetic information is preserved permanently in LCLs while the development of transmitochondrial cybrids provide ex-vivo cellular platform to study molecular mechanism of mitochondrial DNA-based diseases. The cytoplasmic donor cells for previous transmitochondrial cybrids come from patient’s tissue or platelet directly. Here, we depicted in details the principle, methods and techniques to establish LCLs from frozen peripheral bloods harboring mitochondrial 4401G>A mutation by infection of Epstein Barr virus, and then to generate cybrids using ρ0 206 and LCLs. The process of establishing these two cellular models was summarized into four steps as follows: (1) Generation of LCLs; (2) Transformation; (3) Selection; (4) Verification. To faithfully represent the function of mtDNA mutation, we analyzed and identified the sites of mtDNA mutations and copy numbers of each cellular models as well as the karyotype of transmitochondrial cybrids. Those clones with consistent parameters were selected for preservation and future analysis of the function of point mutations of mtDNA. Although these two cellular models play important roles in understanding molecular mechanism of mitochondrial DNA-based diseases on the cellular level, their limitations should be considered when elucidating the character of tissue specificity of mitochondrial DNA-based diseases.

Key words: mitochondria disease, lymphoblastoid, transmitochondrial cybrids, transformation, fusion