人线粒体DNA缺失宫颈癌细胞及其转线粒体细胞的建立与初步分析

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摘要目的：探讨溴化乙锭（EB）诱导法建立人线粒体DNA（mtDNA）缺失宫颈癌Hela S3细胞，以及再转入线粒体构建融合细胞的可行性，并对转线粒体细胞进行初步分析。方法：采用低剂量（100 ng/mL）EB诱导法建立mtDNA缺失的ρ0 Hela S3细胞，通过普通PCR、荧光定量PCR（qPCR）进行mtDNA缺失鉴定。采用聚乙二醇（PEG）介导细胞融合法，以正常人血小板为mtDNA供体转入ρ0 Hela S3细胞，构建转线粒体细胞（transmitochondrial cybrids），并通过PCR、qPCR和透射电镜观察进行初步分析和鉴定。结果：普通PCR和qPCR结果证实EB诱导法能够成功建立ρ0 Hela S3细胞，同时结合透射电镜证明转线粒体细胞能够恢复线粒体正常结构。结论：采用EB诱导法可成功建立ρ0 Hela S3细胞，且通过细胞融合构建的转线粒体细胞能够恢复mtDNA复制和正常线粒体结构，为研究mtDNA突变与线粒体功能异常相关疾病的关系提供可靠的实验基础。

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	温超玮
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	周怀彬
	吕建新
	李伟

关键词 ：线粒体DNA缺失细胞, 转线粒体细胞, 线粒体DNA拷贝数, 线粒体嵴

Abstract：Objective: To generate mtDNA-depleted Hela S3 cells and the transmitochondrial cybrids and investigate mtDNA replication and mitochondrial structure. Methods: Hela S3 cells were incubated with 100 ng/mL ethidium bromide (EtBr). Polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) were performed in ρ0 Hela S3 cells. Platelet-mediated transmitochondrial cybrids were established on the basis of ρ0 Hela S3 cells and identified by PCR, qPCR and transmission electron microscopy (TEM). Results: ρ0 Hela S3 cells had a low level of mtDNA replication and distorted cristae of mitochondria, whereas transmotichondrial cybrids had a normal mtDNA replication and regular arranged cristae of mitochondria. Conlusion: The generation of transmitochondrial cybrids provides a viable method for the further research of the effect of mtDNA mutations on mitochondrial respiratory function associated with diseases.

Key words： mtDNA-depleted cells transmitochondrial cybrids mtDNA copy numbers cristae of mitochondria

收稿日期: 2015-03-07

基金资助:国家自然科学基金资助项目（81271918）。

通讯作者: 李伟，副教授，Email：liweiwzmc@163.com。

作者简介: 温超玮（1987-），女，浙江苍南人，实验员。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2015/V45/I6/402

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