JoVE Journal > Cancer Research
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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암 연구학

使用癌细胞系的传递软骨Cybrid生成

Published: March 17, 2023
doi:
10.3791/65186
, , , , ,
1Department of Biochemistry,University of Zaragoza, 2Institute for Biocomputation and Physics of Complex Systems,University of Zaragoza, 3Sequencing and Functional Genomics Unit,University of Zaragoza-IACS

Summary

该协议描述了一种从悬浮生长的癌细胞生成cybrid的技术，作为研究线粒体在致瘤过程中作用的工具。

Abstract

近年来，致力于确定线粒体与癌症之间联系的研究数量显着增加。然而，仍然需要做出更多的努力来充分了解涉及线粒体改变和肿瘤发生的联系，以及识别与肿瘤相关的线粒体表型。例如，为了评估线粒体在肿瘤发生和转移过程中的贡献，必须了解线粒体在不同核环境中肿瘤细胞的影响。为此，一种可能的方法是将线粒体转移到不同的核背景中以获得所谓的cybrid细胞。在传统的细胞化技术中，缺乏mtDNA（ρ0，核供体细胞）的细胞系被来自去核细胞或血小板的线粒体重新填充。然而，剜除过程需要良好的细胞粘附在培养板上，在许多情况下，侵袭性细胞会部分或完全失去这一特征。此外，传统方法中发现的另一个困难是从线粒体受体细胞系中完全去除内源性mtDNA，以获得纯核和线粒体DNA背景，避免在生成的cybrid中存在两种不同的mtDNA物种。在这项工作中，我们提出了一种应用于悬浮生长癌细胞的线粒体交换协议，该方案基于罗丹明6G预处理细胞与分离线粒体的重新繁殖。这种方法使我们能够克服传统方法的局限性，因此可以用作扩展对线粒体在癌症进展和转移中的作用的理解的工具。

Introduction

重编程能量代谢是癌症1的标志，这是奥托·沃伯格（Otto Warburg）在1930年代首次观察到的2。在有氧条件下，正常细胞将葡萄糖转化为丙酮酸，然后产生乙酰辅酶A，为线粒体机器提供燃料并促进细胞呼吸。然而，Warburg证明，即使在常氧条件下，大多数癌细胞将从糖酵解过程中获得的丙酮酸转化为乳酸，从而改变其获取能量的方式。这种代谢调节被称为“Warburg效应”，使一些癌细胞能够提供其快速生长和分裂的能量需求，尽管产生ATP的效率低于有氧过程3，4，5。近几十年来，许多工作支持代谢重编程在癌症进展中的含义。因此，肿瘤能量学被认为是对抗癌症的有趣靶标1。作为能量代谢和必需前体供应的中心枢纽，线粒体在这些细胞适应中起着关键作用，迄今为止，我们只部分了解这些细胞。

根据上述情况，线粒体DNA（mtDNA）突变已被提出为这种代谢重编程的可能原因之一，这可能导致电子传递链（ETC）性能受损6，并解释为什么一些癌细胞增强其糖酵解代谢以存活。事实上，据报道mtDNA在癌细胞内积累突变，至少存在于50%的肿瘤中7。例如，Yuan等人最近进行的一项研究报告称，肾脏癌，结直肠癌和甲状腺癌中存在超突变和截短的mtDNA分子8。此外，许多研究表明，某些mtDNA突变与更具侵袭性的肿瘤表型和癌细胞转移潜力的增加有关9，10，11，12，13，14，15，16。

尽管线粒体基因组与癌症进展有明显的相关性，但由于目前可用的实验模型和技术的局限性，对这些突变及其对疾病的贡献的研究一直具有挑战性17。因此，需要新技术来了解线粒体DNA对癌症疾病发展和进展的真正影响。在这项工作中，我们介绍了一种从悬浮生长的癌细胞中传输软骨细胞生成的协议，该协议基于罗丹明6G预处理细胞与分离线粒体的重新繁殖，克服了传统胞化方法的主要挑战18，19。该方法允许使用任何细胞核供体，无论其相应的ρ0 细胞系的可用性如何，以及线粒体从遵循传统技术难以去核的细胞（即非贴壁细胞系）转移。

Protocol

注意： 表1中指定了所有培养基和缓冲液组合物。在cybrid生成之前，必须对来自供体和受体细胞的线粒体和核DNA图谱进行分型，以确认细胞系之间两个基因组中存在遗传差异。在这项研究中，使用了市售的L929细胞系及其衍生的细胞系L929dt，该细胞系是我们实验室自发产生的（更多信息见13 ）。这些细胞系在其 mt-Nd2 基因序列中存在两种差异，一旦细胞化过程完…

Representative Results

遵循上述方案后，应获得具有保守核背景但具有新线粒体基因型的同质cybrid细胞系，如图1和图2中的示意图所示。存在于cybrid中的线粒体和核DNA的纯度可以通过RFLP和核DNA基因分型分析（如图4所示）来确认。 如果线粒体转移成功完成，则通过RFLP分析获得的cybrid细胞系的结果必须显示与受…

Discussion

自从Otto Warburg报道癌细胞改变其新陈代谢并增强“有氧糖酵解”3，4，同时减少线粒体呼吸以来，对线粒体在癌症转化和进展中的作用的兴趣呈指数级增长。近年来，mtDNA突变和线粒体功能障碍已被假定为许多癌症类型的标志25。迄今为止，许多研究已经分析了特定肿瘤6，26，27，28，29</…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究由RSA，JMB和AA的授权号PID2019-105128RB-I00以及PFS和RML的PGC2018-095795-B-I00资助，均由MCIN / AEI / 10.13039 / 501100011033和授权号B31_20R（RSA，JMA和AA）和E35_17R（PFS和RML）资助，并由Gobierno de Aragón资助。RSA的工作得到了西班牙反对协会PRDAR21487SOLE的赠款支持。作者要感谢萨拉戈萨大学使用高级调查总局。

Materials

3500XL Genetic Analyzer  ThermoFisher Scientific 4406016
6-well plate Corning 08-772-1B
Ammonium persulfate Sigma-Aldrich A3678
AmpFlSTR Identifiler Plus PCR Amplification Kit ThermoFisher Scientific 4427368
Anode Buffer Container 3500 Series Applied Biosystems 4393927
Boric acid PanReac 131015
Bradford assay Biorad 5000002
Cathode Buffer Container 3500 Series Applied Biosystems 4408256
Cell culture flasks TPP 90076
DMEM high glucose Gibco 11965092
EDTA PanReac 131026
Ethidium Bromide Sigma-Aldrich E8751
Geneticin Gibco 10131027
Homogenizer Teflon pestle Deltalab 196102
L929 cell line ATCC CCL-1
MiniProtean Tetra4 Gel System BioRad 1658004
MOPS Sigma-Aldrich M1254
PCR primers Sigma-Aldrich Custom products
Polyacrylamide Solution 30% PanReac A3626
Polyethylene glycol Sigma-Aldrich P7181
POP-7 Applied Biosystems 4393714
Pyruvate Sigma-Aldrich P5280
QIAmp DNA Mini Kit Qiagen 51306
Rhodamine-6G Sigma-Aldrich R4127
Serum Fetal Bovine Sigma-Aldrich F7524
SspI New England Biolabs R3132
Streptomycin/penicillin PAN biotech P06-07100
Sucrose Sigma-Aldrich S3089
TEMED Sigma-Aldrich T9281
Tris PanReac P14030b
Uridine Sigma-Aldrich U3750
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Tags

Transmitochondrial CybridCancer Cell LinesMitochondrial GenotypesCancer ProgressionMetastasisMitochondrial DNA MutationsAerobic GlycolysisTumorigenesisMitochondrial PhenotypesCybrid Cells
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Soler-Agesta, R., Marco-Brualla, J., Fernández-Silva, P., Mozas, P., Anel, A., Moreno Loshuertos, R. Transmitochondrial Cybrid Generation Using Cancer Cell Lines. J. Vis. Exp. (193), e65186, doi:10.3791/65186 (2023).
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