研究线粒体功能障碍的 体外 方法：一种Cybrid模型
Summary
透软骨圆柱体是通过将线粒体 DNA （mtDNA） 耗尽的细胞 （rho0 细胞） 与来自线粒体疾病患者的细胞形成体（去核细胞）融合而获得的杂交细胞。它们可以确定疾病的核或线粒体起源，评估生化活性，并确认mtDNA相关变异的致病作用。
Abstract
进行氧化磷酸化（OXPHOS）的线粒体呼吸链复合物的缺乏是人类线粒体疾病的生化标志物。从遗传学的角度来看，OXPHOS代表了一个独特的例子，因为它是由两个不同的遗传系统互补产生的：核DNA（nDNA）和线粒体DNA（mtDNA）。因此，OXPHOS缺陷可能是由于影响核和线粒体编码基因的突变引起的。
King和Attardi于1989年发表的开创性工作表明，耗尽mtDNA（名为rho0）的人类细胞系可以被外源线粒体重新填充，以获得所谓的“透射粒体cybrids”。由于这些含有来自线粒体疾病（MD）患者的线粒体和来自rho0 细胞的细胞核的cybrids，因此可以验证缺陷是mtDNA相关还是nDNA相关。这些cybrids也是验证突变的致病性并在生化水平上研究其影响的强大工具。本文提出了一个详细的方案，描述了cybrid的产生，选择和表征。
Introduction
线粒体疾病（MD）是一组多系统综合征，由核（nDNA）或线粒体（mtDNA）DNA 1突变引起的线粒体功能受损引起。它们是最常见的遗传性代谢疾病之一，患病率为1：5，000。mtDNA相关疾病遵循线粒体遗传学的规则：母体遗传，异质和阈值效应以及有丝分裂分离2。人类mtDNA是一个16.6 kb的双链DNA环，其中包含一个短的对照区，其中包含复制和转录所需的序列，13个蛋白质编码基因（呼吸链的所有亚基），22个tRNA和2个rRNA基因3。
在健康个体中，存在一个单一的mtDNA基因型（同质），而在病理条件下存在多个基因型（异质体）。有害的异质突变必须克服一个关键阈值，以破坏OXPHOS并引起可能影响任何器官的疾病，在任何年龄4。OXPHOS的双重遗传学决定了遗传：常染色体隐性遗传或显性遗传，nDNA突变X连锁，母体mtDNA突变，以及nDNA和mtDNA的散发病例。
在线粒体医学时代开始时，King和Attardi5 的一项具有里程碑意义的实验通过创建含有肿瘤细胞系细胞核的杂交细胞，其中mtDNA完全耗尽（rho0 细胞）和来自MD患者的线粒体，为理解导致MD的突变的起源奠定了基础。 并且确定核或线粒体基因组中是否存在突变并不容易。该方法于1989年描述，然后被在线粒体医学领域的几位研究人员使用6，7，8，9;最近发布了10个详细的协议，但尚未制作视频。如今，当NGS可以精确而快速地识别突变的位置时，为什么这样的协议应该具有相关性？答案是，cybrid生成仍然是最先进的方案，可以了解任何新型mtDNA突变的致病作用，将异质体的百分比与疾病的严重程度相关联，并在没有患者自身核背景贡献的同质核系统中进行生化研究11，12，13.
该协议描述了如何从在35mm培养皿中生长的融合的，患者来源的成纤维细胞中获得细胞形成体。在细胞查拉辛B存在下离心培养皿允许分离去核细胞体，然后在聚乙二醇（PEG）存在下与rho0 细胞融合。然后将得到的圆孢子在选择性培养基中培养，直到克隆出现。代表性结果部分显示了所得cybrids的分子表征示例，以证明mtDNA与供体患者的成纤维细胞相同，并且nDNA与肿瘤rho0 细胞系的核DNA相同。
Protocol
注意：使用人类成纤维细胞可能需要道德批准。本研究中使用的成纤维细胞来自MD患者，并按照伦理要求储存在机构生物库中。对细胞的使用提供了知情同意。在室温（RT，22-25°C）的无菌层流柜下执行所有细胞培养程序。使用适用于细胞培养和无菌设备的无菌过滤溶液。在37°C的加湿培养箱中用5%CO2生长所有细胞系。应每周进行一次支原体检测，以确保无支原体培养。143BTK-rho0的细…
Representative Results
产生cybrids需要3天的实验室工作加上一个选择期（约2周）和额外的1-2周用于克隆的生长。关键步骤是细胞质细胞的质量和选择期。环杂蚴的形态类似于rho0 供体细胞的形态。在环杂交中分配正确的mtDNA和nDNA是确认细胞身份所必需的。 图 2 给出了一个示例。在这种情况下，我们从来自携带异质m.3243A>G的患者的成纤维细胞开始产生cybrids，这是与线粒体肌病，脑病，乳酸?…
Discussion
与nDNA相比，mtDNA具有非常高的突变率，因为缺乏保护性组蛋白并且其位置靠近呼吸链，这使得分子暴露于修复系统16无法有效抵消的破坏性氧化作用。第一个致病性mtDNA突变是在1988年发现的17，18，从那时起，已经描述了大量的突变。NGS技术是筛选整个线粒体基因组并轻松识别变异14的相关方法。然而，评估从未描述过…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究是在由马里亚尼基金会资助的线粒体儿科疾病研究中心（http://www.mitopedia.org）进行的。VT是欧洲罕见神经肌肉疾病参考网络（ERN EURO-NMD）的成员。
Materials
| 5-Bromo-2'-Deoxyuridine | Sigma-Aldrich (Merck) | B5002-500MG | |
| 6 well Plates | Corning | 3516 | |
| 96 well Plates | Corning | 3596 | |
| Blood and Cell Culture DNA extraction kit | QIAGEN | 13323 | |
| Centrifuge | Beckman Coulter | Avanti J-25 | 7,200 rcf, 37 °C |
| Centrifuge bottles, 250 mL | Beckman Coulter | 356011 | |
| Cytochalasin B from Drechslera dematioidea | Sigma-Aldrich (Merck) | C2743-200UL | |
| Dialyzed FBS | Gibco | 26400-036 100mL | |
| DMEM High Glucose (w/o L-Glutamine W/Sodium Pyruvate) | EuroClone | ECB7501L | |
| Dulbecco's Phosphate Buffered Saline – PBS (w/o Calcium w/o Magnesium) | EuroClone | ECB4004L | |
| Ethanol Absolute Anhydrous | Carlo Erba | 414601 | |
| FetalClone III (Bovine Serum Product) | Cytiva – HyClone Laboratories | SH30109.03 | |
| Glass pasteur pipettes | VWR | M4150NO250SP4 | |
| Inverted Research Microscope For Live Cell Microscopy | Nikon | ECLIPSE TE200 | |
| JA-14 Fixed-Angle Aluminum Rotor | Beckman Coulter | 339247 | |
| Laboratory autoclave Vapormatic 770 | Labotech | 29960014 | |
| L-Glutamine 200 mM (100x) | EuroClone | ECB 3000D | |
| Minimum Essential Medium MEM | Euroclone | ECB2071L | |
| MycoAlert Mycoplasma Detection Kit | Lonza | LT07-318 | |
| PEG (Polyethylene glicol solution) | Sigma-Aldrich (Merck) | P7181-5X5ML | |
| Penicillin-Streptomycin (solution 100x) | EuroClone | ECB3001D | |
| Primo TC Dishes 100 mm | EuroClone | ET2100 | |
| Primo TC Dishes 35 mm | EuroClone | ET2035 | |
| Sodium Pyruvate 100 mM | EuroClone | ECM0542D | |
| Stereomicroscope | Nikon | SMZ1000 | |
| Trypsin 2.5% in HBSS | EuroClone | ECB3051D | |
| Uridine | Sigma-Aldrich (Merck) | U3003-5G |
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Citar este artigo
Cavaliere, A., Marchet, S., Di Meo, I., Tiranti, V. An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model. J. Vis. Exp. (181), e63452, doi:10.3791/63452 (2022).