内源性表达人RYR1变异体的功能表征
Summary
这里描述了用于研究RYR1突变的功能效应的方法，这些突变在爱泼斯坦巴尔病毒中内源性表达，使人B淋巴细胞永生化，肌肉活检衍生的卫星细胞分化成肌管。
Abstract
RYR1基因的700多种变异已经在患有不同神经肌肉疾病的患者中被发现，包括恶性高热易感性，核心肌病和中心核肌病。由于与RYR1突变相关的表型多种多样，因此表征其功能效应以对患者携带的变异进行分类以用于未来的治疗干预并识别非致病性变异是至关重要的。许多实验室一直对开发功能表征患者细胞中表达的RYR1突变的方法感兴趣。这种方法具有许多优点，包括：突变是内源性表达的，RyR1不会过度表达，避免使用异源RyR1表达细胞。然而，由于患者可能在RYR1以外的不同基因中表现出突变，因此比较具有不同遗传背景的具有相同突变的个体的生物材料的结果非常重要。本手稿描述了为研究内源性表达的RYR1变体的功能效应而开发的方法：（a）爱泼斯坦巴尔病毒永生化的人B淋巴细胞和（b）来自肌肉活检并分化成肌管的卫星细胞。然后监测由添加药理学RyR1激活剂触发的细胞内钙浓度的变化。所选细胞类型加载比例荧光钙指示剂，并通过荧光显微镜在单细胞水平或使用光谱荧光计监测细胞内[Ca2 +]变化。然后比较来自健康对照组的细胞和携带RYR1变异的患者之间的静息[Ca2 +]激动剂剂量反应曲线，从而深入了解给定变异的功能效应。
Introduction
迄今为止，已在人群中鉴定出700多种RYR1变体，并与各种神经肌肉疾病有关，包括恶性高热易感性（MHS），运动诱导的横纹肌溶解，中枢核心疾病（CCD），多微核疾病（MmD），中心核肌病（CNM）1，2，3;然而，表征其功能效应的研究滞后，只有大约10%的突变进行了功能测试。可以使用不同的实验方法来评估给定RyR1变体的影响，包括用编码WT的质粒和突变的RYR1 cDNA4，5转染异源细胞，如HEK293和COS-7细胞，转导呼吸不良的小鼠成纤维细胞与质粒和编码WT的载体和突变的RYR1 cDNA，然后转导肌-D并分化成肌管6，产生携带突变RyR1s的转基因动物模型7，8，9，表征来自内源性表达RYR1变异的患者的细胞10，11，12。这些方法有助于确定不同的突变如何在功能上影响RyR1 Ca2 +通道。
在这里，描述了为评估RYR1突变的功能效应而开发的方法。在内源性表达RyR1钙通道的人细胞中研究了细胞内钙稳态的各种参数，包括肌管和爱泼斯坦巴尔病毒（EBV）永生化的B淋巴细胞。从患者身上获得细胞，在培养物中扩增并加载成比例的荧光钙指示剂，如Fura-2或indo-1。据报道，由于致病性RYR1突变而改变的参数，包括静息[Ca2 +]，对不同药理激动剂的敏感性以及细胞内Ca2 +储存的大小，可以在单细胞水平上，使用荧光显微镜或使用荧光计在细胞群中测量。然后将从突变携带者的细胞中获得的结果与从健康对照家族成员获得的结果进行比较。这种方法已经证明：（i）许多与MHS相关的突变导致静息[Ca2 +]的增加，并且剂量反应曲线向左移动，以KCl诱导的去极化或药理学RyR1激活与4-氯-m-甲酚10，11，12，13;（ii）与CCD相关的突变导致RyR1的药理学激活释放的峰[Ca2 +]降低，并且如果细胞内Ca2 +存储12，13，14，15，则尺寸减小;（iii）某些变体不影响Ca2 +稳态13。这种实验方法的优点是：RyR1蛋白不过度表达并且存在生理水平，细胞可以被永生化（肌肉细胞和B淋巴细胞）提供含有突变的细胞系。一些缺点与患者可能携带参与钙稳态和/或激发收缩偶联（ECC）的多个编码蛋白的基因突变有关，这可能会使实验结论复杂化。例如，在MHS和对照人群中鉴定出两种JP-45变体，并且它们的存在被证明会影响二氢吡啶受体（DHPR）对激活16的敏感性。患者需要可用，生物材料需要新鲜收集，并且需要从当地道德委员会获得道德许可。
Protocol
下面描述的协议符合Ethikkommission Nordwest- und Zentralschweiz EKNZ的道德准则。 1. 爱泼斯坦巴尔永生化B淋巴细胞系的制备11 在知情同意后，在EDTA处理的无菌管中收集30毫升全血，这些无菌管来自携带RYR1突变的先证者和没有突变的健康家庭成员。注意：保持所有溶液无菌，并在组织培养罩中工作。 通过密度梯度离心培养基（例如，Ficoll-Hypaque，0.077…
Representative Results
[Ca2+]i 在EBV永生化B淋巴细胞群体中的测量原代B淋巴细胞表达RyR1同种型，其在B细胞抗原受体刺激的信号传导过程中起到Ca2+释放通道的作用17。用EBV对B细胞进行永生化，这是遗传学家经常用于获得含有患者基因组信息的细胞系的程序，它提供了在携带RYR1突变的患者中产生表达突变RyR1 Ca…
Discussion
本文中描述的方案已被几个实验室成功用于研究RYR1突变对钙稳态的影响。本文概述的方法的关键步骤涉及无菌性，细胞培养技能和技术以及生物材料的可用性。原则上，EBV永生化的B淋巴细胞的使用更简单，并允许产生含有突变RyR1通道的细胞系。细胞可以冷冻并在液氮中储存多年，并且可以随时重新开始培养。此外，可以选择是在细胞群中还是在单细胞水平上监测钙稳态。前一种方法更简单，不?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本手稿中描述的工作得到了瑞士国家科学基金会（SNF）和瑞士肌肉基金会的资助。
Materials
| 4-chloro-m-cresol | Fluka | 24940 | |
| Blood collection tubes | Sarstedt | 172202 | |
| Bovine serum albumin (BSA) | Sigma-Aldrich | A7906 | |
| caffeine | Merk | 102584 | |
| Cascade 125+ CCD camera | Photometrics | ||
| Cascade 128+ CCD | Photometrics | ||
| Creatine | Sigma-Aldrich | C-3630 | |
| DMEM | ThermoFisher Scientific | 11965092 | |
| DMSO | Sigma | 41639 | |
| EGTA | Fluka | 3778 | |
| Epidermal Growth Factor (EGF) | Sigma-Aldrich | E9644 | |
| Ficoll Paque | Cytiva | 17144002 | |
| Foetal calf serum | ThermoFisher Scientific | 26140079 | |
| Fura-2/AM | Invitrogen Life Sciences | F1201 | |
| Glutamax | Thermo Fisher Scientific | 35050061 | |
| HEPES | ThermoFisher Scientific | 15630049 | |
| Horse serum | Thermo Fisher Scientific | 16050122 | |
| Insulin | ThermoFisher Scientific | A11382II | |
| Ionomycin | Sigma | I0634 | |
| KCl | Sigma-Aldrich | P9333 | |
| Laminin | ThermoFisher Scientific | 23017015 | |
| Lanthanum | Fluka | 61490 | |
| Microperfusion system | ALA-Scientific | DAD VM 12 valve manifold | |
| Origin Software | OriginLab Corp | Software | |
| Pennicillin/Streptomycin | Gibco Life Sciences | 15140-122 | |
| Perfusion chamber POC-R | Pecon | 000000-1116-079 | |
| poly-L-lysine | Sigma-Aldrich | P8920 | |
| RPMI | ThermoFisher Scientific | 21875091 | |
| Spectrofluorimeter | Perkin Elmer | LS50 | |
| Thapsigargin | Calbiochem | 586005 | |
| Tissue culture dishes | Falcon | 353046 | |
| Tissue culture flask | Falcon | 353107 | |
| Tissue culture inserts | Falcon | 353090 | |
| Trypsin/EDTA solution | ThermoFisher Scientific | 25300054 | |
| Visiview | Visitron Systems GmbH | Software | |
| Zeiss Axiovert S100 TV microscope | Carl Zeiss AG | ||
| Zeiss glass coverslips | Carl Zeiss AG | 0727-016 |
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Cite This Article
Treves, S., Girard, T., Zorzato, F. Functional Characterization of Endogenously Expressed Human RYR1 Variants. J. Vis. Exp. (172), e62196, doi:10.3791/62196 (2021).