一种半自动且可重现的基于生物学的体外定量钙沉积的方法
Summary
心血管疾病是全世界死亡的主要原因。血管钙化大大加重了心血管发病率和死亡率的负担。该协议描述了一种通过荧光成像在 体外 量化血管平滑肌细胞介导的钙沉淀的简单方法。
Abstract
血管钙化涉及一系列退行性病变，包括炎症、细胞表型改变、细胞死亡和钙化抑制剂的缺乏，伴随导致血管弹性和功能丧失。血管钙化是许多疾病（包括慢性肾脏病、糖尿病和动脉粥样硬化）发病率和死亡率的重要因素。目前研究血管钙化的研究模型是有限的，仅在体内钙化发展的后期才可行。用于研究血管钙化的体外工具使用终点测量，增加了对生物材料的需求，并有可能将变异性引入研究。我们展示了一种新型荧光标记探针的应用，该探针与人体血管平滑肌细胞的体外钙化发展结合，并确定体外钙化的实时发展。在该协议中，我们描述了我们新开发的钙化测定的应用，这是一种具有潜在转化应用的疾病建模新工具。我们设想该测定法与更广泛的矿物沉积研究相关，包括骨骼、软骨或牙科研究的应用。
Introduction
血管钙化 （VC） 是心血管疾病发病率和死亡率的独立危险因素1，2，3。长期以来被认为是异位矿物沉积的被动化学过程，现在它似乎是一种可改变的组织愈合反应，涉及各种细胞的积极贡献，包括活化的血管平滑肌细胞（hVSMC）作为疾病的驱动因素4，5。体内VC可以通过多层CT扫描来测量，作为动脉粥样硬化负荷的评估6，7，8。目前，范式转变正在进行中，其中VC严重程度被认为是心血管疾病，II型糖尿病，慢性肾病和衰老的危险因素9，10，11，12，13，14，15。
hVSMC是心血管系统中最丰富的细胞类型，也是VC发展的主要参与者。 体外 hVSMC诱导的钙化是研究心血管疾病的广泛使用的疾病模型16，17。然而，大多数用于体 外 钙化检测的方案使用终点测量，这可能会限制数据采集，需要更多地使用细胞材料，并且可能会减慢研究速度。检测 体外 hVSMC钙化的常用方法包括邻甲酚酞测定法，该测定法测量溶解钙沉积与总蛋白的关系，需要细胞裂解18。此外，使用茜素红染色，其直接与固定细胞或组织上的钙沉积物结合19。要研究邻甲酚酞或茜素红随时间推移的hVSMC钙化，每个时间点需要多次重复，这增加了对生物材料的需求，进而增加了变异性的机会。
在本文中，我们详细介绍了一种新型测定的应用方法，该测定利用hVSMC和荧光成像探针来确定 体外 VC进展，并用作单一终末期钙化测定。我们之前证明，该测定可与邻甲酚酞和茜素红方法直接相媲美，可用于区分不同的培养条件20。除了实时测量外，该测定还可用于确定血清或血浆样品作为临床VC开发的替代标志物的倾向20。这将有助于应用心血管科学和疾病建模的生物学策略。该测定的进一步应用可能是作为转化生物混合系统来评估VC的严重程度或血液成分（如血清或血浆）的进展。
Protocol
1. 细胞接种、维持和钙化诱导 要培养原代细胞，请使用层流气流柜、手套和无菌设备。在进行任何工作之前和之后对手和工作区进行消毒。将所有原代细胞和培养基视为潜在的生物危害，除非另有证明。优选在处置前高压灭菌剩余细胞和培养基。不要化学灭活和高压灭菌，因为这会释放有毒烟雾。 在未包被的细胞培养板上培养hVSMC。 在补充有10%-20S和1%Pen / Str…
Representative Results
结果包括HOECHST染色细胞核的原始图像，RFP标记的钙化和明场图像。可以检测和分析从低（图2）到高（图3）的不同钙化阶段。钙化通常可以使用光学显微镜发现为黑色斑点（图 2D 和图 3B，箭头表示钙化），这对于初步评估和确定何时开始影像学检查很有用。为了提高信噪比，应分析处理后的RFP图像以量化钙…
Discussion
在本手稿中，我们描述了一种用于 体外 钙化测定的半自动方法。对于这种方法，应优化hVSMC钙化的三个关键步骤。首先，细胞密度对于hVSMC钙化的发展至关重要。由于缺乏细胞间接触以及在钙化条件下诱导的应激，hVSMC的低密度将导致缓慢或无钙化和细胞死亡21。高细胞密度导致过度融合，之后细胞衰老22 ，钙化发育停止。在孔板中播种大约70%的汇合度至…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究由欧盟的地平线2020研究和创新计划资助，根据玛丽·斯克洛多夫斯卡-居里赠款协议第722609和764474，NWO ZonMw（MKMD 40-42600-98-13007）。这项研究得到了BioSPX的支持。WJ-D获得德国研究基金会TRR219项目ID 322900939和项目ID 403041552的资助
Materials
| Calcium chloride, 93%, anhydrous | Thermo Fisher Scientific | 349615000 | |
| Costar 6-well Clear TC-treated well plates | Corning | 3516 | |
| Cytation 3 System | BioTek, Abcoude, The Netherlands | ||
| Fetal Bovine Serum | Merck | F7524-100ML | |
| Fetuin-A-Alexa Fluor-546 | Prepared in-house | ||
| Gen5 Software v3.10 | BioTek | ||
| Gibco Medium 199 | Thermo Fisher Scientific | 11150059 | |
| Hoechst 33342, Trihydrochloride | Thermo Fisher Scientific | H3570 | |
| PBS (10X), pH 7.4 | Thermo Fisher Scientific | 70011044 | |
| Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | |
| Trypsin-EDTA (0.05%), phenol red | Thermo Fisher Scientific | 25300062 |
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Citar este artigo
Jaminon, A. M. G., Rapp, N., Akbulut, A. C., Dzhanaev, R., Reutelingsperger, C. P., Jahnen-Dechent, W., Schurgers, L. J. A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro. J. Vis. Exp. (184), e64029, doi:10.3791/64029 (2022).