肝组织3D细胞培养模型中组蛋白翻译后修饰的全球水平定量
Summary
该协议概述了如何使用三维细胞培养系统在接近生理状态下对染色质修饰进行建模，治疗和分析。
Abstract
哺乳动物细胞的平面培养是一种广泛用于了解细胞生理学 的体外 方法，但由于细胞复制不自然的快速，该系统在模拟固体组织方面受到限制。在模拟成熟染色质时，这尤其具有挑战性，因为快速复制的细胞经常参与DNA复制并且具有异质多倍体群体。下面介绍的是使用三维（3D）细胞培养系统建模，处理和分析静态染色质修饰的工作流程。使用该协议，肝细胞癌细胞系在培养箱中作为可重复的3D球体生长，提供活性营养扩散和低剪切力。用丁酸钠和琥珀酸钠治疗分别诱导组蛋白乙酰化和琥珀酰化的增加。组蛋白乙酰化和琥珀酰化水平的增加与更开放的染色质状态有关。然后收集球状体以分离细胞核，从中提取组蛋白以分析其翻译后修饰。组蛋白分析 通过 液相色谱法在线与串联质谱法相结合，然后进行内部计算管道。最后，显示了数据表示的示例，以研究组合组蛋白标记的频率和发生。
Introduction
自19世纪末 以来，细胞培养系统已被用作研究人体外细胞生长和发育的模型1，2。它们的使用也已扩展到研究组织和器官如何在健康和患病环境中发挥作用1，3。悬浮细胞（例如，血细胞）在培养皿或烧瓶中无缝且可互换地生长，因为它们 在体内不以三维（3D）结构组装。来自实体器官的细胞可以在二维（2D）或3D培养系统中生长。在2D培养中，细胞在粘附在平坦表面的单层中生长2，4。2D细胞培养系统的特点是指数增长和快速倍增时间，通常为24小时至几天5。3D系统中的细胞生长形成复杂的细胞 – 细胞相互作用，更紧密地模拟组织样砾岩，并且它们的特征在于它们能够达到动态平衡，其中它们的倍增时间可以达到1个月或更长时间5。
本文介绍了一种在模拟重力降低的旋转细胞培养系统中培养3D球体的创新方法6。这是NASA在1990年代推出的细胞培养系统的简化衍生物7。这种方法可以最大限度地减少现有方法（如旋转烧瓶）中发生的剪切力，并提高球体再现性6。此外，旋转生物反应器增加了活性营养物质扩散，最大限度地减少了在悬挂式滴细胞培养等系统中发生的坏死形成，其中培养基交换是不切实际的6。通过这种方式，细胞生长大多不受干扰，允许形成与组织中生长的细胞相关的结构和生理特征。以这种方式培养的C3A肝细胞（HepG2 / C3A）不仅具有超微结构细胞器，而且还产生与体内观察到的水平相当的ATP，腺苷酸激酶，尿素和胆固醇的量1，2。此外，在2D与细胞培养系统中生长.3D细胞培养系统表现出不同的基因表达模式8。作为3D球体生长的C3A肝细胞的基因表达分析表明，这些细胞表达广泛的肝脏特异性蛋白，以及参与调节肝功能的关键途径的基因8。先前的出版物证明了2D培养物中指数增长细胞的蛋白质组与3D球体培养物中动态平衡的细胞之间的差异5。这些差异包括细胞代谢，这反过来又会影响细胞5的结构，功能和生理学。在2D培养物中生长的细胞的蛋白质组在参与细胞复制的蛋白质中更富集，而3D球体的蛋白质组在肝功能中更丰富5。
作为3D球体生长的细胞的较慢复制速率更准确地模拟与染色质状态和修饰相关的特定现象（例如组蛋白剪切9）。组蛋白剪切是一种不可逆的组蛋白翻译后修饰（PTM），可导致组蛋白N端尾部部分蛋白水解裂解。虽然它的生物学功能仍在讨论10，11，12，13，但很明显，它在原代细胞和肝脏组织中的存在是由作为球状体生长的HepG2 / C3A细胞模拟的，而不是扁平细胞9。这是至关重要的，因为染色质状态和修饰主要通过调节基因的可及性来调节DNA读数，从而调节它们的表达14。组蛋白PTM要么通过影响组蛋白组装的核小体的净电荷来直接影响染色质状态，要么通过招募染色质写入者，读取器和橡皮擦间接影响染色质状态14。迄今为止，已经鉴定出数百个组蛋白PTM15，这加强了染色质具有细胞用来解释DNA16的“组蛋白代码”的假设。然而，无数PTM组合15的鉴定，以及组蛋白PTM组合通常与孤立存在的PTM具有不同的生物学功能的发现（例如Fischle等人17），强调了需要更多的工作来解密“组蛋白代码”。
目前，组蛋白 PTM 分析要么基于利用抗体（例如，蛋白质印迹、免疫荧光或染色质免疫沉淀，然后进行测序 [ChIP-seq]）的技术，要么基于质谱 （MS） 的技术。基于抗体的技术具有高灵敏度，可以提供关于组蛋白标记全基因组定位的详细信息，但在研究以组合形式存在的罕见PTM或PTM时通常受到限制18，19，20。MS更适合于单一和共存蛋白质修饰的高通量和无偏倚的鉴定和定量，特别是组蛋白18，19，20。由于这些原因，该实验室和其他几个实验室已经优化了MS管道，用于分析组蛋白肽（自下而上的MS），完整的组蛋白尾巴（中下MS）和全长组蛋白（自上而下的MS）21，22，23。
下面详细介绍了用于生长HepG2 / C3A球体的工作流程， 并通过 纳米液相色谱法在线结合串联质谱（nLC-MS / MS）制备它们以进行组蛋白肽分析（自下而上的MS）。培养2D细胞培养物，收获细胞并将其转移到生物反应器中，在那里它们将开始形成球体（图1）。培养18天后，用丁酸钠或琥珀酸钠处理球状体，以增加组蛋白乙酰化和琥珀酰化相对丰度。值得注意的是，3D培养物可以用遗传毒性化合物及其扁平培养物等价物进行处理;事实上，最近的出版物强调，3D培养物中细胞的毒理学反应比2D平板培养物中的细胞更类似于原代组织24，25。然后在指定的时间点收集细胞并进行核分离。然后，根据Garcia等人首先开发的方案，在胰蛋白酶消化之前和之后提取组蛋白并用丙酸酐衍生化。该过程产生适当大小的肽，用于反相色谱（C18）的在线分离和MS检测。最后，组蛋白肽被鉴定和定量，并且生成的数据以多种方式表示，以获得更完整的生物学解释。
Protocol
1. 缓冲液和试剂的制备 细胞生长培养基（用于HepG2 / C3A细胞）：将胎牛血清（FBS）（10%v / v），非必需氨基酸（1%v / v），L-谷氨酰胺补充剂（1%v / v）和青霉素/链霉素（0.5%v / v）添加到Dulbecco的改良鹰培养基（DMEM，含有4.5 g / L葡萄糖）中。生长培养基在4°C下储存最多2周。 200 mM丁酸钠（NaBut）溶液：制备10 mL，将220.18mg NaBut重悬于10 mL ddH2O中，在-20°C下储存1mL等分?…
Representative Results
在该方案中，用20 mM NaBut和10 mM NaSuc处理HepG2 / C3A球体，两者都影响组蛋白PTM的全局水平（图3A）。然后 通过 MS / MS采集在单个残留水平上鉴定和定量组蛋白PTM（图3B）。 当样品在重复中运行时，可以进行统计分析以评估样品之间PTM的倍变富集以及观察的可重复性。显示的数据表明，用乙酰化修饰的肽在用NaBut处理的球状体中?…
Discussion
组蛋白PTM的分析与典型的蛋白质组学分析管道有根本的不同。大多数组蛋白PTM仍然具有神秘的生物学功能;因此，诸如基因本体论或通路数据库之类的注释不可用。存在几种资源将组蛋白修饰与负责其催化的酶或含有结合这些PTM的结构域的蛋白质（例如，HISTome36）相关联。同样，当组蛋白PTM的全球水平受到调节时，可以推测染色质的整体状态。例如，组蛋白乙酰化或其他酰化（如…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Sidoli实验室感谢白血病研究基金会（Hollis Brownstein新研究员研究资助），AFAR（Sagol Network GerOmics奖），Deerfield（Xseed奖），Relay Therapeutics，Merck和NIH主任办公室（1S10OD030286-01）。
Materials
| 0.05% trypsin-EDTA solution | Gibco | 25300054 | |
| 0.5-20 µL pipet tips | BRAND | 13-889-172 (Fisher Scientific) | |
| 1.5 mL microcentrifuge tubes | Bio-Rad | 2239480 | |
| 10 µL multi-channel pipette | BRAND | BR7059000 (Millipore Sigma) | |
| 10 mL syringe | Henke Sass Wolf | 14-817-31 (Fisher Scientific) | Luer lock tip, graduated to 12 mL |
| 10, 20, 200, and 1000 µL single-channel pipettes | Eppendorf | 14-285-904 (Fisher Scientific) | |
| 1000 µL pipet tips | Rainin | 30389164 | |
| 18 G syringe needle | Air-Tite | 14-817-100 (Fisher Scientific) | 3" length, 0.05" diameter |
| 200 µL multi-channel pipette | Corning | 4082 | |
| 2-200 µL pipet tips | BRAND | Z740118 (Millipore Sigma) | |
| 24-well ultra-low attachment microplate | Corning | 07-200-602 | |
| 75 cm2 U-shaped cell culture flask | Corning | 461464U | Untreated, with vent cap |
| 96-well skirted plate | Axygen | PCR-96-FS-C (Corning) | |
| Acetone | Fisher Scientific | A949-1 | Acetone should be used cold |
| Ammonium bicarbonate (NH4HCO3) | Sigma-Aldrich | A6141-25G | |
| Ammonium hydroxide solution | Fisher Scientific | AC423300250 | |
| Cell culture grade water | Corning | 25-055-CV | |
| ClinoReactor | CelVivo | 10004-12 | Bioreactor for 3D cell culture |
| ClinoStar | CelVivo | N/A | Clinostat CO2 incubator for 3D cell culture |
| Control unit | CelVivo | N/A | Tablet for ClinoStar settings |
| Dulbecco's Modified Eagle's Medium (DMEM) | Corning | 17-205-CV | 1X solution with 4.5 g/L glucose and sodium pyruvate, without L-glutamine and phenol red |
| Fetal bovine serum (FBS) | Corning | 35-010-CV | |
| Formic acid | Thermo Scientific | 28905 | |
| Fume hood | Mott | N/A | Model 7121000 |
| Glass Pasteur pipette | Fisher Scientific | 13-678-8B | 9", cotton-plugged, borosilicate glass, non-sterile |
| Glutagro supplement | Corning | 25-015-CI | 200 mM L-ananyl-L-glutamine |
| Hank’s Balanced Salt Solution (HBSS) | Corning | 21-022-CV | 1X solution without calcium, magnesium, and phenol red |
| HPLC grade acetonitrile | Fisher Scientific | A955-4 | |
| HPLC grade water | Fisher Scientific | W6-1 | |
| Hydrochloric acid (HCl) | Fisher Scientific | A481-212 | |
| Ice | N/A | N/A | |
| MEM non-essential amino acids | Corning | 25-025-CI | 100X solution |
| Oasis HLB resin | Waters | 186007549 | Hydrophilic-Lipophilic-Balanced (HLB) Resin with 30µm particle size |
| Orbitrap Fusion Lumos Tribrid mass spectrometer | Thermo Fisher Scientific | IQLAAEGAAPFADBMBHQ | High resolution mass spectrometer |
| Oro-Flex I polypropylene filter plate | Orochem | OF1100 | 96-well polypropylene filter plate w/ 10 µM PE frit |
| Penicillin-Streptomycin | Corning | 30-002-CI | 100X solution |
| pH paper | Hydrion | Z111848 (Sigma-Aldrich) | 0-13 pH test paper |
| Pipette gun | Eppendorf | Z666467 (Millipore Sigma) | |
| Polymicro capillary | Molex | 50-110-7740 (Fisher Scientific) | Flexible fused silica capillary tubing with polymide coating, 75 µM ID x 363 µM OD |
| Polystyrene 10 mL serological pipets, sterile | Fisher Scientific | 1367549 | |
| Propionic anhydride | Sigma-Aldrich | 240311-50G | |
| Refrigerated centrifuge | Thermo Scientific | 75-217-420 | |
| Reprosil-Pur resin | MSWIL | R13.AQ.0003 | 120 Å pore size, C18-AQ phase, 3 µM bead size |
| Rotator | Clay Adams | 25477 (American Laboratory Trading) | Nutator Mixer 1105 |
| Sequencing grade modified trypsin | Promega | V5111 | |
| Sodium butyrate | Thermo Scientific | A11079 | |
| Sodium succinate dibasic | Sigma-Aldrich | 14160-100G | |
| SpeedVac vacuum concentrator (1.5 mL microcentrifuge tubes) | Savant | 20249 (American Laboratory Trading) | |
| SpeedVac vacuum concentrator (96-well) | Thermo Scientific | 15308325 | Savant SPD1010 |
| Sterile hood | Thermo Scientific | 1375 | Class II, Type A2 |
| Sulfuric acid (H2SO4) | Fisher Scientific | 02-004-375 | Baker Analyzed ACS reagent |
| Tissue-culture treated 100 mm x 20 mm dish | Fisher Scientific | 08-772-23 | |
| Trichloroacetic acid (TCA) | Thermo Scientific | AC421451000 | Resuspend 100% w/v in HPLC grade water |
| Trifluoroacetic acid (TFA) | Fisher Scientific | PI28904 | Sequencing grade |
| Vacuum manifold 96-well | Millipore | MAVM0960R | |
| Vortex | Sigma-Aldrich | Z258415 | |
| Water bath | Fisher Scientific | FSGPD10 | |
| Wide bore pipet tips 1000 µL | Axygen | 14-222-703 (Fisher Scientific) | |
| Wide bore pipet tips 200 µL | Axygen | 14-222-730 (Fisher Scientific) |
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Cite This Article
Joseph-Chowdhury, J. N., Stransky, S., Graff, S., Cutler, R., Young, D., Kim, J. S., Madrid-Aliste, C., Aguilan, J. T., Nieves, E., Sun, Y., Yoo, E. J., Sidoli, S. Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue. J. Vis. Exp. (183), e63606, doi:10.3791/63606 (2022).