慢性炎症状态个体的单核细胞轮回和泡沫形成的定量研究
Summary
我们描述了一项协议, 以测量单核细胞在人的内皮单分子膜的轮回, 并随后成熟成泡沫电池。这提供了一种通用的方法来评估的动脉粥样硬化性质的人单核细胞分离的不同疾病的情况下, 并评估血液中的因素, 可能会提高这种倾向。
Abstract
冠状动脉疾病 (CAD) 是全球发病率和死亡率的主要原因。动脉粥样硬化是 CAD 的一个主要原因, 它是由先天免疫单核细胞的轮回所引发的, 它被称为脂肪条纹的脂质的炎症部位, 它们存在于中到大动脉的动脉壁上。动脉粥样硬化早期病变的主要致病特征是单核细胞的成熟, 它会迁移到动脉形成泡沫或脂质巨噬细胞。大量的证据支持这样的假说: 动脉粥样硬化的风险是由慢性炎症性疾病, 如类风湿关节炎和 HIV, 以及一般的老化, 以及这种风险是由单核细胞预测激活.尽管鼠标模型提供了一个很好的平台来研究单核细胞在动脉粥样硬化体内的作用, 但它们需要对天然胆固醇代谢进行基因改变和对正常小鼠饮食的剧烈改变, 并且对人类共疾病的动脉粥样硬化影响研究。这促使我们开发了一个人的体外模型, 以测量孤立于定义疾病状态的个体的单核细胞的动脉粥样硬化潜能。目前, 人类的体外模型是限制的, 他们评估单核细胞的轮回和泡沫形成的孤立。在这里, 我们描述了一个协议, 其中单核细胞从病人的血液刺字跨人的内皮细胞膜到1型胶原基质, 他们的倾向成熟成泡沫细胞存在或没有外源性脂质测量。该议定书已得到验证, 用于使用从艾滋病毒感染者和老年艾滋病毒感染者身上纯化的人单核细胞。这个模型是多才多艺的, 并且允许单核细胞的移居和泡沫形成被评估使用显微镜或流式细胞仪并且允许评估动脉粥样硬化因素存在于血清或血浆。
Introduction
单核细胞轮回是动脉粥样硬化斑块发展的关键一步, 可能导致血栓、中风和心肌梗塞。动脉粥样硬化斑块发育的脂肪条纹, 一般目前在低振荡血流在中到大的动脉, 在那里沉积脂质有助于内皮细胞活化和局部炎症1。单核细胞通过单核趋蛋白 (如 CCL2) 和刺字进入内膜2, 被吸收到脂肪条纹中的内皮。在轮回之后, 单核细胞可能形成动脉粥样硬化的、脂质的巨噬细胞, 称为脂质的摄取、脂质合成、胆固醇外流调节或上述因素的综合作用的结果。单核细胞也可以在循环中积聚脂质, 并有一个泡沫状的表型, 可能是发泡细胞形成的诱因3,4。泡沫细胞是脂肪条纹和早期动脉粥样硬化斑块的决定性特征, 其形成受脂质和炎症介质的影响5。另外, 单核细胞有能力逆转刺字从动脉进入血流6, 从而消除脂质从内膜和行动, 以保持健康的动脉。
确定单核细胞在动脉内皮刺字的倾向, 在内膜内形成泡沫细胞, 或逆转刺字并携带脂质, 是了解细胞活化在增加动脉粥样硬化风险。小鼠模型的 cad, 如动脉粥样硬化是重要的阐明 real-time在体内信息的脂肪条纹/动脉粥样硬化斑块的发展。然而, 这些模型需要基因改变这些动物的自然胆固醇处理能力通常加上剧烈的改变饮食 (如载脂蛋白/西方型饮食模式)7,8, 从而,诱导 non-physiological 积累的循环脂水平, 推动斑块的发展。这些模型可能与慢性炎症性人类疾病的相关性有限, 如 HIV 感染, 而不与增加循环胆固醇或低密度脂蛋白 (LDL) 水平有关。此外, 人与小鼠之间单核细胞生物学的差异, 使得对单核细胞亚群 (如中间单核细胞 (CD14++CD16+)) 的相关性的免疫学问题的测试,9困难.这是重要的, 当研究的机制驱动心血管疾病作为中间单核细胞计数独立预测心血管事件10,11。虽然化验存在顺序测量单核细胞的轮回或泡沫形成的隔离, 没有体外试验已验证, 以量化两方面的早期动脉粥样硬化使用相同的细胞从临床组。Transwell 模型利用一个改进的博伊登两室系统, 其中细胞被加载到顶部室和刺字跨多孔塑料屏障或细胞单层到一个较低的会议厅, 通常包含具有趋化因子12的媒体,13. 虽然广泛用于分析白细胞的轮回, 但这些模型一般不包含一层代表内膜, 导致轮回细胞迁移到溶液中, 不允许测量泡沫细胞相同细胞的形成或反向轮回。相反, 泡沫细胞形成的模型不解释任何 transmigratory 引起的单核细胞的改变或内皮激活的影响, 这是已知的贡献泡沫电池形成14。此外, 这些系统诱导泡沫细胞形成从附着在细胞培养板上的饱和浓度的外源氧化低密度脂蛋白 (oxLDL)15,16, 一个关键的诱导剂泡沫细胞的形成。这些模型中使用的 LDL 通常被 non-physiologically 相关的过程 (如丘索4治疗17) 所氧化, 因此, 使用这些模型来质疑研究的生理学重要性。
在这里, 我们描述了一个定量分析, 量化单核细胞的轮回和泡沫细胞的形成, 不需要添加外源性 oxLDL, 从而更好地模拟单核在泡沫细胞形成中的作用。这个模型最初是由威廉. 穆勒教授 (西北大学, 芝加哥)18开发的, 并且在我们的实验室中得到了进一步的改进, 以评估ex 体内atherogenicity 下单核细胞在 non-activating 下的分离与患动脉粥样硬化的风险增加相关的疾病, 如 HIV 感染19以及衰老的20的患者的条件。该模型还提供了一个平台, 回答基本的生物学问题的倾向, 不同的单核细胞亚群形成泡沫电池20, 细胞因子, 如 TNF 对泡沫细胞形成的影响, 内皮激活14, 以及在凝胶中轮回的深度和速度等单核细胞的迁移特性19。此外, 单核细胞的轮回和泡沫形成可以量化使用标准显微镜, 活细胞成像, 流式细胞仪和成像流式细胞仪, 因此, 提供了一个多用途的方法来评价单核在动脉粥样硬化的作用。
Protocol
Representative Results
Discussion
这里描述的协议提供了一个多才多艺的和生理学相关的方法来评估的 atherogenicity 单核细胞从人的临床组, 结合单核的轮回和泡沫形成。这种模式提供了比其他泡沫细胞形成的方法的优势, 因为它考虑到单核细胞的轮回对泡沫形成的影响, 并允许测量反向轮回6除了固有的在外源性因素存在或缺乏的情况下, 单核细胞倾向于成熟成泡沫。此外, 内皮活化的作用也被考虑到, 因为我们已?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者衷心感谢 Prof. 和 Dr. 克莱尔 Westhorpe 的工作, 因为他们在开发早期的迭代模型中扮演了关键的角色。作者还要感谢 AMREP 流式细胞仪核心的分单核分子子集和阿尔弗雷德医院传染病组的临床研究护士招募艾滋病毒 + 个人的一些研究。作者衷心感谢伯内特研究所收到的维多利亚运营基础设施支持计划的这项工作的贡献。TAA 得到 RMIT 大学校长博士后奖学金的支持。这项工作得到了澳洲项目补助金1108792授予 AJ 和 AH。传统知识得到 nih 资助 nih K08AI08272、nih/NCATS 补助金 UL1TR000124。
Materials
| Gel preparation reagents | |||
| NaOH | Sigma-Aldrich | 221465-500G | 0.1 M NaOH diluted in H20 |
| 10X M199 | Sigma-Aldrich | M0650 | |
| AcCOOH | Sigma-Aldrich | 695092-100ML | 20 mM Acetic acid diluted in H20 |
| Cultrex Bovine Collagen I | R&D Systems | 3442-050-01 | Type I Fibrous Collagen |
| Name | Company | Catalog Number | Comments |
| Cell culture | |||
| M199 | Life Technologies | 11150-059 | M199 media containing Earle's salts, L-glutamine and 2.2 g/L Sodium Bicarbonate. Media supplemented with 100 µg/mL L-glutamine and 100 U/mL penicillin/streptomycin |
| M20 | Supplemented M199 containing 20% heat-inactivated pooled or donor serum. Individual lipid species such as LDL can be added to M199. |
||
| HUVEC | Primary human umbilical cord endothelial cells (HUVEC) can be isolated from umbilical cords donated with informed consent and ethics approval. Isolated HUVEC may also be purchased commercially. | ||
| Human coronary artery endothelial cells | Primary human coronary artery endothelial cells can be isolated from arteries donated with informed consent and ethics approval. Isolated cells may also be purchased commercially. | ||
| EDTA | BDH Merck | 10093.5V | Ethylenediaminetetraacetic acid (EDTA) – 0.5 M, pH 8.0 |
| EGTA | Sigma-Aldrich | E3889-500G | Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) – 1 mM, pH 8.0 |
| 0.05% trypsin EDTA | Gibco | 25300-054 | 0.05% trypsin/0.53 EDTA (1X) |
| L-glutamine | Gibco | 25030-081 | L-glutamine (200 mM) |
| Penicillin/streptomycin | Gibco | 15140-122 | Penicillin/streptomycin (10,000 units/mL Penicillin and 10,000 µg/mL Streptomycin) |
| New born calf serum | Gibco | 16010-142 | New born calf serum: New Zealand origin |
| Fibronectin | Sigma-Aldrich | F1056-1MG | Fibronectin – 50 µg/mL aliquots prepared and stored |
| PBS (1X) | Gibco | 14200-075 | Dulbecco's Phosphate Buffered Saline (10X): Dilute to 1X with sterile H20 |
| 0.1% TNF | Gibco | PHC3015 | Recombinant human TNF – Reconstituted in H20 and stored in 10 µg/mL aliquots |
| Low-density lipoprotein | Merck Millipore | LP2-2MG | Low-density lipoprotein (LDL) |
| Name | Company | Catalog Number | Comments |
| Microscopy | |||
| 1 or 2% formaldehyde | Polysciences | 4018 | 1 or 2% formaldehyde diluted with sterile H20 |
| 50% and 78% methanol | Ajax Finechem | 318-2.5L GL | 50% or 78% v/v methanol, diluted with H20 |
| Oil Red O stain | Sigma-Aldrich | O0625-25G | Dilute to 2 mg/mL in 22% 1M NaOH and 78% (v/v) methanol |
| Microscope slides | Mikro-Glass | S41104AMK | Twin frosted 45 degree ground edge microscope slides (25 X 76 mm) |
| Cover slips | Menzel-Gläser | MENCS224015GP | 22 x 40 mm #1.5 size glass cover slips |
| Double-sided tape | 3M Scotch | 4011 | Super strength exterior mounting tape (25.4 mm x 1.51 m) |
| Giemsa stain | Merck Millipore | 1.09204.0500 | Giemsa's azur eosin methylene blue solution (dilute stock 1:10 in H20) |
| Hole punch | Hand-held single hole punch (6.35 mm punch) | ||
| Name | Company | Catalog Number | Comments |
| Flow cytometry | |||
| Collagenase D | Roche Diagnostics | 11088858001 | Collagenase D diluted in M199 media to 1 mg/mL |
| 35 µm nylon mesh capped polystyrene FACS tubes | BD Biosciences | 352235 | 35 µm nylon mesh capped polystyrene FACS tubes |
| Live/Dead Fixable Yellow Dead Cell stain | Life Technologies | L34959 | Live/Dead Fixable Yellow Dead Cell stain |
| FACS wash | Prepare by mixing 1 X PBS–, 2 mM EDTA and 1% New born calf serum | ||
| Name | Company | Catalog Number | Comments |
| Plasticware | |||
| 96 well plate | Nunclon | 167008 | Delta surface flat-bottomed 96 well plate |
| 10 cm Petri Dish | TPP | 93100 | Sterile 10 cm Petri dish |
| 1.5 mL Eppendorf tubes | Eppendorf | 0030 125.150 | Eppendorf tubes |
| Transfer pipette | Samco Scientific | 222-20S | Sterile transfer pipette (1 mL, large bulb) |
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