线粒体功能在癌症相关疲劳中的作用评价
Summary
我们的目标是制定一个实用的协议, 以评估线粒体功能障碍与疲劳相关的癌症患者。这种创新的协议是优化的临床使用只涉及标准放血和基本的实验室程序。
Abstract
疲劳是影响大多数癌症患者的常见和衰弱的条件。到目前为止, 疲劳仍然很低的特点, 没有诊断测试, 客观地测量这种情况的严重性。在这里, 我们描述了一个最优化的方法来评估 PBMCs 的线粒体功能收集从疲劳的癌症患者。利用紧凑的胞外通量系统和顺序注射呼吸抑制剂, 我们通过测量基底线粒体呼吸、剩余呼吸能力和能量表型来检查 PBMC 线粒体功能状态, 描述应对压力的首选能量通路。新鲜 PBMCs 是现成的临床设置使用标准放血。本议定书所描述的整个化验方法在不到4小时内就可以完成, 而不需要复杂的生化技术。此外, 我们还描述了获取可重现数据所需的规范化方法。所提出的简单程序和规范化方法允许重复样本收集从同一病人和生成可重复的数据, 可以比较时间点, 以评估潜在的治疗效果。
Introduction
疲劳是一种普遍和令人痛心的情况, 对癌症患者的生活质量有负面影响1。到目前为止, 癌症疲劳的定义仍然很差, 仅依靠患者的主观报告2。因此, 迫切需要确定一个易于适应的诊断实验室测试, 客观地描述疲劳的临床设置3,4。
许多潜在的机制, 包括线粒体功能障碍, 已被建议导致疲劳的5。线粒体是发电站的细胞器, 通过氧化磷酸化提供95% 的蜂窝能量需求, 并在钙信号、凋亡、免疫信号和调节其他细胞内信号事件中发挥重要作用6.因此, 线粒体生物能学受损和能源生产缺陷可能导致疲劳。支持这个假说, 以前的研究观察了线粒体 DNA 在慢性疲劳综合征患者中的突变7。尽管目前尚不清楚疲劳的病理生理学起源是否存在于中枢神经系统或周边组织中, 例如骨骼肌8,9, 当前还没有直接的方法来准确评估线粒体功能障碍与活着的 respiring 细胞的疲劳有关。
利用外周血单个核细胞 (PBMCs) 研究线粒体功能提供了一些优势。首先, PBMCs 是现成的在临床设置使用标准的放血, 可以快速隔离使用基本的实验室技术。其次, 血液收集比收集其他组织如肌肉活检更少侵入。因此, 血液样本可以收集从同一病人反复一段时间, 这有助于纵向评估治疗效果。有趣的是, PBMCs 的线粒体功能在动物模型10中似乎与肾脏线粒体状态有很好的相关性。此外, 免疫细胞线粒体已被用于检测不同疾病条件下的系统性变化11,12。循环免疫细胞中的线粒体对免疫功能和免疫信号分子的变化特别敏感, 如细胞因子13,14,15。例如, 有人观察到, 急性风湿性炎症疾病患者的 PBMCs 表现为高基线耗氧量14。相比之下, PBMCs 与全身炎症性疾病 (包括脓毒症16) 隔离的患者的氧耗减少。在炎症条件下, 功能失调的线粒体产生的自由基可能进一步促进氧化应激和长时间炎症17。线粒体在能源生产和氧化应激中的中心作用表明, 利用线粒体功能作为研究癌症患者疲劳的一个重要因素, 可能是13。
以前的研究检测线粒体功能使用生物化学技术, 线粒体膜电位测量, 或隔离特定的细胞群体, 可能不容易适应在临床设置5, 14,18。近年来, 细胞外通量检测的发展使研究人员能够轻松准确地检查氧气消耗率 (OCR) 对呼吸抑制剂自动注射的影响19,20,21,22. 然而, 大多数这些研究都是针对特定的细胞类型而设计的, 而大容量的高通量格式可能不适用于临床设置。在这篇手稿中, 我们描述了一个优化的协议, 以检查线粒体功能的临床使用。
Protocol
目前的研究 (NCT00852111) 得到了马里兰国家卫生研究院 (NIH) 机构审查委员会的批准。参加这项研究的参与者为18岁或以上的 euthymic 男性, 他们被诊断为无转移性前列腺癌, 有或没有事先前列腺切除术, 并计划接受外照射治疗 (外照射)。潜在的参与者被排除, 如果他们有一个渐进性疾病, 可能导致严重的疲劳, 在过去五年的精神疾病, 有未矫正甲状腺功能减退或贫血, 或有第二次恶性肿瘤。使用镇静剂、?…
Representative Results
在对不同的呼吸抑制剂进行序贯注射后, 图面应力测试依赖于测量氧气消耗率 (OCR) 来映射一个完整的线粒体剖面。每种药物注射后的 OCR 测量可用于计算以下与线粒体健康相关的参数:基底 OCR首先在任何药物注射前测量, 以评估满足静止水平 ATP 需求所需的氧气消耗。基础呼吸是通过在寡霉素注射液前减去基线 OCR 的非线粒体呼吸率来计算的。其次, 寡霉素被注?…
Discussion
癌症患者的疲劳是一个虚弱的状况, 没有很好的定义或特征1。疲劳诊断完全依赖于主观报告, 目前还没有诊断标准或治疗, 这主要是由于在其病理2中缺乏了解。在癌症患者所提出的疲劳机制中, 线粒体功能受损是治疗多弹头的最主要途径之一。因此, 我们开发了一种快速实用的方法来测量临床样品中的线粒体功能, 可用于主动识别患癌症治疗相关毒性包括疲劳的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到了马里兰国家卫生研究院国立护理研究所内部研究司的充分支持。
Materials
| CPT Mononuclear Cells Preparation Tube | BD Biosciences | 362761 | For isolating PBMCs following phlebotomy |
| RPMI-1640 | Corning | 10-040 | For making growth media for PBMCs |
| Fetal bovine serum (FBS) | Corning | 35-010-CV | For making growth media for PBMCs |
| Penicillin/Streptomycin | ThermoFisher | 15140122 | For making growth media for PBMCs |
| Cell-Tak | Corning | 354240 | Cell and Tissue adhesive solution; allows suspension cells to adhere to the surface |
| Seahorse XF Calibrant Solution | Agilent | 103059-000 | For hydrating cartridges |
| XFp Fluxpak (miniplates and sensor cartridges) | Agilent | 103022-100 | Contains XFp cell culture miniplates and sensor cartridges |
| XF base media | Agilent | 103335-100 | For making XF assay media |
| 45% cell culture D-(+)-Glucose solution | Corning | 25-037-CI | For making XF assay media |
| Sodium pyruvate solution | Corning | 25-000-CI | For making XF assay media |
| L-glutamine solution | ThermoFisher | 25030081 | For making XF assay media |
| Seahorse XFp Mito Stress Test Kit | Agilent | 103010-100 | Contains oligomycin, FCCP, antimycin A/rotenone |
| CyQUANT Direct Cell Proliferation Assay | ThermoFisher | C35011 | For quantification of live cells and data normalization |
| Seahorse XFp Analyzer | Agilent | S7802AEA | For measuring mitochondrial function in live cells |
| Cytation 5 Cell Imaging Multi-Mode Reader (or any instrument that can quantify fluorescent cells in a plate) | BioTek | BTCYT5PV | For quantification of live cells and data normalization |
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Citar este artigo
Feng, L. R., Nguyen, Q., Ross, A., Saligan, L. N. Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue. J. Vis. Exp. (135), e57736, doi:10.3791/57736 (2018).