原发性白血病细胞代谢谱的评价
Summary
在这里, 我们提出了一个方案, 从白血病患者骨髓中分离白血病细胞, 并分析他们的代谢状态。对原代白血病细胞代谢状况的评估有助于更好地确定原代细胞的需求, 并可导致更个性化的药物。
Abstract
癌细胞的代谢需求会对生存和治疗效果产生负面影响。目前, 药物靶向代谢途径已在多种类型的肿瘤中进行了测试。因此, 对癌细胞代谢设置的描述是不可避免的, 以便针对正确的途径, 提高患者的整体结局。不幸的是, 在大多数癌症中, 恶性细胞的数量相当困难, 需要组织活检。白血病是一个例外, 在这种情况下, 可以从骨髓中分离出足够数量的白血病细胞。在这里, 我们提供了一个详细的方案, 从白血病患者骨髓中分离白血病细胞, 并随后分析他们的代谢状态使用细胞外通量分析仪。白血病细胞是由密度梯度隔离, 这不影响他们的生存能力。下一个培养步骤帮助他们再生, 因此代谢状态测量是细胞在最佳条件下的状态。该协议可以实现一致、标准化的结果, 可用于个性化治疗。
Introduction
代谢谱是细胞的主要特征之一, 改变生物能量现在被认为是癌症1、2、3 的特征之一。此外, 代谢设置的变化可用于癌症的治疗, 靶向信号转导途径或酶机制的癌细胞 4,5,6。因此, 了解癌细胞的代谢倾向是一个优势, 可以帮助改善目前的治疗。
已经有很多方法可以评估细胞在培养中的代谢活性。关于糖酵解, 葡萄糖的摄取可以通过放射性标记来测量, 使用 2-枯草 (2-(n-(n-(n-(n-(7-松-2-osca-1, 3-二唑-4-基) 氨基酸) 或细胞外乳酸水平测量酶7, 8。脂肪酸氧化速率是由同位素标记的棕榈酸9,10测定的另一个代谢参数。氧消耗率是一种广泛用于测定细胞11,12中线粒体活性的方法, 同时还可与线粒体膜电位评估13,14、atp/adp (腺苷)5′-triphosphate/Adenosine 5 ‘-二磷酸) 比测量15或全细胞内 atp 测量16。已知的调节代谢过程的信号通路可以通过蛋白质定量来确定, 并且可以提高对代谢测量的理解17,18,19.
然而, 所有这些方法只测量一个或在最好的情况下, 一个样本中的几个代谢参数。重要的是, 通过海马 xfp 分析仪等细胞外流量分析, 可以同时测量耗氧率 (ocr) 和细胞外酸化率 (ecar)。ocr 是线粒体呼吸的指标, ecar 主要是糖酵解的结果 (我们不能忽视 co2 的产生可能会提高具有高氧化磷酸化活性的细胞的 ecar)20。到目前为止, 已经使用这些分析仪研究了各种细胞类型 21、22、23。
在这里, 我们描述了细胞外通量分析的方案, 从未成熟的造血阶段的原生细胞 (白血病细胞) 从白血病患者。据我们所知, 目前还没有关于一次爆炸的具体协议。
Protocol
所有样本都是在儿童父母或监护人知情同意和捷克共和国布拉格查尔斯大学道德委员会批准的情况下获得的, 研究结果为。nv15-28848a。 1. 试剂的制备 通过溶解 137 mm ncl、2.7 mm kcl、4.3 mm na 2 hpo 4、1.47 mm kh kh2 po 4, 在 ddh2o中,用 hcl 将 ph 值调整到 7.4, 并通过自压灭菌, 准备500毫升的 pbs. 制备100毫升 rpmi 培养基: rpmi-1640 培养基, ?…
Representative Results
图 3显示了糖酵解应激试验和细胞米托压力试验测量 bcp-all (b 细胞前体急性淋巴细胞白血病) 和 aml (急性髓系白血病) 患者白血病爆炸后的曲线。还指出了这些测量的代谢参数的计算。每口井播种50万细胞, 所有测量均以六胞胎进行。 在糖酵解应激试验中, 使用的是唯一的基础培养基, 使细胞失去营养。得到…
Discussion
上述方案允许测量急性淋巴细胞白血病 (all) 或急性髓系 (aml) 患者的原发性白血病细胞中 ocr 和 ecar 值评估的代谢活性。使用细胞外助焊剂分析仪进行测量的优点是, 它能够实时检测活细胞中的代谢分布。从本质上讲, 所提供的协议中的每一步都可以根据你计划研究的细胞类型进行调整。在这里, 我们将讨论可能影响结果并可能提供不太理想的值的最重要参数。
优化的第一步是?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢捷克儿科血液学中心。这项工作得到了卫生部赠款 (nv15-28848a)、捷克共和国卫生部、捷克共和国布拉格莫托大学医院00064203和教育、青年和体育部 nr 的支持。lo1604。
Materials
| RPMI 1640 Medium, GlutaMAX Supplement | Gibco, ThermoFisher Scientific | 61870-010 | |
| Fetal Bovine Serum | Biosera | FB-1001/100 | |
| Antibiotic-Antimycotic (100X) | Gibco, ThermoFisher Scientific | 15240-062 | |
| Sodium bicarbonate | Sigma-Aldrich | S5761-500G | |
| D-(+) Glucose | Sigma-Aldrich | G7021-100G | |
| Oligomycin A | Sigma-Aldrich | 75351-5MG | |
| 2-Deoxy-D-glucose | Sigma-Aldrich | D8375-1G | |
| FCCP | Sigma-Aldrich | C2920-10MG | |
| DMSO | Sigma-Aldrich | D8418-100ML | |
| Rotenone | Sigma-Aldrich | R8875-1G | |
| Antimycin A from Streptomyces sp. | Sigma-Aldrich | A8674-25MG | |
| Seahorse XF Base Medium, 100 mL | Agilent Technologies | 103193-100 | |
| L-glutamine solution, 200 mM | Sigma-Aldrich | G7513-100ML | |
| HEPES solution, 1 M, pH 7.0-7.6 | Sigma-Aldrich | H0887-100ML | |
| Sodium pyruvate | Sigma-Aldrich | P5280-25G | |
| Bovine Serum Albumin | Sigma-Aldrich | A2153-10G | |
| Ficoll-Paque Plus | Sigma-Aldrich | GE17-1440-02 | Density gradient medium |
| Seahorse XFp FluxPak | Agilent Technologies | 103022-100 | |
| Corning™ Cell-Tak Cell and Tissue Adhesive | ThermoFisher Scientific | CB40240 | |
| Seahorse Analyzer XFp | Agilent Technologies | S7802A | |
| Seahorse XFp Cell Culture Miniplate | Agilent Technologies | 103025-100 |
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Citar este artigo
Hlozková, K., Starková, J. Assessment of the Metabolic Profile of Primary Leukemia Cells. J. Vis. Exp. (141), e58426, doi:10.3791/58426 (2018).