Name: evaluating the role of mitochondrial function in cancer-related fatigue
Uploaded: 2018-05-17T12:30:00.000+00:00
Duration: 8 min 56 s
Description: Watch this Scientific Journal Video about Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue at JoVE.com

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。

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、类固醇或非甾体抗炎药物的个体也被排除在外。健康的对照血液样本是在 NIH 批准的协议 (NCT00001846) 下由健康的捐赠者在国立卫生研究院输血医学部门获得的。所有参与者都在 NIH 的马格努森临床研究中心招聘。在学习参与之前获得了书面知情同意书。
1. 线粒体功能测量准备 (实验1天)
<ol><li>水合物传感器墨盒 (请参阅材料表; 大约持续时间: 5 分钟)。<ol>
<li>从包中卸下传感器墨盒。添加 200 ul Calibrant 解决方案的每个井的公用事业板块, 然后填补每个护城河与 400 ul Calibrant 解决方案。</li>
		<li>将传感器板返回到实用新型板上, 现在有 Calibrant 解决方案。将墨盒在非 CO2 37 °c 孵化器过夜。 注: 传感器墨盒需水合至少4小时, 最大可达72小时。</li>
	</ol>
</li>
</ol>2. 临床样品准备 (实验2天)
<ol><li>使用慢性病治疗的13项功能评估-疲劳 (FACIT)2,23,24在基线 (外照射启动之前), 中点和完成外照射, 1 年外照射后, 测量疲劳。<ol>
<li>对每个项目的响应使用 0-4 刻度, 其中0代表 "不完全", 4 表示答辩人与相应的声明 "非常" 相关。总分应从 16-53 不等, 低分数反映高疲劳强度。</li>
		<li>将疲劳定义为 FACIT 分数低于 43, FACIT 评分≥43表示没有或没有临床意义的疲劳2。 注: FACIT 评分为 43, 最佳划分癌症患者和一般人群的疲劳评分为2。</li>
		<li>包括以下13项 FACIT 疲劳等级: 1) 我感到疲倦;2) 我感到身体虚弱;3) 我感到无精打采 ("冲出");4) 我感到疲倦;5) 我有困难开始的事情, 因为我累了;6) 因为我累了, 所以我很难完成事情;7) 我有活力;8) 我能做我平时的活动;9) 白天我要睡觉。10) 我太累了, 不能吃;11) 我需要帮助做我通常的活动;12) 我因太累而无法做我想做的事而感到沮丧;13) 我必须限制我的社交活动, 因为我累了。</li>
	</ol>
</li>
	<li>从新鲜收集的血液样本中分离 PBMC (大约持续时间: 1 小时)。<ol>
<li>收集8毫升血液进入单个核细胞制备管 (见材料表)。 注: 血液样本应在收集2小时内处理。如果样品收集后的血液样品处理超过2小时, PBMCs 的质量可能会受到损害。</li>
		<li>离心机在 1750 x g 在室温下30分钟 (18-25 °c)。将混浊层转移到15毫升锥形管上。加15毫升 PBS, 反转5次。</li>
		<li>离心机在 300 x g 15 分钟在4摄氏度。小心地除去和丢弃上清, 不干扰颗粒。通过添加多达10毫升 PBS, 并反转5次, 重新悬浮颗粒。</li>
		<li>离心机在 300 x g 10 分钟在4摄氏度。在1毫升 PBS 中丢弃液体上清和重新悬浮细胞。将 PBMCs 转移到1.5 毫升离心管。</li>
		<li>离心机在 610 x g 10 分钟。小心地移除上清和并用重悬丸在完全 RPMI-1640 (RPMI-1640 补充10% 血清, 10 毫米青霉素/链霉素)。</li>
	</ol>
</li>
	<li>非黏附细胞的涂层细胞板 (大约持续时间:30 分钟)。<ol>
<li>通过稀释0.1 米碳酸氢钠 pH 值8.0 中的库存溶液, 制备细胞和组织粘合剂溶液 (参见材料表)。工作解决方案应位于表面积的3.5 微克/厘米2处。 注意: 此解决方案包含从海洋贻贝中提取的多酚类蛋白,贻。这些蛋白质是由贻贝分泌的胶水的关键成分, 以锚定自己的表面。我们发现, 细胞-德的工作与 PBMCs 最好的。</li>
		<li>添加 100 ul 稀释胶粘剂溶液的每一个井和孵化至少20分钟室温。洗三次, 用 DI 水和空气干燥。</li>
	</ol>
</li>
</ol>3. 线粒体功能测量
<ol><li>测定培养基的制备 (大约持续时间:10 分钟)。 注意: 化验介质必须在实验当天进行新的准备。<ol>
<li>将 l-谷氨酰胺、丙酮酸和葡萄糖添加到基介质中 (与 Dulbecco 的修饰鹰的培养基 (DMEM) 相同, 但不含碳酸氢钠、葡萄糖、谷氨酰胺或丙酮酸钠) 来制作化验介质。暖媒体高达37摄氏度, 然后调整 pH 值为7.4。 注: l-谷氨酰胺、丙酮酸和葡萄糖的浓度通常与正常生长培养基中的浓度相同, 但可根据该检测进行调整。</li>
	</ol>
</li>
	<li>为图美的应力测试准备 PBMCs (大约持续时间: 2 小时)。<ol>
<li>板材足够 PBMCs 从步2入每个井到达 80-90% 融合。根据我们的经验, 1.5 x 105单元格/很好地产生了最一致的结果。 注: 井 A 和 H 为背景井, 只应包含无细胞的化验介质。在井 B 到 G, 我们建议电镀至少 3-6 井每个患者样品为了考虑异常点。</li>
		<li>旋转板以 200 x g 为2分钟, 允许细胞粘在井底。用化验介质清洗细胞。这一步从生长培养基中去除血清和碳酸氢钠。 注意: 按照我们的经验, 洗涤步骤通常会去除 20-30% 的细胞。</li>
		<li>在每个井中添加 180 ul 化验介质, 包括背景井 a 和 h. 在非 CO2 37 °c 孵化器中孵化 45-60 分钟。</li>
	</ol>
</li>
	<li>运行户内应力测试<ol>
<li>用化验介质在图美的应力试剂盒中重建药物, 如下所示:<ol>
<li>寡霉素: 添加 252 ul 化验介质到瓶子, 以产生一个股票溶液在50微米。</li>
			<li>FCCP: 在小瓶中加入 288 ul 的化验介质, 生成50微米的库存溶液。</li>
			<li>Antimycin/鱼藤酮: 添加 216 ul 的化验介质到瓶子, 以产生一个股票溶液在25微米。</li>
		</ol>
</li>
		<li>漩涡重组药物约1分钟。稀释成工作解决方案。 注意: 工作溶液的浓度应在实验前根据细胞类型滴定和预先确定。对于 PBMCs, 我们发现1微米的寡霉素, 1 微米 FCCP, 0.5 微米 antimycin A/鱼藤酮的工作最好。</li>
		<li>按顺序将药物吸管插入传感器墨盒中的每个端口:<ol>
<li>端口 A: 吸管 20 ul 10 微米寡霉素, 为最终浓度在每井1微米。</li>
			<li>端口 B: 吸管 22 ul 10 微米 FCCP, 为最终浓度在每井1微米。</li>
			<li>C 港: 吸管 25 ul 5 微米 antimycin/鱼藤酮为最终浓度在每井0.5 微米。</li>
		</ol>
</li>
	</ol>
</li>
	<li>在胞外通量仪上选择 "图美应力测试"。按照仪表提示插入传感器墨盒。该仪器将自动执行传感器校准。在传感器校准后插入细胞板, 仪器将完成其余的化验。氧气动力学被测量在530毫微米 (励磁)/650 毫微米 (发射), 并且质子浓度被测量在470毫微米 (励磁)/530 毫微米 (放射)。</li>
</ol>4. 线粒体功能数据的规范化
<ol><li>准备细胞增殖测定溶液 (大约持续时间: 5 分钟)。<ol>
<li>添加 48 ul 核酸染色 (500x) 和 240 ul 背景抑制器11.7 毫升 PBS (2x)。核酸染色是一种细胞 permeant DNA 结合染料, 背景抑制剂是一种掩蔽染料, 它阻止死细胞或细胞受损细胞膜完整性的染色。DNA 结合染料和背景抑制剂的结合确保只有活细胞被染色。</li>
		<li>在图中应力测试完成后, 将2x 工作溶液 (180 ul) 的相等容积直接添加到每个井中的介质中。</li>
	</ol>
</li>
	<li>在37摄氏度孵化器的细胞增殖试验溶液中孵化细胞, 用于 45-60 分钟. 用 508 nm (励磁)/527 nm (发射) 和规范化 OCR 数据 (近似持续时间:10 分钟) 量化板读器上活细胞 (荧光) 的数量。. 注意: 除了数字活细胞外, 用户还可以选择使用细胞总数、核酸量、蛋白质浓度、等来规范化数据。</li>
</ol>

<ol>
	<li>Berger, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-Related+Fatigue,+Version+2.2015.">Cancer-Related Fatigue, Version 2.2015.</a> Journal of the National Comprehensive Cancer Network. 13 (8), 1012-1039 (2015).</li><li>Feng, L. R., Dickinson, K., Kline, N., Saligan, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+phenotyping+approaches+lead+to+dissimilar+biologic+profiles+in+men+with+chronic+fatigue+following+radiation+therapy.">Different phenotyping approaches lead to dissimilar biologic profiles in men with chronic fatigue following radiation therapy.</a> Journal of Pain and Symptom Management. 52 (6), 832-840 (2016).</li><li>Feng, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Predictors+of+Fatigue+in+Men+With+Non-Metastatic+Prostate+Cancer+Receiving+External+Beam+Radiation+Therapy.">Clinical Predictors of Fatigue in Men With Non-Metastatic Prostate Cancer Receiving External Beam Radiation Therapy.</a> Clinical Journal of Oncology Nursing. 19 (6), 744-750 (2015).</li><li>Feng, L. R., Wolff, B. S., Lukkahatai, N., Espina, A., Saligan, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploratory+Investigation+of+Early+Biomarkers+for+Chronic+Fatigue+in+Prostate+Cancer+Patients+Following+Radiation+Therapy.">Exploratory Investigation of Early Biomarkers for Chronic Fatigue in Prostate Cancer Patients Following Radiation Therapy.</a> Cancer Nursing. 40 (3), 184-193 (2017).</li><li>Myhill, S., Booth, N. E., McLaren-Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+fatigue+syndrome+and+mitochondrial+dysfunction.">Chronic fatigue syndrome and mitochondrial dysfunction.</a> International Journal of Clinical and Experimental Medicine. 2 (1), 1-16 (2009).</li><li>Pernas, L., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mito-Morphosis:+Mitochondrial+Fusion,+Fission,+and+Cristae+Remodeling+as+Key+Mediators+of+Cellular+Function.">Mito-Morphosis: Mitochondrial Fusion, Fission, and Cristae Remodeling as Key Mediators of Cellular Function.</a> Annual Review of Physiology. 78 (1), 505-531 (2016).</li><li>Vecchiet, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+characterization+of+somatic+parietal+tissues+in+humans+with+chronic+fatigue+syndrome.">Sensory characterization of somatic parietal tissues in humans with chronic fatigue syndrome.</a> Neuroscience Letters. 208 (2), 117-120 (1996).</li><li>Jones, D. E. J., Hollingsworth, K. G., Taylor, R., Blamire, A. M., Newton, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+in+pH+handling+by+peripheral+muscle+and+potential+regulation+by+the+autonomic+nervous+system+in+chronic+fatigue+syndrome.">Abnormalities in pH handling by peripheral muscle and potential regulation by the autonomic nervous system in chronic fatigue syndrome.</a> Journal of Internal Medicine. 267 (4), 394-401 (2010).</li><li>Feng, L. R., Suy, S., Collins, S. P., Saligan, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+TRAIL+in+fatigue+induced+by+repeated+stress+from+radiotherapy.">The role of TRAIL in fatigue induced by repeated stress from radiotherapy.</a> Journal of Psychiatric Research. 91, 130-138 (2017).</li><li>Karamercan, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+peripheral+blood+mononuclear+cells+be+used+as+a+proxy+for+mitochondrial+dysfunction+in+vital+organs+during+hemorrhagic+shock+and+resuscitation.">Can peripheral blood mononuclear cells be used as a proxy for mitochondrial dysfunction in vital organs during hemorrhagic shock and resuscitation.</a> Shock. 40 (6), (2013).</li><li>Ijsselmuiden, A. J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+white+blood+cells+and+platelets+amplify+oxidative+stress+in+heart+failure.">Circulating white blood cells and platelets amplify oxidative stress in heart failure.</a> Nature Clinical Practice Cardiovascular Medicine. 5, 811 (2008).</li><li>Kong, C. -. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+mitochondrial+alterations+after+cardiac+surgery+involving+cardiopulmonary+bypass:+Clinical+correlations.">Leukocyte mitochondrial alterations after cardiac surgery involving cardiopulmonary bypass: Clinical correlations.</a> Shock. 21 (4), 315-319 (2004).</li><li>Straub, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+and+immune+system+prompt+energy+shortage+in+chronic+inflammation+and+ageing.">The brain and immune system prompt energy shortage in chronic inflammation and ageing.</a> Nature Reviews Rheumatology. 13, 743 (2017).</li><li>Kuhnke, A., Burmester, G., Krauss, S., Buttgereit, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetics+of+immune+cells+to+assess+rheumatic+disease+activity+and+efficacy+of+glucocorticoid+treatment.">Bioenergetics of immune cells to assess rheumatic disease activity and efficacy of glucocorticoid treatment.</a> Annals of the Rheumatic Diseases. 62 (2), 133-139 (2003).</li><li>Kramer, P. A., Ravi, S., Chacko, B., Johnson, M. S., Darley-Usmar, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+mitochondrial+and+glycolytic+metabolism+in+human+platelets+and+leukocytes:+Implications+for+their+use+as+bioenergetic+biomarkers.">A review of the mitochondrial and glycolytic metabolism in human platelets and leukocytes: Implications for their use as bioenergetic biomarkers.</a> Redox Biology. 2, 206-210 (2014).</li><li>Garrabou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Sepsis+on+Mitochondria.">The Effects of Sepsis on Mitochondria.</a> The Journal of Infectious Diseases. 205 (3), 392-400 (2012).</li><li>Mittal, M., Siddiqui, M. R., Tran, K., Reddy, S. P., Malik, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+in+inflammation+and+tissue+injury.">Reactive oxygen species in inflammation and tissue injury.</a> Antioxidants &amp; Redox Signaling. 20 (7), 1126-1167 (2014).</li><li>Adrie, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Membrane+Potential+and+Apoptosis+Peripheral+Blood+Monocytes+in+Severe+Human+Sepsis.">Mitochondrial Membrane Potential and Apoptosis Peripheral Blood Monocytes in Severe Human Sepsis.</a> American Journal of Respiratory and Critical Care Medicine. 164 (3), 389-395 (2001).</li><li>Traba, J., Miozzo, P., Akkaya, B., Pierce, S. K., Akkaya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+protocol+to+analyze+glycolysis+and+mitochondrial+respiration+in+lymphocytes.">An optimized protocol to analyze glycolysis and mitochondrial respiration in lymphocytes.</a> Journal of Visualized Experiments. (117), e54918 (2016).</li><li>Nicholls, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+profile+experiment+using+C2C12+myoblast+cells.">Bioenergetic profile experiment using C2C12 myoblast cells.</a> Journal of Visualized Experiments. (46), e2511 (2010).</li><li>Van den Bossche, J., Baardman, J., de Winther, M. P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+characterization+of+polarized+M1+and+M2+bone+marrow-derived+macrophages+using+real-time+extracellular+flux+analysis.">Metabolic characterization of polarized M1 and M2 bone marrow-derived macrophages using real-time extracellular flux analysis.</a> Journal of Visualized Experiments. (105), e53424 (2015).</li><li>Boutagy, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+isolated+mitochondria+from+minimal+quantities+of+mouse+skeletal+muscle+for+high+throughput+microplate+respiratory+measurements.">Using isolated mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements.</a> Journal of Visualized Experiments. (104), e53216 (2015).</li><li>Yellen, S. B., Cella, D. F., Webster, K., Blendowski, C., Kaplan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+fatigue+and+other+anemia-related+symptoms+with+the+Functional+Assessment+of+Cancer+Therapy+(FACT)+measurement+system.">Measuring fatigue and other anemia-related symptoms with the Functional Assessment of Cancer Therapy (FACT) measurement system.</a> Journal of Pain and Symptom Management. 13 (2), 63-74 (1997).</li><li>Yost, K. J., Eton, D. T., Garcia, S. F., Cella, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+important+differences+were+estimated+for+six+Patient-Reported+Outcomes+Measurement+Information+System-Cancer+scales+in+advanced-stage+cancer+patients.">Minimally important differences were estimated for six Patient-Reported Outcomes Measurement Information System-Cancer scales in advanced-stage cancer patients.</a> Journal of Clinical Epidemiology. 64 (5), 507-516 (2011).</li><li>Kang, J. -. G., Wang, P. -. y., Hwang, P. M., Galluzzi, L., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 542, 209-221 (2014).</li><li>Chacko, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+defining+distinct+bioenergetic+profiles+in+platelets,+lymphocytes,+monocytes,+and+neutrophils,+and+the+oxidative+burst+from+human+blood.">Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood.</a> Laboratory Investigation. 93 (6), 690-700 (2013).</li><li>Jones, L. J., Gray, M., Yue, S. T., Haugland, R. P., Singer, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+determination+of+cell+number+using+the+CyQUANT&#174;+cell+proliferation+assay.">Sensitive determination of cell number using the CyQUANT&#174; cell proliferation assay.</a> Journal of Immunological Methods. 254 (1), 85-98 (2001).</li><li>Hartman, M. -. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+mitochondrial+oxygen+consumption+in+peripheral+blood+mononuclear+cells+to+vascular+function+in+type+2+diabetes+mellitus.">Relation of mitochondrial oxygen consumption in peripheral blood mononuclear cells to vascular function in type 2 diabetes mellitus.</a> Vascular Medicine. 19 (1), 67-74 (2014).</li></ol>

作者没有什么可透露的。

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?...

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。

<table><tbody><tr><td>CPT Mononuclear Cells Preparation Tube&amp;nbsp;</td><td>BD Biosciences</td><td>362761</td><td>For isolating PBMCs following phlebotomy</td></tr><tr><td>RPMI-1640&amp;nbsp;</td><td>Corning</td><td>10-040</td><td>For making growth media for PBMCs</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Corning</td><td>35-010-CV</td><td>For making growth media for PBMCs</td></tr><tr><td>Penicillin/Streptomycin</td><td>ThermoFisher</td><td>15140122</td><td>For making growth media for PBMCs</td></tr><tr><td>Cell-Tak</td><td>Corning</td><td>354240</td><td>Cell and Tissue adhesive solution; allows suspension cells to adhere to the surface</td></tr><tr><td>Seahorse XF Calibrant Solution</td><td>Agilent</td><td>103059-000</td><td>For hydrating cartridges</td></tr><tr><td>XFp Fluxpak (miniplates and sensor cartridges)</td><td>Agilent</td><td>103022-100</td><td>Contains XFp cell culture miniplates and sensor cartridges</td></tr><tr><td>XF base media</td><td>Agilent</td><td>103335-100</td><td>For making XF assay media</td></tr><tr><td>45% cell culture D-(+)-Glucose solution</td><td>Corning</td><td>25-037-CI</td><td>For making XF assay media</td></tr><tr><td>Sodium pyruvate solution</td><td>Corning&amp;nbsp;</td><td>25-000-CI</td><td>For making XF assay media</td></tr><tr><td>L-glutamine solution</td><td>ThermoFisher</td><td>25030081</td><td>For making XF assay media</td></tr><tr><td>Seahorse XFp Mito Stress Test Kit</td><td>Agilent</td><td>103010-100</td><td>Contains oligomycin, FCCP, antimycin A/rotenone</td></tr><tr><td>CyQUANT Direct Cell Proliferation Assay</td><td>ThermoFisher</td><td>C35011</td><td>For quantification of live cells and data normalization</td></tr><tr><td>Seahorse XFp Analyzer</td><td>Agilent</td><td>S7802AEA</td><td>For measuring mitochondrial function in live cells</td></tr><tr><td>Cytation 5 Cell Imaging Multi-Mode Reader (or any instrument that can quantify fluorescent cells in a plate)</td><td>BioTek</td><td>BTCYT5PV</td><td>For quantification of live cells and data normalization</td></tr></tbody></table>

evaluating the role of mitochondrial function in cancer-related fatigue

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。

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?...

Watch this Scientific Journal Video about Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue at JoVE.com

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

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。

线粒体功能在癌症相关疲劳中的作用评价

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

evaluating-role-mitochondrial-function-cancer-related

Research

JoVE Journal

Cancer Research

9.1K 次观看.  National Institutes of Health. 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。

视频: Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

原发性白血病细胞代谢谱的评价

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

科研

癌症研究

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

跑步机疲劳试验:疲劳类似的行为，为鼠标操作简单，高通量检测

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

行为学

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule synthesis. Genetic and functional adaptations of mitochondria to the tumor environment drive proliferative and metastatic potential. The advent of DNA and RNA sequencing removed critical barriers to the evaluation of genetic mediators of tumorigenesis. However, to date, methodological approaches to evaluate tumor mitochondrial function remain elusive and require technical proficiency limiting the feasibility, ultimately diminishing diagnostic and prognostic value in both experimental and clinical settings. Here, we&#160;outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates using high-resolution respirometry. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways. Using this protocol, we demonstrate that mice bearing a luminal B mammary cancer exhibit defective nicotinamide adenine dinucleotide-linked respiration and reliance on succinate to generate adenosine triphosphate via OXPHOS.

We developed a practical protocol and analytical approach to evaluate mitochondrial oxidative phosphorylation and electron transfer capacity in fresh tumor homogenates. This protocol can be easily adapted to survey various mitochondrial functions that contribute to cancer initiation, progression, and treatment response.

分析测定切除实体瘤匀浆物的线粒体功能

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule ...

来自悬浮生长癌细胞的传输软骨 Cybrid 生成

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (&#961;0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

作者聚焦:使用癌细胞系进行传递软骨 Cybrid 生成  

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

使用癌细胞系的传递软骨Cybrid生成

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

生物化学

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

医学

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

实时测量中性粒细胞的线粒体生物能量特征

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and ...

免疫与感染

Use of Minimally Invasive Methods to Assess Fuel Utilization and Circadian Rhythms in Older Adults

Aging is associated with multiple physiological changes that contribute synergistically and independently to physical disability and the risk of chronic disease. Although the etiology of age-related physical disability is complex and multifactorial, the decline in mitochondrial function appears to coincide with the progression of functional decline in many older adults. The reason why there is a decrease in mitochondrial function with aging remains elusive, but emerging science indicates that both fuel metabolism and circadian rhythms can influence mitochondrial function.
Recent studies have established that circadian rhythms become disturbed with aging, and that disrupted circadian rhythms have pathological consequences that impact mitochondrial function and overlap with many age-associated chronic diseases. Current quantitative methods for direct assessment of mitochondrial function are invasive and typically require a muscle biopsy, which can pose difficulties with participant recruitment and study adherence, given the perceived levels of potential pain and risk. Thus, an innovative and relatively noninvasive protocol to assess changes in mitochondrial function at the cellular level and circadian patterns in older adults was adapted. Specifically, a real-time metabolic flux analyzer is used to assess the mitochondrial bioenergetic function of white blood cells under differential substrate availability.
The expression of circadian clock genes in white blood cells to cross-correlate with the mitochondrial bioenergetics and circadian rhythm outcomes are also analyzed. It is believed that these innovative methodological approaches will aid future clinical trials by providing minimally invasive methods for studying mitochondrial substrate preference and circadian rhythms in older adults.

A novel and minimally invasive protocol is presented to evaluate the synergistic impact of fuel utilization and circadian rhythms on aging individuals, utilizing peripheral blood mononuclear cells.

Aging is associated with multiple physiological changes that contribute synergistically and independently to physical disability and the risk of chronic ...

线粒体功能在癌症相关疲劳中的作用评价

摘要

探索更多视频

此视频中的章节

原发性白血病细胞代谢谱的评价

跑步机疲劳试验:疲劳类似的行为，为鼠标操作简单，高通量检测

分析测定切除实体瘤匀浆物的线粒体功能

使用癌细胞系的传递软骨Cybrid生成

使用癌细胞系的传递软骨Cybrid生成

使用癌细胞系的传递软骨Cybrid生成

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

磷31磁共振波谱分析:一种测量工具

实时测量中性粒细胞的线粒体生物能量特征

Use of Minimally Invasive Methods to Assess Fuel Utilization and Circadian Rhythms in Older Adults