Name: calorespirometry: a powerful, noninvasive approach to investigate cellular energy metabolism
Uploaded: 2018-05-31T14:30:00
Description: Watch this Scientific Journal Video about Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism at JoVE.com

这项工作部分是由国家科学基金会授予玛丽 e. Konkle 和迈克尔. Menze 的 CHE-160944 资助的。

1. 细胞培养
<ol><li>维持人肝癌 (HepG2) 细胞在 Dulbecco 的改良鹰的培养基 (DMEM) 含有10% 胎牛血清 (血清) 和附加基质 (10 毫米葡萄糖, 2 毫米谷氨酰胺, 1 毫米丙酮酸盐) 在37°c 在细胞培养孵化器 (5% CO2, 95% 空气, 100% 湿度)。 注: 在 respirometry 和量热仪的后续步骤中引用 DMEM 时, 请使用上述 DMEM 公式。</li>
	<li>将单元格在 1 x 106细胞每100毫米培养板上, 每7天或在到达90% 融合之前, 每 3-4 天更...

<ol>
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calorespirometry 的目的是定量评价有氧和厌氧途径对代谢活动的贡献, 并获得细胞能量通量的综合观点。这是通过同时测量散热和氧通量, 然后用计算的 CR 比与理论 oxycaloric 当量的比较来完成的。对于可重现和可靠的数据, 必须考虑几个关键步骤。维持一种无菌、健康的细胞培养是至关重要的。细菌或真菌污染可能导致被挪用的热量生产或氧气消耗量, 使 CR 比率没有意义。此外, 细胞计数不当, 样本混...

在生物系统中, 新陈代谢过程中的热释放或焓变化通常直接通过 O 2消耗量和/或 CO2 (通过 respirometry) 来监测。不幸的是, 当这些技术被隔离使用时, 关键信息就会丢失, 例如厌氧途径对细胞代谢的贡献。Calorespirometry 是一种强大的技术, 依赖于同时测量散热和呼吸。开创性的 calorespirometric 工作研究了完全氧合哺乳动物细胞的厌氧代谢, 并证明了有氧和厌氧途径同时贡献能量动态平衡, 尽管转化的细胞在完全氧合环境1。Calorespirometry 已经被应用到各种各样的生物学问题上。一些例子包括研究低氧水平的动物热力学, 除草剂和雌激素对双壳贝类鳃的影响, 陆地有机体的新陈代谢, 以及有机土壤物质的微生物分解2,3,4,5,6. 此外, calorespirometry 还揭示了冷冻前的代谢预处理改善了哺乳动物细胞的冷冻保存7。每种方法, 无论是量热和 respirometry, 都独立增加了我们对细胞和生物体生物能学的认识。然而, 可以通过使用 calorespirometry 来回答的基本生物学问题仍然是相对未探索的。
赫斯定律指出, 反应的总焓变化与初始和最终状态之间的通路无关。例如, 生化通路的总焓变化是通路内所有反应焓的变化总和。量热法提供了一种测量细胞热量的实时方法, 它不加区别地检测有氧和厌氧通路。这是基于没有在系统中交换能量的基础, 除非通过实验安瓿8的墙壁。散热的变化等于安瓿中所有代谢反应释放出的焓的变化。因此, 负焓与系统的热量损失相关。在过去四年里, 详尽的研究描绘了分解代谢和代谢的热力学景观。这是由美国国立医学图书馆 (NLM) 在国家卫生研究院 (PubMed) 索引的 "生物" 和 "量热" 搜索词下的研究文章的稳步增加所代表的。搜索结果显示, 在1970之前, 共有27份出版物参考生物量热仪;与此同时, 仅在 2016年, 就有546份出版物利用了这项技术。
热方法的建立, 以确定热生产。但是, 对于解决 respirometric 值, 还是给予了更大的灵活性。respirometric 度量可以由 o2、CO2或 o2和 co2组成。此外, O2或 CO2的测量可以通过各种技术完成, 其中包括 optrodes、克拉克型电极和可调谐二极管激光吸收光谱7、9、10 ,11。虽然 CO2生产在许多 respirometric 研究中是一个有价值的指标, 但培养细胞的培养基通常利用 bicarbonic 缓冲系统来控制 pH 值1213。为避免在碳酸氢钠系统中 CO2测量的并发症, 以下用于区域性单元格 calorespirometry 的协议利用 O2作为唯一 respirometric 参数。
在测量氧通量的同时, 某些 respirometers (参见材料表) 是为了详细评估线粒体功能而设计的。衬底-uncoupler 抑制剂-滴定 (套装) 协议是很好的建立和兼容的实验设计, 以测量膜电位或活性氧 (ROS) 形成14。所提出的完整细胞 calorespirometry 协议与化学 uncouplers 的引入相兼容, 如羰基氰化物-trifluoromethoxyphenylhydrazone (FCCP) 和 f0f1-ATP 合成酶抑制剂寡霉素。通过添加 FCCP, 氧消耗可以从 ATP 生产中拆解, 这有助于评估潜在疗法对线粒体绩效15的影响。此外, 寡霉素的添加也照亮了泄漏呼吸的程度。因此, 在 calorespirometry 期间进行的 respirometric 测量与广泛的协议兼容, 旨在进一步阐明线粒体生理学。
同时测量散热和氧通量可以计算 calorespirometric (CR) 比。这个比率然后被比较对桑顿的恒定或理论 oxycaloric 等值, 范围在-430 到-480 焦摩尔-1之间根据细胞线或组织利益和被补充的碳基体1, 16. 因此, 一个更负的 CR 比率揭示了从厌氧途径到整体新陈代谢活动的贡献增加。例如, 常规肌肉组织呼吸的 CR 比率没有工作的活动性能范围从-448 到-468 焦 mol-1, 这是在理论 oxycaloric 等效17,18的范围内。同时, 在高葡萄糖的培养基中培养的哺乳动物癌细胞, 在糖酵解和相对低线粒体接触19的情况下增强了乳酸发酵。这种表型结果在-490 至-800 焦摩尔-1范围内的 CR 比值, 显示了更多的负 CR 比值1,7, 在细胞代谢中的厌氧通路的增加参与, 16,20。
商业和非营利细胞和组织分销商目前提供150多个物种的细胞线, 近4000个细胞系来源于人类。永生化细胞系是快速评估潜在疗法毒性的方便工具, 其中许多可能直接或间接干扰线粒体功能。在药物筛选过程中使用转换后的细胞可能具有有限的预测价值, 部分原因是因为华宝效应是许多癌症的标志。通常, 癌症产生 ATP 从基质层磷酸化和保持氧化还原平衡, 通过生产乳酸, 而不充分参与线粒体下有氧条件下19。制药业的发展是众所周知的昂贵和低效的, 约8的9化合物测试在人体临床试验未能达到市场批准21。虽然潜在的治疗可能通过初步筛选由于细胞株的细胞毒性低, 有可能这些化合物中的一些 mitotoxic。如果没有一个合适的方法来检测这些毒素如何损害不显示华宝效应的初级细胞的能量平衡, 关键信息往往是过度观察, 瓶颈治疗发展的早期阶段。
Calorespirometry 是一种实用的、无创的方法来分析各种生物样品中的代谢活动, 包括细胞和组织。所提出的协议的核心与一系列应用程序兼容。然而, 已经发现了一个并发症。永生化细胞通常培养在无葡萄糖培养基中补充半乳糖, 以增加氧化磷酸 (OXPHOS) 对能源生产的贡献, 以便使细胞敏感到潜在的 mitotoxins22, 23。当样品放在热量计15使用的不锈钢安瓿中时, 这种代谢重新编程似乎不清楚分析。在葡萄糖培养基中培养的细胞继续从事高代谢活动几个小时。同时, 在半乳糖培养基中培养的细胞在安瓿的30分钟内减少了热量的产生, 使测量仅限于早期实验时间点。不幸的是, 这种行为阻碍了评估细胞增殖的机会。尽管有这一特定限制, 大多数应用都与 calorespirometric 分析兼容, 并且可以通过这种方法获得详细的代谢信息。

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HepG2 Cells</td>
			<td style="width:125px;">American Type Culture Collection</td>
			<td style="width:119px;">HB-8065</td>
			<td style="width:167px;">Cells used for calorespirometry</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">O2k-Respirometer</td>
			<td style="width:125px;">Oroboros Instruments</td>
			<td style="width:119px;">10022-02</td>
			<td>Respirometer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LKB 2277 thermal activity monitor (TAM)</td>
			<td style="width:125px;">Thermometric AB</td>
			<td style="width:119px;"></td>
			<td>Thermometric was purchased by TA Instruments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Pyruvate (100 mM)</td>
			<td style="width:125px;">Thermofisher Scientific</td>
			<td style="width:119px;">11360070</td>
			<td>100x solution added to DMEM medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum - Premiuim Select</td>
			<td style="width:125px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11550</td>
			<td>Added to 10% in DMEM medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsn-EDTA (0.25%)</td>
			<td style="width:125px;">Thermofisher Scientific</td>
			<td style="width:119px;">25200072</td>
			<td>Cell dissociation reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Oligomycin from Streptomyces diastatochromogenes</td>
			<td style="width:125px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">O4876</td>
			<td>Mitochondrial Inhibitor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td>
			<td style="width:125px;">Sigma Aldrich</td>
			<td style="width:119px;">C2920</td>
			<td>Mitochondrial Uncoupler</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning 100 mm TC-Treated Culture Dish</td>
			<td style="width:125px;">Corning Corporation</td>
			<td style="width:119px;">430167</td>
			<td>Tissue culture dish</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glucose, powder</td>
			<td style="width:125px;">Thermofisher Scientific</td>
			<td style="width:119px;">15023021</td>
			<td>Glucose for DMEM medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Galactose, powder</td>
			<td style="width:125px;">Fischer Scientific</td>
			<td style="width:119px;">BP656500</td>
			<td>Galactose for DMEM medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-Glutamine (200 mM)</td>
			<td style="width:125px;">Thermofisher Scientific</td>
			<td style="width:119px;">25030081</td>
			<td>Glutamine for DMEM medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM, no glucose</td>
			<td style="width:125px;">Thermofisher Scientific</td>
			<td style="width:119px;">11966025</td>
			<td>Cell culture medium</td>
		</tr>
	</tbody>
</table>

calorespirometry: a powerful, noninvasive approach to investigate cellular energy metabolism

在基本生物和生物医学研究中使用的许多细胞系通过有氧和厌氧呼吸的结合来维持能量平衡。然而, 这两种途径在多大程度上促进了细胞能源生产的前景一直被忽视。在饱和葡萄糖中培养的转化细胞往往会减少对 ATP 生产的氧化磷酸化的依赖性, 这是由于基质层磷酸化的增加而得到补偿的。这种新陈代谢平衡的转变允许细胞增殖, 尽管存在线粒体毒素。在忽视转化细胞的新陈代谢平衡时, 药物筛选的结果可能会被误解, 因为在高葡萄糖存在下培养的模型细胞株可能无法检测到潜在的 mitotoxic 效应。浓度。该协议描述了两种强大的技术, respirometry 和量热, 这使得对细胞 ATP 生产的有氧和厌氧贡献的定量和无创评估。有氧和厌氧呼吸产生热量, 可以监测通过量热。同时, 测定氧耗率可以评估有氧呼吸的程度。当同时测量散热和耗氧量时, 可以确定 calorespirometric 比。实验得到的值可以与理论 oxycaloric 等效, 可判断厌氧呼吸的程度。因此, calorespirometry 提供了一种独特的方法来分析广泛的生物学问题, 包括药物的发展, 微生物的生长, 和基本生物能学在常氧和缺氧条件下。

calorespirometric 测量的重现性取决于适当和一致的样品准备。如果板块生长过度, 细胞培养所制备的样品不应使用, 因为细胞计数可能由于结块而变得不准确。此外, 由于在丛生细胞中的基底扩散有限, 可能会发生热量流动减少。因此, 在使用粘附细胞时, 选择一个融合在 60-80% 之间的板并在实验之前改变中等 24 h 是至关重要的。
适当的仪器校准和维护对于信息和可重现的 calorespiro...

Watch this Scientific Journal Video about Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism at JoVE.com

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Calorespirometry: 一种功能强大、无创的研究细胞能量代谢的方法

该协议描述 calorespirometry, 直接和同时测量散热和呼吸, 这提供了一种无创的方法来评估能量代谢。该技术用于通过监测总的细胞能量流来评估有氧和厌氧途径对能源利用的贡献。

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

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