Name: establishment of an extracellular acidic ph culture system
Uploaded: 2017-11-19T14:00:00
Description: Watch this Scientific Journal Video about Establishment of an Extracellular Acidic pH Culture System at JoVE.com

我们感谢东京大学 RCAST 基因组科学和系统生物学和医学实验室的成员。我们特别感谢 Dr. h. Aburatani、Dr. t 儿、(LSBM、RCAST、东京大学)、Dr. k. Tomizuka、Dr. t. 吉田和 Dr. A。 国 (协和发酵麒麟 Co., Ltd.) 进行有益的讨论和支持。这项工作得到了部分支持补助金为年轻科学家 (A) (26710005, 培训), 补助金为创新领域的科学研究 (26116711 和 16H01567, 培训) 和补助金为挑战 日本教育、文化、体育、科学和技术部、武田科学基金会 (培训)、小林癌症研究基金会 (培训) 和癌症研究项目的探索性研究 (16K14605, 培训)治疗进化 (P 创造) 和创新的癌症控制的实际研究从日本医疗研究和发展机构, 阿曼德 (培训)。

1. 酸性培养基的制备
<ol><li>
制备控制 (ph 值 7.4) 和 low-pH (ph 6.8) 培养基
	<ol>
<li>在500毫升的水中溶解4.75 克无 l-谷氨酰胺的 DMEM 粉。</li>
		<li>在压紧瓶中准备 0.33 M NaHCO3解决方案。 注意: 由于 NaHCO3在加热和热分解时发出 CO2气体, 因此使用压力紧瓶非常重要。</li>
		<li>高压釜介质和溶液。允许这些缓冲器冷却到25° c。</li>
		<li>添加5毫升的200毫米 l-谷氨酰胺, 5 ?...

<ol>
	<li>Cairns, R. A., Harris, I. S., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+cell+metabolism.">Regulation of cancer cell metabolism.</a> Nature reviews. Cancer. 11 (2), 85-95 (2011).</li><li>Chang, C. H., 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=Metabolic+Competition+in+the+Tumor+Microenvironment+Is+a+Driver+of+Cancer+Progression.">Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression.</a> Cell. 162 (6), 1229-1241 (2015).</li><li>Hsu, P. P., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+metabolism:+Warburg+and+beyond.">Cancer cell metabolism: Warburg and beyond.</a> Cell. 134 (5), 703-707 (2008).</li><li>Gerweck, L. E., Seetharaman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pH+gradient+in+tumor+versus+normal+tissue:+potential+exploitation+for+the+treatment+of+cancer.">Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer.</a> Cancer research. 56 (6), 1194-1198 (1996).</li><li>Webb, B. A., Chimenti, M., Jacobson, M. P., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulated+pH:+a+perfect+storm+for+cancer+progression.">Dysregulated pH: a perfect storm for cancer progression.</a> Nature Rev. Cancer. 11 (9), 671-677 (2011).</li><li>Gatenby, R. A., Gillies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+cancers+have+high+aerobic+glycolysis?.">Why do cancers have high aerobic glycolysis?.</a> Nature reviews. Cancer. 4 (11), 891-899 (2004).</li><li>McBrian, 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=Histone+acetylation+regulates+intracellular+pH.">Histone acetylation regulates intracellular pH.</a> Mol Cell. 49 (2), 310-321 (2013).</li><li>Damaghi, 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=Chronic+acidosis+in+the+tumour+microenvironment+selects+for+overexpression+of+LAMP2+in+the+plasma+membrane.">Chronic acidosis in the tumour microenvironment selects for overexpression of LAMP2 in the plasma membrane.</a> Nat Commun. 6, 8752 (2015).</li><li>Corbet, 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=Acidosis+Drives+the+Reprogramming+of+Fatty+Acid+Metabolism+in+Cancer+Cells+through+Changes+in+Mitochondrial+and+Histone+Acetylation.">Acidosis Drives the Reprogramming of Fatty Acid Metabolism in Cancer Cells through Changes in Mitochondrial and Histone Acetylation.</a> Cell Metab. 24 (2), 311-323 (2016).</li><li>Chen, J. 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=The+genomic+analysis+of+lactic+acidosis+and+acidosis+response+in+human+cancers.">The genomic analysis of lactic acidosis and acidosis response in human cancers.</a> PLoS genetics. 4 (12), e1000293 (2008).</li><li>Kondo, 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=Extracellular+Acidic+pH+Activates+the+Sterol+Regulatory+Element-Binding+Protein+2+to+Promote+Tumor+Progression.">Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.</a> Cell Rep. 18 (9), 2228-2242 (2017).</li></ol>

在这里, 我们描述了一个简单的酸性 pH 培养系统及其评价过程。结合三种培养基酸化方法,即: 即, 降低碳酸氢盐浓度, 添加乳酸和盐酸, 使我们能够深入研究 pH 反应机制, 并对其他肿瘤的细胞反应进行比较。微条件如缺氧或营养饥饿。
该方法的关键是在培养基中测定碳酸氢盐、乳酸和盐酸的适当浓度。在第一次执行此方法时, 用不同的碳酸氢盐浓度测定中 pH 值对于确定?...

肿瘤微环境在肿瘤进展和癌细胞代谢中起关键作用1,2,3。癌细胞经常暴露在缺氧、养分匮乏和酸性细胞外 pH 等条件下。然而, 在肿瘤进展的作用没有被澄清如在缺氧或营养饥饿的广泛。肿瘤组织中的脂肪细胞可以变为酸性, 达到大约 pH 值 6.84,5。酸性 pH 产生于有氧和厌氧酵排泄质子 (H+) 和乳酸的增殖癌细胞5,6。
最近的研究发现, 酸性的组蛋白脱乙酰, 脂肪酸氧化, 和胞酸性溶酶体适应严重酸性环境7,8,9,10。然而, 细胞外酸化影响肿瘤行为的机制和酸性 pH 肿瘤微环境中关键调控因子的身份尚未完全确定。此外, 一些报告描述了各种酸性 pH 介质使用不清楚的碳酸氢盐, 三, 管道, 和 HEPES 缓冲剂或乳酸和 HCl, 但很少有报告显示稳定的调整介质或全面几种独特酸性培养基的比较7,8,9,10
为了阐明细胞外酸化过程中肿瘤细胞的主要调控因子和代谢变化, 我们建立了一个简单的体外培养模型, 以维持酸性细胞的活性, 并研究了在癌细胞中的作用.使用这种方法, 我们维持了一个酸性培养基与6.8 在37° c 以下 5% CO2, 在培养基中使用还原碳酸氢盐浓度。pH 值7.4 被用作正常和控制介质。在 PANC-1 和 AsPC-1 胰腺癌细胞培养过程中, 培养基 pH 值持续至少 24 h, 并逐渐降低 72 h。由于增强的糖酵解加速了不仅质子的排泄, 而且乳酸7,8,9, 我们还建立了一种模拟乳酸诱导酸中毒的培养方法, 添加乳酸, 而不是减少碳酸氢盐浓度。此外, 在培养基中 HCl 诱导的酸性物质允许我们排除细胞对酸性 pH 培养基反应的可能性, 而不是由于碳酸氢盐浓度的降低。此外, 使用不同浓度的 pH 值为6.4 到7.4 的不同介质, 我们可以对细胞反应的影响程度进行评估。

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium&#8220;Nissui&#8221; 2</td>
			<td>Nissui</td>
			<td>05919</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher&#160;</td>
			<td>10438</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Wako</td>
			<td>191-01305</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine solution</td>
			<td>Thermo Fisher&#160;</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Mixed Solution(Stabilized)</td>
			<td>Nacalai</td>
			<td>09367-34</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5g/l-Tripsin/0.53mmol/l-EDTA Solution, with Phenol Red</td>
			<td>Nacalai</td>
			<td>32778-05</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5%-Trypan Blue Stain Solution</td>
			<td>Nacalai</td>
			<td>29853-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>252476</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>H9892</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plus Mini Kit</td>
			<td>QIAGEN</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV First-Strand Synthesis System</td>
			<td>Thermo Fisher&#160;</td>
			<td>18091</td>
			<td></td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix&#160;</td>
			<td>Applied Biosystems&#160;</td>
			<td>4368702&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-1</td>
			<td>ATCC</td>
			<td>CRL-1469</td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1</td>
			<td>ATCC</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
		<tr>
			<td>KMS-6</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB0432</td>
			<td></td>
		</tr>
		<tr>
			<td>TIG</td>
			<td>JCRB Cell Bank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRC9</td>
			<td>ATCC</td>
			<td>CCL-212</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>ATCC</td>
			<td>CRL-1730</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One Microvolume UV-Vis Spectrophotometer with Wi-Fi</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 5 Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of an extracellular acidic ph culture system

肿瘤微环境的条件, 如缺氧或营养饥饿, 在癌症进展和恶性肿瘤中起关键作用。然而, 酸性细胞外 pH 在肿瘤侵袭性中的作用及其机制并没有得到广泛的研究, 与缺氧或营养饥饿的情况。此外, 一个明确的培养方法来模拟酸性细胞外肿瘤微环境没有得到充分报道。
在这里, 我们提出了一个简单的体外培养方法, 以保持酸性细胞外 pH 使用还原碳酸氢盐和增加乳酸或 HCl 浓度的培养基。培养基 pH 值维持至少24小时, 并逐渐减少 72 h 以下的培养 PANC-1 和 AsPC-1 胰腺癌细胞。三独特的酸性介质条件在这项研究中高度上调 pH 反应基因, 如MSMO1, INSIG1和IDI1相比, 缺氧或营养饥饿.这些基因的上调可作为酸性 pH 值的标志物。这些简单的技术有助于阐明酸性肿瘤微环境下肿瘤恶性肿瘤的基本机制。因此, 我们的胞外酸性 ph 培养系统能够发现细胞酸性 ph 反应不仅在癌细胞, 而且在初级细胞, 如肾小管细胞, 与其他酸性疾病, 包括糖尿病酮症酸中毒,乳酸酸中毒, 肾小管酸中毒和呼吸道酸中毒。

为了确定适当的碳酸氢盐的浓度, 我们准备 DMEM 在 0-8 毫米 NaHCO3 (最终浓度在培养基中), 并成功地准备的文化介质 pH 范围从 6.4-7.4 (图 1)。我们准备 DMEM 与8毫米 NaHCO3 (pH 7.4) 作为控制介质, 和2毫米 NaHCO3 (ph 6.8) 作为一个介质与酸性 ph 值根据前一份报告指出, 胞外 ph 值达到 ph 6.8 为固体肿瘤4,<sup class="xref...

Watch this Scientific Journal Video about Establishment of an Extracellular Acidic pH Culture System at JoVE.com

Establishment of an Extracellular Acidic pH Culture System

酸性肿瘤微环境在肿瘤进展中起重要作用。为了评估酸性细胞外 pH 对癌细胞的影响, 在体外,建立了简单的酸性培养体系。

胞外酸性 pH 培养体系的建立

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