Name: apoptosis induction and detection in a primary culture of sea cucumber intestinal cells
Uploaded: 2020-01-21T14:00:00
Description: Watch this Scientific Journal Video about Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells at JoVE.com

作者感谢浙江大学的周宁明教授提供的技术建议和实验室设备可供使用。这项工作得到了国家自然科学基金（资助号41876154、41606150和41406137）和浙江省高校和科研院所基础研究基金的资助[批号2019JZ00007]].

1. 细胞培养培养培养准备
<ol><li>
凝液制备
	<ol>
<li>
口管液收集：在无菌条件下，解剖健康的海参（湿重85-105克），收集凝血液，并将其储存在无菌玻璃瓶中。</li>
		<li>
细胞切除：在50 mL离心管中将胶质液在1，700 x g下离心5分钟，并将上清液转移到新的无菌玻璃瓶中;接下来，收集海参的无细胞凝血液。</li>
		<li>
补充组件失活：在40~50°C水?...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+holothurian+tissues+as+a+source+of+cells+for+long-term+cell+cultures.">Regenerating holothurian tissues as a source of cells for long-term cell cultures.</a> Marine Biology. 146 (5), 915-921 (2005).</li><li>Rastogi, R. P., Richa, R. P., Sinha, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+Molecular+Mechanisms+and+Pathogenicity.">Apoptosis: Molecular Mechanisms and Pathogenicity.</a> Excli Journal. 8, 155-181 (2009).</li><li>Wan, 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=Apoptosis,+proliferation,+and+morphology+during+vein+graft+remodeling+in+rabbits.">Apoptosis, proliferation, and morphology during vein graft remodeling in rabbits.</a> Genetics and Molecular Research. 15 (4), (2016).</li><li>Kasibhatla, S., 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=Staining+of+suspension+cells+with+hoechst+33258+to+detect+apoptosis.">Staining of suspension cells with hoechst 33258 to detect apoptosis.</a> Cold Spring Harbor Protocols. 2006 (3), (2006).</li><li>Mikiewicz, M., Otrocka-Domagala, I., Pazdzior-Czapula, K., Rotkiewicz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+long-term,+high-dose+dexamethasone+administration+on+proliferation+and+apoptosis+in+porcine+hepatocytes.">Influence of long-term, high-dose dexamethasone administration on proliferation and apoptosis in porcine hepatocytes.</a> Research in Veterinary Science. 112, 141-148 (2017).</li><li>Rinkevich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cultures+from+marine+invertebrates:+new+insights+for+capturing+endless+stemness.">Cell cultures from marine invertebrates: new insights for capturing endless stemness.</a> Marine Biotechnology. 13 (3), 345-354 (2011).</li><li>Bello, S. A., Abreu-Irizarry, R. J., Garcia-Arraras, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cell+cultures+of+regenerating+holothurian+tissues.">Primary cell cultures of regenerating holothurian tissues.</a> Methods in Molecular Biology. 1189, 283-297 (2015).</li><li>Yu, 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=Impact+of+water+temperature+on+the+growth+and+fatty+acid+profiles+of+juvenile+sea+cucumber+Apostichopus+japonicus+(Selenka).">Impact of water temperature on the growth and fatty acid profiles of juvenile sea cucumber Apostichopus japonicus (Selenka).</a> Journal of Thermal Biology. 60, 155-161 (2016).</li></ol>

在过去的几十年里，人们一直致力于建立细胞系，然而，在海洋无脊椎动物细胞的长期培养上仍然难以取得进展。据报道，从再生的荷卢图里亚组织的培养细胞在很长一段时间内是可行的，在特定的细胞17、23中可以检测到高活性的增殖。然而，对于正常的海洋无脊椎动物细胞，还没有实际的细胞培养方法被?...

在人工控制条件下，而不是在其自然环境中培育细胞，为生物研究提供了统一的实验材料，特别是对于在实验室环境中不易培养的物种而言。海洋无脊椎动物占所有动物物种1的30%以上，它们为研究特定生物过程的调控机制提供了大量生物材料，如再生2、3、应激反应4、环境适应5、6。
海参，阿波西乔布斯黄瓜，是北太平洋沿岸温带水域中研究最多的棘皮树种之一。众所周知，它是一种具有商业重要性的物种，在东亚，特别是中国，有大规模的海水养殖。关于A.japonicus的许多科学问题，包括8号脑电化后肠道再生的调控机制和免疫性9的退化、代谢控制10、11和免疫反应12、13在热或致病压力下，都引起了研究者的注意。然而，与研究良好的模型动物相比，基础研究，特别是在细胞水平上，受到技术瓶颈的限制，如缺乏先进的细胞培养方法。
研究人员为建立细胞系付出了很大的努力，但他们也面临许多挑战，任何海洋无脊椎动物的细胞系还没有建立起来。然而，海洋无脊椎动物的主要细胞培养在过去十年中已经发展，15，16，他们提供了在细胞水平上的实验机会。例如，从A.japonicus再生的细胞素已被作为长期细胞培养的细胞来源，为海洋无脊椎动物的原性细胞培养提供了实用的方法17。该协议结合和优化了无脊椎动物细胞培养方法，并开发出了一种广泛适用于海参或其他海洋无脊椎动物的初级培养方法。
凋亡是一种由各种外源性刺激和内源性刺激触发的固有细胞自杀程序。协调凋亡对许多生物系统至关重要18，19，它一直与海参的肠道回归在9年。为了研究感兴趣的生物体的凋亡过程，已经建立了一系列方法，包括Hoechst染色和显微镜检测，并成功地应用了20个。在这里，我们在海参原生培养的肠道细胞中进行了凋亡诱导和检测，以评估原生细胞在海洋无脊椎动物生物学研究中的可用性。地马酮是常用的合成糖皮质激素21，用于诱导海参培养肠道细胞凋亡，荧光显微镜在染色细胞中成功检测到显著的Hoechst 33258信号。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 &#956;m filter</td>
			<td>Millipore</td>
			<td>SLVV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Genom</td>
			<td>GNM25200</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m filter</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>4 cm dishes</td>
			<td>ExCell Bio</td>
			<td>CS016-0124</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde solution</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>80096618</td>
			<td>in PBS</td>
		</tr>
		<tr>
			<td>Benchtop Centrifuges</td>
			<td>Beckman</td>
			<td>Allegra X-30R</td>
			<td></td>
		</tr>
		<tr>
			<td>BeyoClick EdU-488 kit</td>
			<td>Beyotime</td>
			<td>C0071S</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10005817</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant temperature incubator</td>
			<td>Lucky Riptile</td>
			<td>HN-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>XW00500221</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric thermostatic water bath</td>
			<td>senxin17</td>
			<td>DK-S28</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>80176961</td>
			<td>75%</td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor(FGF)</td>
			<td>PEPROTECH</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Leica DMI3000B</td>
			<td>DMI3000B</td>
			<td></td>
		</tr>
		<tr>
			<td>Garamycin</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>XW14054101</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>63005518</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33258 Staining solution</td>
			<td>Beyotime</td>
			<td>C1017</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>XW1106168001</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin like Growth Factor(IGF)</td>
			<td>PEPROTECH</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10016308</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15</td>
			<td>Genom</td>
			<td>GNM41300</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine (100 mg/mL)</td>
			<td>Genom</td>
			<td>GNM-21051</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>XW77863031</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2SO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10020518</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019308</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019718</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>P1020</td>
			<td>pH7.2-7.4</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Genom</td>
			<td>GNM15140</td>
			<td></td>
		</tr>
		<tr>
			<td>PH meter</td>
			<td>Bante</td>
			<td>A120</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>SIGMA</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>VE</td>
			<td>Seebio</td>
			<td>185791</td>
			<td></td>
		</tr>
	</tbody>
</table>

apoptosis induction and detection in a primary culture of sea cucumber intestinal cells

初级培养细胞在各种科学学科中使用，作为生物物质的功能评估或特定生物活动表征的特别重要的工具。然而，由于缺乏普遍适用的细胞培养基和协议，对海洋生物的细胞培养方法描述得也有限。同时，海洋无脊椎动物细胞普遍存在的微生物污染和多热带特性进一步阻碍了海洋无脊椎动物有效细胞培养策略的建立。在这里，我们描述了一种易于处理的方法，用于从海参的黄豆骨细胞培养肠道细胞;此外，我们还提供了一个在原发性培养的肠道细胞中诱导和检测体外凋亡的例子。此外，本实验还详细介绍了适当的培养培养基和细胞收集方法。所述协议与来自海洋生物（包括Echinodermata、Mollusca和甲壳类）的各种广泛可用的组织样本兼容，它可以为多种体外实验应用提供足够的细胞。这项技术将使研究人员能够有效地操作来自海洋无脊椎动物的原生细胞培养物，并促进对细胞靶向生物材料的功能评估。

在这里，我们建立了A.japonicus的原发性肠道细胞培养，并传递了细胞。图1显示了不同培养阶段的圆形细胞。EdU染色测定提供了直接证据，揭示了这些圆细胞在后期的增殖活性（图2）。我们还稍微调整了协议，培养切碎的组织块，而不是过滤细胞;此外，主轴单元类型可以成功培养。这种细胞类型发生在肠道组织块周围，在培养四天后可以观察到;?...

Watch this Scientific Journal Video about Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells at JoVE.com

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

该协议提供了一种易于处理的方法，用于培养海参黄斑狼的肠道细胞，并与来自海洋生物（包括Echinodermata、Mollusca和甲壳类）的各种广泛可用的组织样本兼容。

海参肠细胞原培养的凋亡诱导与检测

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...

该协议为海洋无脊椎动物提供了一种易于处理和广泛兼容的初级细胞培养方法。这项技术为研究人员提供了一种在相对较短的时间内培养海洋无脊椎细胞的方法。细胞培养应具有一致的遗传背景，用于海洋生物学研究中不同的生物实验应用。对于这一领域的新研究，我们认为，指示的简化协议应该是锻炼的好选择。麻醉后，解剖和收集前肠，然后垂直分部组织样本，去除内层。在PBS中清洗组织样本两次，并在75%乙醇溶液中浸入消毒，持续不超过两秒钟。然后将PBS中的组织样品洗涤三次，取出乙醇，将约100毫克的组织样品转移到两毫升无菌微离心管中。将1.5毫升预优化培养基加入海参肠道组织块，用灭菌手术剪刀切碎块，直到溶液浑浊。添加 400 微升 0.25% 的 trypsin。通过反转混合溶液，并在室温下孵育五分钟。然后使用100微米细胞滤株过滤溶液，并在新的无菌两毫升微型离心管中收集滤液。对于可选的简化协议，培养培养基中切割的组织块可以直接转移到培养皿中，然后孵育。在1700xG下离心管3分钟，丢弃上先液，然后用抗生素重新悬浮培养介质中的颗粒，然后用培养介质洗涤两次。为细胞培养预先设置培养箱，并在18摄氏度的温度和饱和的湿度下至少提前运行24小时。然后使用指示介质的200微升重新悬浮细胞，并将它们转移到四厘米的盘子中。培养孵化器中的细胞。六小时后，将它们从培养箱中取出，并在细胞培养皿中加入两毫升指示的培养剂。每12小时，更换一半的介质。五到七天后，细胞开始分裂。通过将指示的抗生素、青霉素、链霉素和根霉素在培养基中的浓度减少一半，减少抗生素的不利影响。每两到三天更换细胞培养培养。当原细胞密度达到60%传导细胞传通时。首先，在室温下使用PBS清洗培养细胞两次。在每个菜中加入200微升0.25%的三辛溶液，并手动搅拌菜肴，确保覆盖整个底部。丢弃三辛溶液，在室温下孵育细胞五分钟。通过移液和重新悬浮细胞，用一毫升新鲜培养剂清洗细胞。将0.5毫升的细胞悬浮液转移到新盘中。加入1.5毫升的新鲜介质，在18摄氏度下孵育细胞。12小时后，改变介质，在显微镜下观察细胞，以评估条件。准备三个实验组，包括一个控制组和两组溶液，其中一个微摩尔和100微摩尔德西梅萨酮。在0小时、24小时或48小时内有或没有二氧基酮的孵育后，用PBS清洗细胞三次。关灯后，将每孔300微升的铲33258溶液加入12孔板，轻轻搅拌板以覆盖所有细胞，确保其染色。在18摄氏度下孵育30分钟。然后去除铲渍溶液，将 PBS 中 4% 的 4% 的半成甲醛溶液中 300 微升，修复细胞。轻轻搅拌 15 分钟。之后，在PBS中清洗固定细胞三次。上次洗涤后保持 PBS，以保持细胞覆盖。打开荧光显微镜硬件，包括汞灯电源、荧光灯电源和 PC。登录到操作系统帐户，启动软件，并检查其配置。将准备好的板放在显微镜舞台上。使用舞台控制器将样品放置在目标透镜上。在光学显微镜下找到感兴趣的细胞。切换到荧光显微镜，将激发和发射参数分别调整到约352纳米和461纳米，用于核DNA观察。捕获图像。本研究建立和传递了海参的原发性肠道细胞培养。此处显示了不同培养阶段的圆形细胞。这里是在第三天，种子后附着在盘子底部的细胞。这里是第七天的细胞增殖。这是传传后附着在盘子底部的细胞。和细胞失去活性，并在10个通道后死亡。EDU 说明分析提供直接证据，揭示这些圆形细胞在后期阶段的增殖活性。与对照和100微摩尔德西梅萨酮刺激相比，使用一微摩尔德西美沙酮的刺激以更快的速度增加凋亡细胞速率。培养细胞在不同时间段内用德西梅萨酮进行处理，随后进行33258染色，用于凋亡细胞检测。孵化前的细胞治疗对长期控制至关重要。当单元格增长不好时，可以提醒尝试可选的简化协议。副甲醛通过皮肤接触或吸入而具有中度毒性，并记录为可能为人类致癌物质。

Research

JoVE Journal

Biology

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

科研

生物学

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

小学组织两个胸苷类似物的免疫荧光检测（CldU IDU）

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

Induction and Testing of Hypoxia in Cell Culture

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Isolation and Primary Culture of Rat Hepatic Cells

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Induction and Analysis of Epithelial to Mesenchymal Transition

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

逆转录病毒感染的原发性肠化培养的视频协议

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...