海参肠细胞原培养的凋亡诱导与检测
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 细胞培养培养培养准备 凝液制备 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。 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。 补充组件失活：在40~50°C水?…
Representative Results
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;?…
Discussion
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?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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]].
Materials
| 0.1 μm filter | Millipore | SLVV033RS | |
| 0.22 μm filter | Millipore | SLGP033RB | |
| 0.25% Trypsin | Genom | GNM25200 | |
| 100 μm filter | Falcon | 352360 | |
| 4 cm dishes | ExCell Bio | CS016-0124 | |
| 4% paraformaldehyde solution | Sinopharm Chemical Reagent | 80096618 | in PBS |
| Benchtop Centrifuges | Beckman | Allegra X-30R | |
| BeyoClick EdU-488 kit | Beyotime | C0071S | |
| CaCl2 | Sinopharm Chemical Reagent | 10005817 | |
| Constant temperature incubator | Lucky Riptile | HN-3 | |
| Dexamethasone | Sinopharm Chemical Reagent | XW00500221 | |
| Electric thermostatic water bath | senxin17 | DK-S28 | |
| Ethanol | Sinopharm Chemical Reagent | 80176961 | 75% |
| Fibroblast Growth Factor(FGF) | PEPROTECH | 100-18B | |
| Fluorescent microscope | Leica DMI3000B | DMI3000B | |
| Garamycin | Sinopharm Chemical Reagent | XW14054101 | |
| Glucose | Sinopharm Chemical Reagent | 63005518 | |
| Hoechst33258 Staining solution | Beyotime | C1017 | |
| Insulin | Sinopharm Chemical Reagent | XW1106168001 | |
| Insulin like Growth Factor(IGF) | PEPROTECH | 100-11 | |
| KCl | Sinopharm Chemical Reagent | 10016308 | |
| Leibovitz's L-15 | Genom | GNM41300 | |
| L-glutamine (100 mg/mL) | Genom | GNM-21051 | |
| MgCl2 | Sinopharm Chemical Reagent | XW77863031 | |
| Na2SO4 | Sinopharm Chemical Reagent | 10020518 | |
| NaCl | Sinopharm Chemical Reagent | 10019308 | |
| NaOH | Sinopharm Chemical Reagent | 10019718 | |
| PBS | Solarbio | P1020 | pH7.2-7.4 |
| Penicillin-Streptomycin | Genom | GNM15140 | |
| PH meter | Bante | A120 | |
| Taurine | SIGMA | T0625 | |
| VE | Seebio | 185791 |
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Diesen Artikel zitieren
Wang, T., Chen, X., Xu, K., Zhang, B., Huang, D., Yang, J. Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells. J. Vis. Exp. (155), e60557, doi:10.3791/60557 (2020).