的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识
Summary
Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.
Abstract
细胞凋亡奄奄一息，也被称为调控细胞死亡，获得多种新的活动，使他们能够影响相邻的活细胞的功能。重要的活动，如存活，增殖，生长，分化，是由凋亡细胞所调制的许多细胞功能中。识别并响应凋亡细胞的能力似乎是所有单元的普遍特征，无论谱系或始发的器官。然而，多样性和应对凋亡细胞的任务是非常谨慎的解剖负责任何特定结果的信号事件和途径采取的复杂性。特别是，必须由凋亡细胞可以存活应答细胞内影响细胞内信号通路，包括多种机制区分：受体介导的识别凋亡细胞的，由神经细胞凋亡的可溶性介质的释放和/或接合的吞噬细胞性机械。这里，我们提供了用于鉴定正在追踪其暴露于细胞凋亡诱导存活应答细胞的细胞内信号事件的协议。该协议的主要优点在于它支付由细胞凋亡调节信号反应细胞内事件的机制，解剖的注意。而该协议是专用于一个条件永生鼠标近端肾小管细胞系（BU.MPT细胞），它很容易适应于处于原点非上皮和/或从除肾脏等器官的细胞系。作为刺激使用死细胞的引入，可以阻碍细胞内信号传导事件的检测几个独特的因素。这些问题，以及策略来最小化或规避它们，在协议范围内进行讨论。该协议中的应用应援我们的广泛影响力的死亡或濒临死亡的细胞在他们的现场发挥的邻居扩展知识，无论是在健康和disea本身。
Introduction
细胞凋亡，或调控细胞死亡1，有助于在一个基本的方式组织的维持和发展。看过最简单地说，细胞凋亡证岁，损坏或不损害被淘汰到周围组织2,3多余的细胞。凋亡组织稳态的贡献，但是，是相当多的动态和变化。细胞凋亡垂死采集多个新的活动，既分泌和细胞相关，这使他们能够影响相邻活细胞4-10的功能。早期的研究集中于凋亡细胞以抑制炎症11-16的能力，但凋亡细胞也调节范围广泛的细胞功能，包括诸如生命活动作为生存4,9,10，增殖4,9,10，分化17迁移18和19的增长。而且，这些效果并不限于专业吞噬细胞，巨噬细胞样S，但是延伸到几乎所有类型的细胞和谱系，包括传统的非吞噬细胞，例如上皮细胞和内皮细胞7,9,10,18-21。
相邻活细胞暴露于细胞凋亡的特异性响应取决于涉及两个活应答细胞和凋亡细胞本身多种因素。例如，虽然暴露于凋亡细胞同时抑制小鼠巨噬细胞，肾近端小管上皮细胞（PTECs）的增殖，这两种类型的细胞显着地在他们的生存响应于凋亡细胞4,6,9,10不同。凋亡细胞巨噬细胞促进的生存，但诱发PTECs 4,6,9,10的凋亡。值得注意的是，与凋亡细胞的反应可能响应之间一脉相承的细胞，取决于产地10（ 例如 ，肾PTECs 对乳腺上皮的细胞反应的器官，甚至不同细胞）或激活状态22（ 如中性粒细胞）。相反，凋亡细胞可以在视细胞凋亡的性质非常相同的细胞唤起的响应不同刺激10,23或凋亡10,14的阶段。
鉴于响应于凋亡细胞的多种多样和复杂性，大必须小心在解剖负责任何特定结果的信号转导事件和通路。首先，需要可行和凋亡细胞之间的直接物理相互作用的反应必须从那些由凋亡细胞3,6-10释放或生成可溶性介质引起的分化。如果需要的物理相互作用，然后进一步分化应该执行。信号事件可能取决于结合的凋亡细胞的凋亡细胞，随后独立的吞噬，或巨噬细胞吞噬，独立于特定的受体的受体介导的认可 3-7,9,10,19。在后一种情况下，响应不特定于凋亡细胞，并且可以通过任何吞噬材料4,6被触发。
这些区别的重要性可以再次通过对比巨噬细胞和肾PTECs的反应可以理解。对于这两种类型的细胞，暴露于凋亡细胞改变了促存活激酶Akt的活性。调制是依赖于物理作用，因为响应的分离和细胞凋亡由0.4微米的聚碳酸酯膜废除响应4,9,10,19。然而，PTECs的响应是受体介导的和独立的吞噬作用的，而巨噬细胞的反应是由吞噬4,9,10,19驱动。这个结论是通过以下事实暴露于乳胶珠，中性细胞吞噬的刺激，对在PTECs Akt活性没有影响，但模拟凋亡细胞在巨噬细胞4,9的效果增强。
“>虽然少很好的研究，细胞坏死，或意外的细胞死亡1垂死，还调制附近活细胞的功能3-10,19一样凋亡细胞，坏死细胞通过各种机制，特别是在泄漏发挥它们的作用通过其破裂细胞膜3,5,6,9,24细胞内的内容。很多细胞，包括PTECs和巨噬细胞，具有明显的非竞争受体细胞死去坏死5,9。这些受体参与诱导是信号事件常相反的那些细胞凋亡4-10,19诱导的受体的接合。例如，在PTECs，坏死细胞增加的Akt的磷酸化，而凋亡细胞减少的磷酸化9,10,19。在这里，我们描述了一种协议，用于识别通过相邻凋亡PTECs的受体介导的识别诱导存活肾PTECs细胞内信号传导事件<sup> 9,10,19。而该协议是专用于称为BU.MPT细胞9,10,19,25,26一个条件永生PTEC细胞系，它很容易适应于处于原点非上皮和/或从大于其它器官的细胞系肾。重要的是，使用细胞凋亡作为细胞刺激造成不存在与可溶性配体的某些固有的实验困难。最重要的是凋亡细胞必须加入以悬浮液，而不是作为一种解决方案。这些困难，以及策略来最小化或规避它们，在协议范围内进行讨论。几乎所有的在协议中描述的技术简单，标准的细胞培养。此协议的优势在于它关注由凋亡细胞应答调节细胞内的信号转导多种机制。这些机制包括经由表面决定簇的结合，或对重桥接分子的特异性受体应的小区，可溶性介质的释放，和/或吞噬机械的接合。坏死细胞被包括在所有的实验中，以确保结果是特定细胞死亡的模式，而不是广义响应于死细胞。细心的方法，因为在这个协议中建议，是对我们不断扩大的影响力正在死亡的细胞上发挥他们的现场的邻居，在健康和疾病的认识至关重要。
Protocol
Representative Results
Discussion
我们在此提供用于表征下列其暴露于经历凋亡，或其他形式的细胞死亡的细胞的存活细胞诱导胞内信号事件的协议。协议强调由暴露于凋亡细胞可调节活应答细胞内的细胞内信号传导途径的多种机制。这些机制包括：互动（通过对死亡细胞或其棚泡和微泡架桥分子或表面决定，通常被称为凋亡小体）与应答细胞的特异性受体;可溶性介质（甚至他们的外代27）的释放;和吞噬机械的接合。下?…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported, in whole or in part, by institutional funds from Dr. José A. Arruda and the Section of Nephrology, University of Illinois at Chicago (to J. S. L.); and funding from the Department of Medicine of McGill University and the Research Institute of the McGill University Health Centre (to J. R.)
Materials
| Cell culture materials | |||
| DMEM, 1X with [4.5 g/L] glucose, L-glutamine & sodium-pyruvate | Mediatech (Manassas, VA) | 10-013-CV | |
| HyClone Fetal Clone II, fetal bovine serum | GE Healthcare (Logan, UT) | SH30066.03 | add 10% (v/v) to the culture medium A |
| Penicillin-Streptomycin-Glutamine, 100X | Life Technologies (Grand Island, NY) | 10378-016 | add 100 units/mL to the culture media A and B |
| γ-Interferon, mouse recombinant, from Escherichia coli | Calbiochem, Millipore (Billerica, MA) | 407303 | add 10 units/mL to the culture medium A |
| Falcon polystyrene tissue-culture treated dishes, non-pyrogenic | Corning (Durham, NC) | 353003 | sterile, 100 mm diameter |
| Falcon polystyrene centrifuge tubes, conical bottom | Corning (Durham, NC) | 352095 | sterile, 15 mL |
| Trypsin-EDTA, 0.05%, Gibco | Fisher (Waltham, MA) | 25300062 | sterile, 100 mL |
| Name | Company | Catalog | Comments |
| Chemicals and inhibitors | |||
| Staurosporine, from Streptomyces sp. | Sigma (St. Louis, MO) | S4400 | use at [1 μg/mL] for 3 h to induce apoptosis |
| Epidermal growth factor (EGF), [1 mg/ml] | Millipore (Billerica, MA) | EA140 | use at 50 nM to stimulate responder cells |
| UltraPure, 0.5 M EDTA, pH 8.0 | Life Technologies (Grand Island, NY) | 15575-038 | use 5 mM to detach adherent cells |
| Trypan blue solution, 0.4% | MP Biomedicals (Santa Ana, CA) | 91691049 | for dead cells staining |
| Paraformaldehyde Solution, 4% in DPBS | Affymetrix (Santa Clara, CA) | 19943 | use 0.4% (v/v) in 1× DPBS for cell fixation |
| Dulbecco's phosphate-buffered saline (DPBS), 1×, Gibco | Life Technologies (Grand Island, NY) | 14040133 | stock solution |
| Dulbecco's phosphate-buffered saline (DPBS), Ca+2- and Mg+2-free, 1×, Gibco | Life Technologies (Grand Island, NY) | 14190367 | stock solution |
| Cytochalasin D, 4 mM in dimethyl sulfoxide (DMSO), from Zygosporium mansonii | Sigma (St. Louis, MO) | C8273 | use at [4 μg/mL] to inhibit phagocytosis |
| Dimethyl sulfoxide (DMSO) | Sigma (St. Louis, MO) | D2650 | solvent for cytochalasin D |
| Name | Company | Catalog | Comments |
| Cell lysis buffer ingredients | |||
| Tris-buffered saline (TBS), 10×, pH 7.4 | BioRad (Hercules, CA) | 1706435 | for cell lysis buffer |
| Sodium pyrophosphate | Sigma-Aldrich (St. Louis, MO) | 221368 | 10 mM for cell lysis buffer |
| Sodium deoxycholate | Sigma-Aldrich (St. Louis, MO) | D6750 | 0.5% w/v for cell lysis buffer |
| Sodium dodecyl sulfate (SDS) | Sigma-Aldrich (St. Louis, MO) | L3771 | 0.1% w/v for cell lysis buffer |
| Glycerol | Sigma-Aldrich (St. Louis, MO) | G5516 | 10% for cell lysis buffer |
| Sodium fluoride | Sigma-Aldrich (St. Louis, MO) | 450022 | 25 mM for cell lysis buffer |
| Complete mini EDTA-free protease inhibitor cocktail tablet | Roche Diagnostics (Indianapolis, IN) | 4693159001 | for cell lysis buffer, 1 tablet per 10 mL of buffer |
| Triton X-100 | Sigma-Aldrich (St. Louis, MO) | T8787 | 10% for cell lysis buffer |
| Dithiothreitol (DTT) | MP Biomedicals (Santa Ana, CA) | 11DTT00005 | 1 mM for cell lysis buffer |
| Phenylmethanesulfonyl fluoride (PMSF) | MP Biomedicals (Santa Ana, CA) | 4800263 | 1 mM for cell lysis buffer |
| Sodium orthovandate | MP Biomedicals (Santa Ana, CA) | 2159664 | 10 mM for cell lysis buffer |
| Name | Company | Catalog | Comments |
| Equipment | |||
| Sorvall T6000B centrifuge | Du Pont (Wilmington, DE) | T6000B | |
| Fisher Tissuemat, water bath | Fisher (Waltham, MA) | ||
| Sonic Dismembrator (sonicator), model 100 | Fisher (Waltham, MA) | ||
| Miscellaneous | |||
| Cell counting chamber, Neubauer | Hawksley (Lancing, Sussex, UK) | AC2000 | |
| Annexin V-FITC Apoptosis Detection Kit II | Millipore (Billerica, MA) | CBA059 | for PI and Annexin V staining to determine the stage of apoptosis |
| Eppendorf microcntrifuge tubes, 1.5 ml | Sigma-Aldrich (St. Louis, MO) | T9661 | |
| Transwell permeable support, 0.4 μm polycarbonate membrane | Corning (Corning, NY) | 3413 | sterile, polystyrene, tissue culture treated |
| Name | Company | Catalog | Comments |
| Antibodies | |||
| Phospho-glycogen synthase kinase-3α/β (GSK-3α/β) (Ser21/9), polyclonal antibody | Cell Signaling Technology | 9331 | rabbit polyclonal antibody |
| Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antobody ( (14C10) ) | Cell Signaling Technology | 2118 | rabbit monoclonal antibody |
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