巨噬细胞吞噬胸腺细胞吞噬的实验分析
Summary
在这里, 我们提出了一个方案, 以编制凋亡胸腺细胞和腹膜巨噬细胞, 并分析了细胞内吞的效率和特定的抑制介导的抑制凋亡胸腺细胞吞没。该协议在包括人工珠子和细菌在内的其他颗粒的细胞介导清除中有着广泛的应用。
Abstract
细胞凋亡是一个自然的过程, 在胚胎发育、稳态调节、免疫耐受诱导和炎症的治疗中起着至关重要的作用。体内凋亡碎片的积累可能会引发慢性炎症反应, 随着时间的推移导致全身自身免疫性疾病。凋亡细胞清除障碍与各种自身免疫性疾病有关。凋亡清除是一个复杂的过程, 在生理条件下很少检测到。它涉及丰富的表面感受器官和信号分子。研究凋亡细胞清除过程提供了深刻的分子机制和随后的生物反应, 这可能导致新的治疗方法的发展。在这里, 我们描述了诱导凋亡胸腺细胞, 腹腔巨噬细胞的制备, 以及流式细胞仪和显微镜分析凋亡细胞清除的协议。所有细胞在一定阶段都会发生凋亡, 许多体内和循环细胞可以吸收凋亡碎片。因此, 这里描述的协议可用于许多应用, 以表征凋亡细胞结合和摄入的许多其他细胞类型。
Introduction
我们的身体每天产生1-10 个凋亡细胞。如此大量的凋亡细胞必须以免疫反应保持静止的方式清除。为了确保及时清除凋亡细胞, 多种组织驻体细胞和循环细胞发展出吞没凋亡细胞的机制 1。细胞凋亡的功能失调调节与各种炎症性疾病的发生和进展有关, 自身免疫 2。细胞凋亡在癌症发展的发病机制及其对常规治疗的抵抗力3,4中也起着至关重要的作用。去除凋亡细胞通常会促进抗炎反应, 这可能与免疫耐受5有关。凋亡细胞清除的干扰推动了自我免疫, 并有助于人类和小鼠全身自身免疫性疾病的发展。
当细胞发生凋亡时, 它们将磷脂酰丝氨酸 (PtdSer) 从内小叶暴露到膜的外小叶。然后, PtdSer 将通过表面受体被吞噬细胞识别。已经发现了十几个受体, 以识别和/或促进凋亡细胞的吞没。一般情况下, 至少有三种类型的表面受体参与凋亡细胞清除: 网络受体, 识别凋亡细胞;发痒的受体, 启动吞没;陪护受体, 促进整个过程7。TAM 受体酪氨酸激酶 (TAM Rtk) 由tyro-3、 axl 和mer 组成, 主要由免疫系统的骨髓细胞表达8。TAM Rtk 的主要功能是作为连接受体, 促进凋亡细胞和碎片的吞噬性去除。本组多年来一直在自身免疫环境下研究 TAM 介导的凋亡细胞清除。维生素 k 依赖性蛋白生长抑制特定蛋白 6 (gas6) 和蛋白 s (pros) 结合并激活 tam 受体9,10。Gas6 产生于心脏、肾脏和肺部。P s 主要是在肝脏中产生的11。TAM 识别凋亡细胞的方式是, Gas6/ProS 的 n 端与凋亡细胞上的 PtdSer 结合, Gas6/ProS 的 c 端与锚定在吞噬细胞表面的 TAM 受体结合。与其他受体一起, 凋亡细胞吞没发生 12。虽然尔可以与配体 ProS 和 Gas6 结合, 但我们发现 Gas6 似乎是 em 介导的凋亡细胞巨噬细胞吞噬的唯一配体, 而这可以被抗莫抗体13阻断.巨噬细胞是专业的吞噬细胞。巨噬细胞快速清除凋亡细胞对于抑制细胞内抗原的炎症和自身免疫反应具有重要意义。莫氏受体酪氨酸激酶是巨噬细胞吞没和凋亡细胞的有效清除14的关键。在小鼠脾脏中, Maer 主要表现为边缘区和有形体巨噬细胞13。
本文介绍的协议描述了诱导细胞凋亡的基本方法, 并演示了检测细胞凋亡过程和效率的方法。这些方案可以很容易地适应研究其他细胞类型的自泡增多症, 在不同来源的凋亡细胞吞没。
Protocol
Representative Results
Discussion
细胞凋亡是一个高度保守的细胞死亡过程, 涉及许多信号级联, 并诱导蛋白质的表达, 分泌和运输。细胞凋亡常与细胞形态变化有关 17。凋亡细胞主动释放细胞因子和趋化因子, 吸引吞噬细胞迁移到该位点, 并在严格控制下启动吞没过程18。另一方面, 坏死细胞死亡释放引发炎症反应的危险信号1。凋亡细胞的有效或长时间清除会导致这些细胞的继?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
邵实验室的研究得到了医学院研究创新奖和辛辛那提大学内科初级教师试点奖的支持, 并获得了 NIDDK/nih 颁发的 DK K01_095067。
Materials
| Ack lysing buffer | GIBCO | A10492 | |
| Annexin V/7-AAD | BD Pharmingen | 559763 | |
| Anti-Mer antibody | R&D Systems | BAF591 | |
| CD11b-PE (clone M1/70) | BD Pharmingen | 553311 | |
| CFSE | Invitrogen | C1157 | |
| DMSO | Sigma-Aldrich | D-2650 | |
| EDTA (0.5 mM) | GIBCO | 15575-020 | |
| FACS tubes | BD Biosciences | 352017 | |
| Frosted slides | Fisher Scientific | 12-552-343 | |
| Horse Serum (Heat-inactivated) | Invitrogen | 26050088 | |
| Lidocaine | Sigma-Aldrich | L-5647 | Prepare 1% buffer in 1x PBS |
| PBS, 1x | Corning | 21040CV | |
| RPMI-1640 | Corning | 10040CV | |
| RXDX-106 | Selleck Chemicals | CEP-40783 | |
| Staurosprine (100mg) | Fisher Scientific | BP2541-100 | Add 214.3 ml of DMSO into 100mg to make 1mM stocking solution |
| Thioglycolate Medium Brewer Modified | BD Biosciences | 243010 | Prepare 3% thioglycolate buffer in 1´PBS, autoclaved, and store in the dark for 3 months. |
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