足月胎盘胎膜中原发性人蜕细胞的分离
Summary
该协议展示了一种从足月胎盘的胎儿细胞膜中分离出的主要人类蜕细胞的方法, 可用于各种应用 (即免疫细胞化学、流式术中、等), 目的是研究不同细胞群在妊娠并发症中的作用。
Abstract
蜕膜, 也称为怀孕子宫内膜, 是至关重要的生殖组织。蜕细胞主要由 decidualized 基质细胞和免疫细胞组成, 负责分泌激素和炎症因子, 这些因素对于成功的囊胚植入、胎盘发育和在在足月和早产时开始分娩。许多妊娠并发症可能是由不同细胞种群的细微平衡的扰动引起的, 包括蜕膜。改变特定蜕细胞类型的比例可能会扰乱这些关键的过程, 并增加发生严重妊娠并发症的风险, 如胚胎植入失败, 宫内生长限制, 先兆子痫和早产。此处概述的协议说明了一种成本和时间有效的方法, 以隔离从足月胎盘的胎儿细胞膜收集的主要人类蜕细胞。结合酶消化和蜕组织的温和机械破坏, 获得了蜕细胞的高产, 几乎没有绒毛膜污染。重要的是, 分离的蜕细胞的特征 (基质细胞 (55-60%), 白细胞 (35%), 上皮 (1%) 或滋养层 (0.01%) 细胞) 和维持高生存能力 (80%), 这是由多色成像流式细胞仪检测证实。这个协议是特定于蜕膜颞骨, 可以适应第一和第二个月胎盘。一旦分离, 蜕细胞可用于众多的实验应用, 目的是了解不同蜕细胞亚群在妊娠并发症中的作用。
Introduction
子宫内膜是最活跃的成年女性组织之一, 通过对卵巢激素、雌激素 (E2) 和孕酮 (P4) 的刺激反应, 对每个月经周期进行戏剧性的重塑。蜕膜, 也称为怀孕子宫内膜, 是一个至关重要的生殖组织, 是由 postovulatory 阶段结束后形成的 P4-driven 分化后, E2-dominant 增生阶段。蜕细胞负责分泌的荷尔蒙因素, 成功的囊胚植入和发展的子宫-胎盘界面, 以保持产妇耐受胎儿移植。
蜕膜化是需要植入和随后重塑蜕螺旋动脉。子宫内膜基质细胞接受蜕膜化, 在 P4 和营地的控制下, 在月经周期的晚期黄体期1。这一过程是围绕血管和传播在整个基质, 表明其在血管重塑和白细胞贩运规则的作用。这种细胞转化的特点是圆形形态学, 增加核尺寸, 并扩大粗内质网和高尔基仪器2。Decidualized 基质细胞能够产生支持囊胚植入的分泌因子, 其特点是多种激素分泌 (即催乳素)、血管生成因子、胰岛素生长因子结合 protein-1(IGFBP-1), 前列腺素 (PG) E (细胞内的刺激器), 细胞因子, 细胞外基质成分和营养素为胎盘植入和发展所必需的3,4,5,6.
蜕细胞的数量不完全由 decidualized 基质细胞组成, 但也含有大量的、妊娠特异的蜕白细胞种群。蜕膜化包括短暂的局部水肿和自然杀伤细胞、T 细胞、树突状细胞和巨细胞的涌入。最大的白细胞亚群是子宫 NK 细胞, 包括大约50-70% 的所有母体白细胞浸润的蜕膜细胞因子和血管生成因子的来源, 这可能有助于蜕膜化过程中, 增加整个怀孕期间的数字7。巨噬细胞是免疫细胞的第二大亚群, 在植入部位周围发现并在妊娠期间增加8。它们是细胞因子和生长因子的来源, 如蜂群刺激因子 (CSF-1)9, 肿瘤坏死因子α (TNFα)10和前列腺素 (PG) E11。
在整个妊娠期和足月分娩之前, 蜕膜是细胞因子和趋化因子的主要来源, 负责孕产妇周围白细胞活化, 随后移入子宫组织以启动分娩。动物研究表明, 在分娩过程中, 老鼠蜕膜中有许多促炎性细胞因子被调节, 如 TNF a、IL-6、IL-12 和 IL-1b12。在人类蜕膜中, 促炎性细胞因子 IL-1b、IL-6 和 IL-8 (中性粒细胞趋化蛋白) 在分娩过程中表现出较高的表达, 而不是在劳动中13。这些分泌的细胞因子导致细胞的活化和流入蜕组织14;蜕巨噬细胞和中性粒细胞浸润在人和大鼠中的增加在足月劳动期间被看见, 与蜕入前浸润4倍更大, 表明在这两个相邻的子宫组织之间的激活级联15. 这些浸润白细胞产生的 PGs 能够激活子宫16, 基质金属蛋白酶 (基质) 的同步收缩, 以启动膜破裂17,18, 以及促炎性细胞因子放大子宫活化过程 (“细胞因子风暴”)。
由于蜕细胞的许多重要功能, 如在植入过程中发挥关键作用, 在早期妊娠中保持孕产妇耐受性和参与分娩的激活, 不同的病理可能出现在妊娠。例如, (1) 因反复植入失败和复发性妊娠丢失而导致的不孕, 可能是蜕成熟失败所致;(2) 宫内生长限制 (IUGR) 和先兆子痫, 因发育不良和蜕膜/胎盘功能障碍或蜕-浸润交界的血管改变而受损;以及 (3) 早产可能是由于过早蜕激活所致。
鉴于这些主要疾病, 加上人类体内研究的伦理和实际局限性, 建立主要的人蜕细胞系对于体外分析是必不可少的, 目的是更好地了解和改善妊娠并发症的临床处理。因此, 我们的研究目的是制定一项协议, 允许隔离的人原蜕细胞高细胞产量和生存能力从胎儿细胞膜的足月胎盘。本协议明确描述了一种时间和经济高效的方法, 用于隔离用于各种体外分析的蜕细胞的特定子类型。蜕亚群的丰度和表型特征在足月和与第一或第二次妊娠的比较中是确定其在整个人类妊娠中的作用的关键。
Protocol
Representative Results
Discussion
此处描述的协议说明了一种成本和时间有效的方法, 用于隔离从整个人类足月胎盘的胎儿细胞膜中收集的主要蜕细胞, 这是非常容易接近和直接的。该协议的成功取决于两个关键因素, (1) 蜕刮的效率从绒毛膜层的胎儿膜和 (2) 照顾, 蜕细胞处理整个议定书。重要的是, 绒毛膜组织污染的控制, 确保没有意外包括在蜕刮, 并确定和清除任何污染在洗涤步骤 (蜕膜和绒毛膜形态的差异允许容易去除的绒毛膜…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢捐助者、RCWIH 生物库和西奈医院/–减压振动妇产科为本研究使用的人体标本。我们要感谢碱液实验室的成员, 特别是卡罗琳·扣篮博士对她的方法开发的帮助。这项工作得到了宝来欢迎基金 (赠款 #1013759) 的支持。
Materials
| Hank’s balanced salt solution with calcium and magnesium | Prepared in facility (LTRI) | ||
| Hank’s balanced salt solution without calcium and magnesium | Prepared in facility (LTRI) | ||
| Diaper pads | Sigma-Aldrich | D9542 | |
| Large surgical scissors | AL Medical | 2018-12-20. | |
| Large surgical forceps | Fine Science Tools | 11000-18 | |
| Plastic disposable cell scraper (25 cm) | Sarstedt | 83.183 | |
| 250 mm (size 60 mesh) metal sieve | Sigma-Aldrich | S1020-5EA | |
| Disposable scalpel with plastic handle (#21) | Fisher Scientific | 08-927-5D | |
| Sterile plastic petri dish (diameter 10 cm) | Sarstedt | 82.1473.001 | |
| Sterile specimen container (urine cup, 4.5 oz) | VWR | 25384-146 | |
| Nylon filter (70 mm) | VWR/Corning | 21008-952 | |
| Erythrocyte lysis buffer | Qiagen | 79217 | |
| Trypan blue, 0.4% solution | Lonza | 17-942E | |
| Parafilm | Fisher Scientific | 13-374-10 | |
| Hemocytometer | Reichert | 1490 | |
| Roswell Park Memorial Institute (RPMI) 1640 culture media | Invitrogen | 11835-055 | |
| Fetal bovine serum | Wisent | 080-150 | |
| Normocin (50mg/ mL) | Invivogen | ant-nr-1 | |
| Plastic top filtration unit (0.22 mm membrane, 500 mL) | Millipore | SCGPT05RE | |
| Collagenase 2, lyophilized powder | Sigma-Aldrich | C6885 | |
| Soy bean trypsin inhibitor, powder | Sigma-Aldrich | T9003-250mg | |
| DNase powder | Roche | 10104159001 | |
| Bovine serum albumin (BSA powder) | Fisher Scientific | BP1600-100 | |
| Spinning disc confocal microscope – Leica DMI 6000B | Leica | ||
| Imaging Flow cytometer – Image Stream MK2 | Amnis | ||
| IDEA software | Millipore Sigma | ||
| APC-conjugated Vimentin antibody | R&D Systems | IC2105A | |
| APC H7-conjugated CD45 antibody | BD | 641399 | |
| FITC-conjugated Cytokeratin antibody | MACs Miltenyi Biotec | 130-080-101 | |
| PerCP -conjugated Cytokeratin 7 antibody | Novus | NBP2-47941PCP | |
| eFluor450 Fixable Viability dye | Thermo Fisher Scientific | 65-0863-14 | |
| Vimentin primary antibody | Santa Cruz | sc-7558 | |
| CD45 primary antibody | Dako | M0701 | |
| Cytokeratin primary antibody | Dako | M0821 | |
| Cytokeratin 7 primary antibody | Dako | M7018 | |
| Mouse IgG | Santa Cruz | sc-2025 | |
| Goat IgG | Santa Cruz | sc-2028 | |
| Alexa Fluor 546 secondary antibody | Invitrogen | A10036 | |
| Alexa Fluor 594 secondary antibody | Fisher Scientific | A-11058 | |
| DAPI | Sigma-Aldrich | D9542 |
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