人胎盘滋养层细胞培养模型，研究褪黑素对缺氧的保护作用/复氧引起的中断
Summary
This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.
Abstract
This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.
Introduction
整个人类怀孕，胎盘滋养层细胞，这是单个核干细胞，快速增殖和分化成绒毛或绒毛外滋养层细胞。绒毛外滋养细胞侵入并重塑子宫壁的螺旋动脉。绒毛细胞滋养细胞，另一方面，不断地增殖，分化和融合形成多核合体（合胞体）1。绒毛滋养细胞稳态的维持是胎儿的福祉和健康的怀孕是必不可少的。事实上，绒毛滋养细胞允许氧气和营养物质的母胎交换，并产生必要的荷尔蒙怀孕。此外，该合体是唯一的细胞类型与母体血液循环的直接接触，并提供了一个基本的物理和免疫障碍。因此，必须合体凋亡和替代稳态维护和AVOID胎盘病变2-5。
通过Kliman 等 6在1986年发展到初级绒毛细胞滋养细胞从人胎盘分离的技术中，允许参与绒毛滋养细胞分化的分子机制的研究导致胎盘研究了一场革命。这种古典工艺的基础上，用胰蛋白酶和DNA酶连续酶消化，其次是密度离心分离介质（胶体二氧化硅颗粒涂层由聚乙烯吡咯烷酮，或珀）是目前公认的金标准隔离绒毛细胞滋养层细胞。该技术可以通过磁免疫纯化，即从基于这些细胞的表面上的特异性抗原的差异表达非滋养层细胞分离绒毛细胞滋养层的过程进行优化。我们选择了人类白细胞抗原ABC（HLA-ABC）由于对滋养细胞MEMBRAN没有其表达Ë7,8。
胎盘是妊娠期间发生的氧气含量急剧变化的机构。在第一三个月，充氧比率是生理上非常低的（2％O 2），但增大到在第二和第三个三个月氧合温和水平（8％O 2）。 Tuuli 等人 9描述了胎盘绒毛内的滋养环境的体外再生是在氧合水平的挑战和变化甚至可能导致表型的改变。正是因此，建议采用8％的氧作为常氧模仿怀孕8,9的3个月期间胎盘绒毛中发现的氧张力。 Chen等人 10广泛的研究与在滋养层细胞培养物中的氧张力几个变量和表现出确定在细胞周围环境中的氧含量的重要性。氧在绒毛水平趋向于增加由于血管。在不断胎盘绒毛增加血流和过氧化氢 的水平（一种丰富的活性氧）是控制血管11,12的重要信号。在妊娠并发症，缺乏血管生成的产生缺氧，更重要的是，氧的间歇性变化（称为缺氧/复氧）。这些条件导致氧化应激的异常增加，这影响胎盘和胎儿的生存能力13,14。即滋养层细胞缺氧/复氧发作期间在体内经历的改变可以在体外模仿如下：绒毛细胞滋养细胞中含氧量正常的条件（8％O 2），直到它们分化 成合体下保持。然后将它们进行4小时低氧条件（0.5％O 2），其次是常氧（复氧）的另外的18小时。使用这种缺氧/复氧的做法，滋养细胞前hibit解除管制的氧化还原状态和增加的固有凋亡8的水平，正如在某些妊娠并发症被观察到。因此，这是一个有用的体外模型来评估，以打击胎盘缺氧/复氧关联妊娠并发症的新的预防和治疗的方法。
胎盘细胞产生的褪黑激素，其具有几个重要的功能，如要避免氧化应激和胎盘功能不全15的能力。这里，我们目前使用在分子，细胞和功能8级证明褪黑激素在胎盘滋养层细胞的保护作用的实验方法和细胞模型。
Protocol
Representative Results
Discussion
在哺乳动物中，胎儿发育是直接依赖于足够胎盘功能。健康障碍的发展起源是基于的以后的生活中表现的疾病的病因可追溯到早期的发展，而且胎盘在胎儿编程30-32一种机械作用的假说。胎盘是胎儿生长发育的关键调解人：它调节养分转移，防止有害的曝光，并具有重要的内分泌功能。可重复的方法来隔离可行的主要滋养由Kliman 等的发展。在正常和异常胎盘功能6研?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) (no. 262011-2009) to CV and March of Dimes Social and Behavioral Sciences Research grant (#12-FY12-179) to CV and JTS; by studentships to LSF from the Ministère de l’éducation, de l’Enseignement supérieurs et de la recherche (MEESR)-Fonds de recherche du Québec (FRQ)-Nature et technologies (NT) and the Fondation Universitaire Armand-Frappier INRS, to HC from the Réseau Québécois en Reproduction-NSERC-CREATE, to AAHT from the Canadian Institutes of Health Research (CIHR) and FRQ-Santé, and to JBP from NSERC; by a fellowship to EMAS from the Conselho Nacional de Desenvolvimento Cientìfico e Tecnològico (CNPq) and the Programme de bourses d’excellence pour étudiants étrangers MEESR-FRQNT.
Materials
| Curved Metzenbaum Scissors | Shandon | 9212 | surgical equipment (cell isolation) (2 units) |
| Splinter Forceps Fine 41/2in | Fisherbrand | 13-812-42 | surgical equipment (cell isolation) (2 units) |
| Scissors 4.5 Str Dissection | Fisherbrand | 08-940 | surgical equipment (cell isolation) (2 units) |
| Gauze Sponge 10cm X 10cm | Cardinal Health | 361020733 | |
| Oblong Glass Baking Dish | Pyrex | 1105397 | Glassware (2.8L) |
| Funnel Buchner | Coorstek Inc | 10-356E | Glassware (114MM DIAMeter) |
| Watch Glass | pyrex | 9985100EMD | Glassware |
| Formalin solution, neutral buffered, 10% | Sigma-Aldrich | HT501128-4L | histological tissue fixative solution |
| Trypsinizing Flasks | Wheaton | 355395 | Glassware (1 unit) |
| Disposable Culture Tubes | Kimble | 73750-13100 | Glassware |
| Borosilicate Glass Pasteur Pipet (22.8 Cm) | Fisherbrand | K63B1367820C | Glassware |
| 250 Ml Glass Beakers | Fisherbrand | KFS14005250 | Glassware |
| Glass Media Bottles With Cap | Fisherbrand | KFS14395250 | Glassware (8 units) |
| 50 Ml Corex Tube | Corning | 8422-A | (1 unit) |
| 15 Ml Polystyrene Centrifuge Tube | Corning | 430791 | |
| 50 Ml Polystyrene Centrifuge Tube | Corning | 430829 | |
| 10ml Serological Pipet | Corning | 11415038 | |
| Cell Strainer 100μm Nylon | Corning | 431752 | |
| Absorbant Liner | Scienceware | 1199918 | |
| 500 Ml Bottles Top Filter | Corning | Pore: 0,22 µm / medium and HBSS preparation | |
| 2 Ml Criogenic Vials | Corning | 430488 | |
| Freezing Container, Nalgene Mr. Frosty | Sigma-Aldrich | C1562-1EA | |
| Peristaltic Pump | Pharmacia Fine Chemicals | P3 model | |
| Shaking Water Bath | Fisher | Model 127 | |
| Vacuum Pump | ABM | 4EKFS6CX-4 | |
| Sodium Chloride | Fisherbrand | EC231-598-3 | Saline solution 0.9% |
| Hank’s Buffered Salt Solution (Hbss) | Sigma-Aldrich | H2387 | Quantity: 9.25 (one vial) for 1L of digestion solution |
| Hydroxypiperazineethansulphonic Acid (Hepes) | Life Technologies | 15630-080 | 25mL (1M) for 1L of digestion solution |
| Trypsin Type I | Sigma-Aldrich | T8003 | 9,888U |
| Deoxyribonuclease Type Iv | Roche | 10-104-159-001 | 402,000U |
| Calcium Chloride | Sigma-Aldrich | C4901 | 100mM |
| Magnesium Sulfate | Baker | 2500-01 | 800mM |
| Dulbecco’s Modified Eagle Medium High Glucose (Dmem) | Life Technologies | 10564-045 | |
| Penicillin/Streptomycin Sulphate | Hyclone | SV30010 | |
| Fetal Bovine Serum | Corning | 35-010-CV | |
| Percoll | Sigma-Aldrich | P1644 | Density centrifugation media gradient. Volume: 36mL |
| Isopropanol | Acros | 42383-0010 | 50mL |
| Dimethyl Sulfoxide | Sigma-Aldrich | 472301 | |
| Automacs Magnetic Separator | Miltenyi Biotec | Model 003 | |
| Automacs Columns | Miltenyi Biotec | 130-021-101 | |
| Automacs Running Buffer | Miltenyi Biotec | 130-091-221 | http://www.miltenyibiotec.com/~/media/Images/Products/Import/0001100/IM0001131.ashx?force=1 |
| Automacs Rinsing Solution | Miltenyi Biotec | 130-091-222 | http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/cell-separation-buffers/automacs-rinsing-solution.aspx |
| Anti-Human Hla Abc Purified Clone W6/32 | Affymetrix eBioscience | 14-9983-82 | anti-mouse antibody |
| Anti Mouse Igg Microbeads | Miltenyi Biotec | 130048401 | |
| Multiple Well Plate - 6 Well With Lid | Corning | 3335 | Cell Bind surface |
| Multiple Well Plate - 24 Well With Lid | Corning | 3337 | Cell Bind surface |
| Multiple Well Plate - 96 Well With Lid | Corning | 3300 | Cell Bind surface |
| Modular Incubator Chamber | Billups-Rothenberg | MIC-101 | A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions |
| Single Flow Meter | Billups-Rothenberg | SFM3001 | |
| 50 Mm In-Line Filter | Whatman | 6721-5010 | PTFE, pore: 1.0 µm |
| Gas Regulator | Pro Star | PRS301233 | A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions |
| Gas Hose Class Vi Clear 5/16 | Parker | 100-05070102 | 3 pieces with ~ 0.5 m |
| 17 Mm Adjustable Gas Hose Clamp | Tiewraps | THCSS-16 | |
| Normoxia Gas Cylinder | Praxair | NI CDOXR1U-K | Size K (3rd trimester‘s composition: 5% CO2, 8% O2, Bal. N2) |
| Normoxia Gas Cylinder | Praxair | NI CDOXR1U-K | Size K (3rd trimester‘s composition: 5% CO2, 0.5% O2, Bal. N2) |
| Oxygen Microelectrode Mi-730 | Microelectrodes INC | 84477 | |
| Oxygen Adapter | Microelectrodes INC | 3572 | |
| ROS Detection Reagent: CM-H2DCFDA | Invitrogen | C-400 | |
| β-hCG ELISA kit | DRG internatinal | EIA-4115 | |
| Anti-Vimentin ourified antibody | eBioscience | 14-9897 | Host: mouse |
| Anti-Cytokeratin 7 (FITC) antibody | Abcam | ab119697 | Host: mouse |
| Alexa Fluor 488 Goat Anti-mousse IgG H&L antibody | Life Technologies | A-11029 |
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