メラトニンに対する低酸素/再酸素化誘発性の破壊の保護効果を研究するために、ヒト初代栄養膜細胞培養モデル
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
ヒト妊娠全体にわたって、単核幹細胞である胎盤栄養膜細胞は、急速に増殖し絨毛またはextravillous細胞栄養芽層細胞のいずれかに分化します。 Extravillous細胞栄養芽層が侵入し、子宮壁のらせん動脈を改造します。絨毛細胞栄養層は、他の一方で、多核合胞体（シンシチウム）1を形成するために、増殖し分化し、ヒューズを続けます。絨毛栄養膜の恒常性の維持は、胎児の幸福と健康な妊娠のために不可欠です。実際には、絨毛栄養膜は、酸素と栄養素の母体胎児の交換を可能にし、妊娠に不可欠なホルモンを産生します。また、合胞体栄養細胞は、母体の血液循環と直接接触している唯一の細胞型であり、本質的な物理的および免疫学的障壁を提供します。したがって、合胞体栄養細胞は恒常性維持のため、アポトーシスおよび交換を受けなければならないとAVOしますID胎盤病理2-5。
Kliman らによって開発された技術。1986年の6個のヒト胎盤からの一次絨毛細胞栄養層を分離するためには、絨毛栄養膜分化に関与する分子メカニズムの研究を可能にすることによって、胎盤の研究に革命をもたらしました。密度遠心分離メディア（ポリビニルピロリドン、またはパーコールによってコーティングされたコロイダルシリカ粒子）で分離に続いてトリプシンおよびDNアーゼによる逐次酵素消化に基づいて、この古典的な技術は、現在、絨毛栄養膜細胞を単離するためのゴールドスタンダードとして認識されています。技術は、磁気免疫精製、これらの細胞の表面上の特異的抗原の示差発現に基づいて、非栄養膜細胞から絨毛細胞栄養芽層を分離する手順によって最適化することができます。私たちは、原因絨毛細胞membran上のその発現の欠如にヒト白血球抗原ABC（HLA-ABC）を選びました電子7,8。
胎盤は、妊娠中の酸素レベルの劇的な変化を受ける器官です。妊娠初期では、酸素の比は、第二及び第三のトリメスターに生理学的に非常に低い（2％O 2）が、酸素の穏やかなレベルに増加する（8％O 2）です。 Tuuli ら 9は、胎盤絨毛内部の栄養膜環境のin vitroでの再生が酸素化レベルでの挑戦とばらつきがあっても表現型の変化につながる可能性があることを説明しました。したがって、妊娠8,9の第三期中の胎盤絨毛に見られる酸素分圧を模倣するために正常酸素として8％の酸素を採用することが示唆されます。 Chenら 10は、広く栄養膜細胞培養における酸素分圧に関連するいくつかの変数を検討し、細胞周囲環境の酸素レベルを決定することの重要性を実証しました。絨毛中の酸素の濃度が増加する傾向にあります脈管形成に起因します。常に胎盤絨毛増加の血流および過酸化水素（豊富な反応性酸素種）のレベルは、脈管11,12を制御する重要な信号です。妊娠合併症、脈管形成の欠如は、低酸素状態を生成し、そしてより重要なことには、酸素の間欠的な変形は、（低酸素/再酸素化と呼ばれます）。これらの条件は、胎盤と胎児の生存率13,14を損なう酸化ストレスの異常な増加につながります。次のように栄養膜細胞が低酸素/再酸素化のエピソードの間に生体内で受ける改変は、in vitroで模倣することができる。彼らは合胞体栄養細胞に分化するまで、絨毛細胞栄養層は、酸素正常状態（8％O 2）下に維持されています。次いで、それらを酸素正常状態（再酸素化）の追加の18時間、続いて4時間、低酸素条件（0.5％O 2）に供されます。この低酸素/再酸素化のアプローチを使用して、元のトロホブラスト特定の妊娠合併症で観察されているように、調節解除酸化還元状態と内因性アポトーシス8のレベルの増加をhibit。したがって、これは胎盤性低酸素症/再酸素化に関連した妊娠合併症に対処するために新たな予防・治療法を評価するインビトロモデルに有用です。
胎盤細胞は、例えば、酸化ストレスおよび胎盤機能不全15を回避する能力など、いくつかの重要な機能を有し、メラトニンを生成します。ここで、我々は、分子細胞および機能レベル8で胎盤栄養膜細胞におけるメラトニンの保護効果を実証するために使用される実験的なアプローチとセルモデルを提示します。
Protocol
Representative Results
Discussion
哺乳類では、胎児の発育が十分な胎盤機能に直接依存しています。健康障害の発生起源は、その後の人生で明らかに病気の原因は早期開発に胎盤が胎児プログラミング30-32で機構的な役割を持っていることをさかのぼることができ仮説に基づいています。胎盤は胎児の成長と発展の重要なメディエーターである：それは、栄養移動を規制する有害な暴露から保護し、主要な内分泌機能…
Divulgations
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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