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Introduction
Protocol
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Biologia do Desenvolvimento

İnsan İlköğretim Trofoblast Melatonin Karşı Hipoksi Koruyucu Etkilerinin Eğitim için Hücre Kültürü Modeli / reoksijenasyon kaynaklı bozulması

Published: July 30, 2016
doi:
10.3791/54228
, , , , , , , , ,
1INRS-Institut Armand-Frappier

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

insan gebelik boyunca, mononükleer kök hücreler plasenta sitotrofoblast hücreleri, hızla çoğalır ve villöz veya extravillous sitotrofoblast hücrelerine ya farklılaşırlar. Extravillous sitotrofoblastlar istila ve rahim duvarına spiral arterleri pişmanlık. Villöz sitotrofoblastlar, diğer yandan, çok çekirdekli sinsityotrofoblastik (Sinsityum) 1 oluşturmak için çoğalmaya ayırt ve sigorta devam etmektedir. villöz trofoblast homeostazındaki bakım fetal iyilik ve sağlıklı hamilelik için esastır. Aslında, villus trofoblastlar oksijen ve besin anne-cenin alışverişi sağlar ve gebelik için gerekli hormonları üretmek. Ayrıca, sinsityotrofoblast anne kan dolaşımı ile doğrudan temas halinde, sadece hücre tipi ve gerekli fiziksel ve immünolojik bir engel oluşturur. Bu nedenle, sinsityotrofoblast homeostatik bakım için apoptoz ve değiştirme geçmelidir ve Avo içinid plasental 2-5 yakınmaları.

Insan plasenta primer villus sitotrofoblastlar izole etmek için 1986 yılında Kliman ve ark. 6 tarafından geliştirilen teknik villöz trofoblast farklılaşmasında rol oynayan moleküler mekanizmaların çalışma izin vererek plasental araştırmalarda bir devrim yarattı. yoğunluk santrifüj medyada izole ardından tripsin ve DNaz ile sıralı enzimatik sindirim dayanan bu klasik teknik, (kolloidal silika partikülleri polivinilpirolidon ile kaplanmış, ya da Percoll) artık villus sitotrofoblast hücrelerini izole etmek için altın standart olarak kabul edilmektedir. teknik manyetik immün-, bu hücrelerin yüzeyleri üzerinde spesifik antijenlerin farklı ekspresyonu göre olmayan trofoblastik hücrelerin villöz sitotrofoblastlar ayıran bir prosedürle optimize edilebilir. Biz nedeniyle trofoblastik hücre zarı üzerindeki ifadesinin yokluğu insan lökosit antijeni ABC (HLA-ABC) seçtie 7,8.

Plasenta gebelik sırasında oksijen seviyelerinde dramatik değişimleri uğrar bir organdır. İlk trimesterde, oksijenasyon oranı fizyolojik çok düşük (% 2 O 2), ancak ikinci ve üçüncü üç aylık dönemde oksijenasyonu hafif düzeyde (% 8 O 2) ile artar. Tuuli ve ark., 9 plasental villus içinde trofoblast ortamında in vitro üreme oksijenlenme seviyelerinde bir meydan okuma ve çeşitleri bile fenotipik değişikliklere yol açabilir olduğunu anlattı. Bu nedenle, gebeliğin 8,9 üçüncü trimesterde plasental villus bulunan oksijen gerilimi taklit etmek normoxia olarak% 8 oksijen benimsemeye önerdi olduğunu. Chen ve ark. 10 yoğun trofoblast hücre kültüründe oksijen basıncına ilgili çeşitli değişkenler okudu ve bir hücreyi ortamda oksijen düzeylerinin belirlenmesi önemini gösterdi. villuslarında oksijen seviyeleri artma eğilimindevaskülojenez nedeniyle. Sürekli plasental villus artar kan akımı ve hidrojen peroksit seviyesi (bol bir reaktif oksijen türleri) vaskülojenez 11,12 kontrol eden önemli bir sinyaldir. gebelik komplikasyonları, vaskülojenez eksikliği hipoksi ve daha da önemlisi, oksijenasyon aralıklı varyasyonlar (denilen hipoksi / reoksijenasyonunu) üretir. Bu koşullar plasental ve fetal canlılığı 13,14 ödün oksidatif stres anormal artışa yol açmaktadır. Villöz sitotrofoblastlar da sinsityotrofoblastlarda farklılaşırlar kadar normoksik koşullar (% 8 O 2) altında tutulan aşağıdaki gibidir: trofoblast hücrelerin hipoksi / reoksijenasyonun dönemleri sırasında, in vivo maruz değişiklikler in vitro taklit edilebilir. Daha sonra normoksiyada (reoksijenasyon) arasında ilave bir 18 saat, ardından 4 saat hipoksik koşullar (% 0.5 O 2) tabi tutulur. Bu hipoksi / reoksijenizasyon yaklaşımı kullanarak, ex trofoblastlarBazı gebelik komplikasyonları gözlenmiştir olarak HiBit, redoks durumu ve iç apoptoz 8 daha yüksek düzeyde deregüle. Bu nedenle, bu plasental hipoksi / reoksijenasyon ile ilişkili gebelik komplikasyonları ile mücadele için yeni önleyici ve tedavi yaklaşımları değerlendirmek için in vitro model bir yararlıdır.

Plasenta hücreler oksidatif stres ve plasental disfonksiyon 15 ortadan kaldırmak için bir yeteneği gibi birçok önemli işlevlere sahiptir melatonin üretir. Burada, moleküler, hücresel ve fonksiyonel 8. seviyede plasental trofoblast hücrelerinde melatoninin koruyucu etkisi göstermek için kullanılan deneysel yaklaşım ve hücre modelleri sunuyoruz.

Protocol

Plasenta, bilgilendirilmiş hasta onam ve etik komitelerin (CHUM-St-Luc Hastanesi ve INRS-Institut Armand-Frappier onayıyla, hemen CHUM-St-Luc Hastanesi, Montreal, QC, Kanada'da gebeden gelen spontan vajinal doğum sonrası elde edildi Laval, QC, Kanada). Villöz sitotrofoblast 1. Hücre İzolasyonu ve Saflaştırılması Çözümler ve medya Ilave Nakliye ortamı hazırlamak Dulbecco'nun Eagle Ortamı Yüksek Glikoz% 1 hacim / hacim antibiyotik (DMEM-HG) (10,000 birim / ml …

Representative Results

Vajinal doğum ile elde edilen normal bir terim plasenta İzolasyon ve villöz sitotrofoblast hücrelerinin immünolojik saflaştırma 1 x 10 8 canlı hücreleri elde edildi. Plasenta, 350 g ağırlığında diskoid şekil ve şeffaf membran ile 4 cm boyunda çapı 19 cm idi. Hiçbir kotiledon malformasyon tespit edildi. Göbek kordonu Parasantral lokalizasyon ve 56 cm'lik bir uzunluğa sahipti. saflık vimentin ve sitokeratin-7 belirteçleri kullanarak flow sitometri ile …

Discussion

Memelilerde, fetal gelişim yeterli plasental fonksiyonu üzerine doğrudan bağlıdır. Sağlık bozukluklarının gelişimsel kökenleri sonraki hayatında tecelli hastalıkların nedeni erken gelişmesine ve plasenta, fetal programlama 30-32 bir mekanik role sahip olduğu geri izlenebilmektedir hipotezine dayanmaktadır. Bu besin devrini düzenleyen zararlı maruz karşı korur ve önemli endokrin fonksiyonlara sahiptir: plasenta, fetal büyüme ve gelişmenin temel aracıydı. Kliman ve arkadaşları<…

Declarações

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
DOWNLOAD MATERIALS LIST

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Tags

Keywords: Primary Trophoblast Cell CultureHypoxia/reoxygenationMelatoninPregnancy ComplicationsPlacentaOxidative StressVillous Cytotrophoblast IsolationDensity CentrifugationTrophoblast CultureFirst Trimester Placenta
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Sagrillo-Fagundes, L., Clabault, H., Laurent, L., Hudon-Thibeault, A., Salustiano, E. M. A., Fortier, M., Bienvenue-Pariseault, J., Wong Yen, P., Sanderson, J. T., Vaillancourt, C. Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption. J. Vis. Exp. (113), e54228, doi:10.3791/54228 (2016).
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