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Biologia do Desenvolvimento

인간의 기본 영양막 세포 멜라토닌에 대해 저산소증의 보호 효과를 연구하기 위해 세포 배양 모델 / 재산 소화에 의한 혼란

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

인간 임신 중, 단핵 줄기 세포 인 cytotrophoblast 태반 세포는 빠르게 증식 또는 융모 extravillous cytotrophoblast 세포로 분화하거나. Extravillous cytotrophoblasts는 침략과 자궁 벽의 나선형 동맥을 개조. 융모 cytotrophoblasts 반면에, 다핵는 syncytiotrophoblast합니다 (syncytium) (1)를 형성하기 위해 증식 분화 퓨즈 계속한다. 융모 영양막 세포의 항상성의 유지 보수는 태아 웰빙과 건강한 임신을 위해 필수적이다. 사실, 융모 trophoblasts는 산소와 영양소의 모체 – 태아의 교환을 허용하고, 임신을위한 필수적인 호르몬을 생산하고 있습니다. 또한,는 syncytiotrophoblast는 모체 혈액 순환과 직접 접촉만을 셀형이며 필수적인 물리적 면역 장벽을 제공한다. 따라서,는 syncytiotrophoblast는 항상성 유지를위한 세포 사멸 및 교체를 받아야하고 AVO하기아이디 태반은 2-5 병리.

인간의 태반에서 차 융모 cytotrophoblasts를 분리하기 위해 1986 년 Kliman 등. (6)에 의해 개발 된이 기술은 융모 영양막 세포의 분화에 관여하는 분자 메커니즘 연구를 허용하여 태반 연구에 혁명을 일으켰습니다. 밀도 원심 분리 미디어에서 분리 한 다음 트립신과 DNase를 순차적 효소 digestions에 따라이 고전적인 기술은, (콜로 이달 실리카 입자는 폴리 비닐 피 롤리 돈에 의해 코팅, 또는 퍼콜)는 지금 융모 cytotrophoblast 세포를 분리하기위한 황금 표준으로 인식하고 있습니다. 이 기술은 자기 immunopurification, 이들 세포의 표면에 특이 항원의 발현 차이에 기초하여 비 융모 세포 융모 cytotrophoblasts 분리 절차에 의해 최적화 될 수있다. 우리 때문에 융모 membran 세포에 발현의 부재에 사람 백혈구 항원 ABC (HLA-ABC)를 선택한전자 7, 8.

태반은 임신 중 산소 수준에 극적인 변화를 겪는다 기관이다. 임신 초기에, 산소 비율은 생리 매우 낮음 (2 % O 2) 그러나 두 번째 및 세 번째 임신에서 산소화 온화한 수준 (8 % O 2)로 증가한다. 툴리 등. (9)는 태반 융모 내부 영양막 환경 시험 관내 재생 산소화 수준의 도전 및 변형도 표현형 변화를 초래할 수 있음을 설명했다. 따라서, 임신 8,9의 세 번째 임신시 태반 융모에있는 산소 장력을 모방 normoxia로 8 % 산소를 도입하는 것을 제안한다. Chen 등. (10)는 광범위 영양막 세포 배양 산소 장력과 관련된 여러 변수를 검토하고 pericellular 환경에서의 산소 농도를 결정하는 중요성을 입증 하였다. 융모의 산소 농도가 증가하는 경향혈관 생성에 기인. 계속 태반 융모 증가 혈류량과 과산화수소의 수준 (풍부한 반응성 산소 종) 혈관 형성 (11, 12)을 제어하는 중요한 신호이다. 임신 합병증, 혈관 생성의 결핍은 저산소증, 더욱 중요한 것은, 산소 간헐적 변동 (호출 저산소증 / 재산 소화)를 생성한다. 이러한 조건은 태반과 태아의 생존 13,14 타협 산화 스트레스의 비정상적인 증가로 이어집니다. 융모 cytotrophoblasts 그들이는 syncytiotrophoblast로 분화 될 때까지 정상 산소 상태 (8 % O 2)에서 유지되는 다음과 같이 영양막 세포가 저산소증 / 재산 소화의 에피소드 중 생체 내에서 받아야 변화는 시험 관내에서 모방 할 수 있습니다. 그들은 다음 normoxia (재산 소화)의 추가 18 시간 뒤에, 4 시간 동안 저산소 조건 (0.5 % O 2)을 실시한다. 이 저산소증 / 재산 소화 방식을 사용하여, 예를 trophoblasts특정 임신 합병증에서 관찰 된 바와 같이, 하이드 록시 에틸는, 산화 환원 상태 및 고유 세포 사멸 (8)의 증가 수준을 규제 완화. 따라서, 이는 태반 저산소증 / 재산 소화와 관련 임신 합병증을 방지하기 위해 새로운 예방 및 치료 방법을 평가하기 위해 시험 관내 모델에서 유용하다.

태반 세포는 산화 스트레스와 태반 기능 부전 (15)를 미연에 방지 할 수있는 능력 등 여러 가지 중요한 기능을 가지고 멜라토닌을 생산하고 있습니다. 여기, 우리는, 분자 세포 및 기능 수준 8시 태반의 영양막 세포에서 멜라토닌의 보호 효과를 입증하는 데 사용되는 실험 방법 및 세포 모델을 제시한다.

Protocol

태반은 정보를 환자의 동의와 윤리위원회 (CHUM – 세인트 루크 병원 INRS – 인스 티 투트 아르망 – Frappier의 승인을 즉시 미끼 – 세인트 루크 병원, 몬트리올, QC, 캐나다에서 복잡하지 않은 임신에서 자연 질식 분만 후 얻었다 라발, QC, 캐나다). 산모 Cytotrophoblast 세포의 1. 분리 및 정제 솔루션 및 미디어 보완하여 전송 매체를 준비 둘 베코의 이글의 중간 높은 포도당 1 % 부피 /의 …

Representative Results

질식 분만에 의해 얻어진 일반 용어 태반에서 분리 및 융모 cytotrophoblast 세포의 immunopurification 1 × 10 8 가능한 세포를 얻었다. 태반은 350g 무게 원반 모양과 투명 막에 4cm 높이 직경 19cm였다. 더 자엽 기형은 발견되지 않았다. 탯줄은 paracentral 현지화 및 56cm의 길이를 가지고 있었다. 순도는 멘틴 및 사이토 케라틴-7 마커를 사용하여 유동 세포 계측법에 의해 평가되었?…

Discussion

포유 동물에서 태아의 발달은 적절한 태반 기능에 직접적으로 의존한다. 건강 장애의 발달 기원은 이후의 삶에서 나타난 질환의 원인은 초기 개발 및 태반 태아 프로그래밍 30-32에서 기계적 역할을하고 있음을 다시 추적 될 수 있다는 가설에 기초한다. 그것은 영양 전달을 조절 유해한 노출 방지 및 주요 내분비 기능을 가지고 : 태반은 태아의 성장과 발전의 중요한 매개체이다. Kliman ?…

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
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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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