Name: human primary trophoblast cell culture model to study the protective effects of melatonin against hypoxia/reoxygenation-induced disruption
Uploaded: 2016-07-30T15:00:00
Description: Watch this Scientific Journal Video about Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption at JoVE.com

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 Minist&#232;re de l'&#233;ducation, de l'Enseignement sup&#233;rieurs et de la recherche (MEESR)-Fonds de recherche du Qu&#233;bec (FRQ)-Nature et technologies (NT) and the Fondation Universitaire Armand-Frappier INRS, to HC from the R&#233;seau Qu&#233;b&#233;cois en Reproduction-NSERC-CREATE, to AAHT from the Canadian Institutes of Health Research (CIHR) and FRQ-Sant&#233;, and to JBP from NSERC; by a fellowship to EMAS from the Conselho Nacional de Desenvolvimento Cient&#236;fico e Tecnol&#242;gico (CNPq) and the Programme de bourses d'excellence pour &#233;tudiants &#233;trangers MEESR-FRQNT.

Placenta&#39;s werden verkregen direct na spontane vaginale leveringen van ongecompliceerde zwangerschap met de CHUM-St-Luc ziekenhuis, Montreal, QC, Canada, met geïnformeerde toestemming van de patiënt en de goedkeuring van de ethische commissies (CHUM-St-Luc Ziekenhuis en INRS-Institut Armand-Frappier, Laval, QC, Canada). 1. Isolatie en zuivering van Villous cytotrofoblastcellen <ol><li> Oplossingen en media <ol><li> Bereid transportmiddelen aanvulling Dulbecco&#39;s gemodificeerd Eagle&#39;s Medium hoog g...

In zoogdieren, ontwikkeling van de foetus is direct afhankelijk van een adequate placenta-functie. De oorsprong van ontwikkeling stoornissen zijn gebaseerd op de hypothese dat de oorzaak van ziekten manifesteren op latere leeftijd naar vroege ontwikkeling en dat de placenta een mechanistische rol bij de foetale 30-32 kunnen worden getraceerd. De placenta is de sleutel mediator van de foetale groei en ontwikkeling: het reguleert nutriëntentransfers, beschermt tegen schadelijke blootstelling, en heeft grote en...

Gedurende de zwangerschap, de placenta cytotrofoblastcellen, die mononucleaire stamcellen zijn, zich snel vermenigvuldigen en differentiëren in ofwel villous of extravilleuze cytotrofoblastcellen. Extravilleuze cytotrofoblasten binnen te vallen en de vorm van de spiraal arteriën van de baarmoederwand. Villous cytotrofoblasten, anderzijds blijven prolifereren, differentiëren en zekering multinucleated syncytiotrofoblast (de syncytium) 1 vormen. Het onderhoud van villi trofoblast homeostase is essentieel voor foetale welzijn en gezonde zwangerschap. In feite, villous trofoblasten mogelijk maternale-foetale uitwisseling van zuurstof en voedingsstoffen en hormonen produceren essentieel voor de zwangerschap. Bovendien is het syncytiotrofoblast het enige celtype in direct contact met de maternale bloedsomloop en een essentiële fysische en immunologische barrière. Daarom moet de syncytiotrofoblast apoptose en vervanging ondergaan homeostatische onderhoud en AVOid placenta pathologie 2-5. De door Kliman et al. 6 ontwikkeld in 1986 tot primaire villous cytotrofoblasten isoleren van menselijke placenta&#39;s techniek zorgde voor een revolutie in de placenta onderzoek doordat de studie van de moleculaire mechanismen die betrokken zijn bij villous trofoblast differentiatie. Deze klassieke techniek, waarbij in de enzymatische digesties met trypsine en DNase, gevolgd door isolatie van dichtheidscentrifugatie media (colloïdale silicadeeltjes bekleed met polyvinylpyrrolidon of Percoll) wordt nu erkend als de gouden standaard voor het isoleren villeus cytotrofoblastcellen. De techniek kan worden geoptimaliseerd door magnetische immunozuivering, een procedure die villous cytotrofoblasten scheidt van niet-trofoblastcellen gebaseerd op de differentiële expressie van specifieke antigenen op het oppervlak van deze cellen. We kozen voor het menselijke leukocyt antigen ABC (HLA-ABC) vanwege de afwezigheid van de expressie op het trofoblastische cel membraame 7,8. De placenta is een orgaan dat dramatische verschillen in zuurstofgehalte ondergaat tijdens de zwangerschap. In het eerste trimester is de oxygenatie verhouding fysiologisch zeer laag (2% O 2) maar neemt milde niveaus van oxygenatie (8% O 2) in het tweede en derde trimester. Tuuli et al. 9 beschreven dat het in vitro reproduceren van de trofoblast omgeving in de placenta villi is een uitdaging en variaties oxygenatie niveau kan zelfs leiden tot fenotypische veranderingen. Het is daarom voorgesteld 8% zuurstof vast die normoxia de zuurstofspanning in placenta villi in het derde trimester van de zwangerschap 8,9 nabootsen. Chen et al. 10 uitgebreid bestudeerd verscheidene variabelen in verband met zuurstofspanning in trofoblast celkweek en toonde het belang van het bepalen van het zuurstofgehalte in een pericellulaire omgeving. De niveaus van zuurstof in de villi neiging te stijgendoor vasculogenese. De bloedstroom in placenta villi voortdurend toeneemt en het niveau van waterstofperoxide (een overvloedige reactieve zuurstofspecies) een belangrijk signaal dat vasculogenese 11,12 regelt. In de zwangerschap complicaties, een gebrek aan vasculogenese genereert hypoxie, en nog belangrijker, intermitterende variaties van oxygenatie (genaamd hypoxie / reoxygenatie). Deze omstandigheden leiden tot een abnormale verhoging van de oxidatieve stress, die de placenta en de levensvatbaarheid van de foetus 13,14 compromissen. De veranderingen die trofoblast cellen ondergaan in vivo tijdens episodes van hypoxie / reoxygenation kan worden nagebootst in vitro als volgt: villous cytotrofoblasten onder normoxische omstandigheden (8% O 2) totdat ze differentiëren tot syncytiotrofoblast worden gehandhaafd. Zij worden vervolgens onderworpen aan hypoxische omstandigheden (0,5% O2) gedurende 4 uur, gevolgd door nog 18 h normoxia (reoxygenatie). Met behulp van deze hypoxie / reoxygenation benadering, trofoblasten exHiBit gedereguleerd redox status en verhoogde intrinsieke apoptose 8, zoals in sommige zwangerschap complicaties waargenomen. Daarom is het zinvol om in vitro model om nieuwe preventieve en therapeutische mogelijkheden te evalueren bestrijding zwangerschap complicaties van placenta hypoxie / reoxygenatie. Placenta cellen produceren melatonine, dat een aantal belangrijke functies, zoals het vermogen om oxidatieve stress en placentale dysfunctie 15 overbodig is. Hier presenteren we de experimentele aanpak en mobiele modellen die worden gebruikt om de beschermende effecten van melatonine in de placenta trofoblast cellen vertonen op moleculair, cellulair en functioneel niveau 8.

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human primary trophoblast cell culture model to study the protective effects of melatonin against hypoxia/reoxygenation-induced disruption

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.

Isolatie en immunozuivering van villous cytotrofoblastcellen van een normale term placenta verkregen door vaginale bevalling leverde 1 x 10 8 levensvatbare cellen. De placenta woog 350 g, was 19 cm in diameter 4 cm hoog met schijfvormige vorm en transparant membranen. Geen zaadlob misvorming werd gedetecteerd. De navelstreng had paracentrale lokalisatie en een lengte van 56 cm. De zuiverheid werd beoordeeld door flowcytometrie behulp vimentine en cytokeratine 7-merkers. Meer d...

Watch this Scientific Journal Video about Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption at JoVE.com

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

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.

Human Primary Trofoblast Cell Cultuur aan de beschermende effecten van melatonine tegen hypoxie Studie / reoxygenation-geïnduceerde Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

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The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Isolatie en karakterisatie van primaire Rat klep interstitiële cellen: een nieuw Model voor het bestuderen van verkalking van de aortaklep

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

De isolatie en de cultuur van de primaire Epicardial cellen, afgeleid van menselijke volwassen en foetale hart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Organotypic cultuur methode om te studeren van de ontwikkeling van embryonale kip weefsels

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...