Untersuchungen zur Isolierung, Reinigung und Kultivierung von humanen endometrialen Zellen und zur In-vitro-Expression von Inhibin in glandulären Epithelzellen

Zusammenfassung

Die separate In-vitro-Kultivierung von isolierten und gereinigten humanen Endometriumzellen bietet sicher die attraktivste experimentelle Möglichkeit, um die endometriale Funktion auf zellulärer Ebene zu erforschen. Dazu wurden nach einer ersten Collagenasedigestion stromale- und glanduläre Epithelzellen durch Filtration getrennt. Die Epithelzellen wurden durch zwei weitere Collagenasedigestionen, Filtration, Sedimentation und Ficoll Gradientenzentrifugation gereinigt. Stromale Zellen konnten mit Hilfe von Erythrozytenlyse-Puffer, Filtration und Sedimentation aufgearbeitet werden. Eine signifikant höhere Sezernierung von Inhibin konnte in der späten sekretorischen Phase im Vergleich zur proliferativen und frühen sekretorischen Phase beobachtet werden. Diese Ergebnisse zeigen, dass glanduläre Epithelzellen in vitro ihre ursprüngliche Differenzierung beibehalten. Die gleichbleibende Inhibinkonzentration in der späten sekretorischen Phase könnte dementsprechend eine entscheidende Rolle in der endometrialen Funktion und Reifung spielen. Somit könnte Inhibin als Marker der endometrialen Differenzierung in vitro genutzt werden. Versuche an isolierten glandulären Epithelzellen sollten in den ersten zwei Wochen durchgeführt werden. Die beschriebene Methode erlaubt die In-vitro-Vermehrung von getrennten endometrialen Zellarten welche sowohl zur Untersuchung der endometrialen Funktion als auch zur Klärung von Implantationsmechanismen benutzt werden kann.

Abstract

The separate in vitro cultivation of isolated and purified human endometrial glands and stromal cells seems to be the most attractive experimental way of studying the endometrial function on cellular level. In this paper a new method has been described to establish monolayer cultures of isolated endometrial stromal and epithelial cell populations. After a first collagenase digestion, stromal and epithelial cells were separated by filtration. The glandular epithelial cells were further purified with two collagenase digestion steps, filtration, a differential sedimentation at unity gravity and a Ficoll gradient centrifugation. Stromal cells were isolated with the use of erylyse-buffer, filtration and differential sedimentation at unity gravity. A significant higher inhibin production was observed during late secretory compared to proliferative and early secretory phase. Therefore, glandular epithelial cells maintain in vitro their initial differentiation. The higher inhibin concentration during secretory phase implicates a subsential role in endometrial function and maturation. Therefore, inhibin could be used as a marker of endometrial differenziation. Experiments on isolated glandular epithelial cells should be performed within two weeks. The method described here allows the propagation in vitro of separate endometrium cell types which can be used to study endometrial function as well as implantation mechanisms.

Schlüsselwörter

Endometrium - Zellkultur - glanduläre Epithelzellen - stromale Zellen - Inhibin

Key words

Endometrium - cell culture - epithelial cells - stromal cells - inhibin

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