Untersuchungen zur Regulation von CRH, ACTH und Kortisol in der Trophoblastzelle in vitro

Zusammenfassung

Fragestellung: Die Trophoblastzelle synthetisiert die Peptidhormone CRH und ACTH. Durch ihre besondere enzymatische Ausstattung kann sie die Reaktion Kortisol ↔ Kortison katalysieren. In-vitro-Stimulierungsversuche an isolierten Trophoblastzellen mit CRH, ACTH und Prednisolon sollen den Einfluss auf die ACTH- und Kortisolsekretion zeigen. In einem Langzeitversuch wurde die basale Kortisolsekretionsleistung untersucht.
Methode und Methodik: Die Trophoblastzellisolierung erfolgt nach grober Präparation des Plazentagewebes mittels mehrschrittiger DNAse I und Trypsinverdauung. Die gewonnene Zellsuspension wird auf die definierte Zellkonzentration von 1 × 10⁶ Zellen/ml eingestellt und in Nährmedium kultiviert. Nach jeweils 8 Stunden erfolgt die Zugabe von ACTH, CRH oder Prednisolon. Nach weiteren 20 bzw. 30 Minuten wird die Kortisol- bzw. ACTH-Konzentration gemessen. Parallel werden die Kortisol- bzw. ACTH-Werte einer nichtstimulierten Kultur bestimmt.
Ergebnisse: In Langzeitkulturen zeigt die Kortisolkonzentration einen rhythmischen Verlauf. Die Zugabe von CRH (500 ng/ml, 1 µg/ml) stimuliert die ACTH- und Kortisolkonzentration zeitabhängig. ACTH (500 ng/m-2 µg/ml) stimuliert die Kortisolkonzentration zeitabhängig. Prednisolon stimuliert die ACTH-Sekretion.
Schlussfolgerung: Die Trophoblastzelle weist in vitro einen rhythmischen Verlauf der Kortisolkonzentration auf. Erstmals konnte eine vollständige CRH-ACTH-Kortisol-Feedbackschleife innerhalb der Trophoblastzelle in vitro demonstriert werden.

Abstract

Objective Trophoblast cells synthesise CRH and ACTH, which are peptide hormones. On the strength of special enzymes they are capable of catalyzing the reaction cortisol ↔ cortisone. In vitro experiments should give a proof of influence to ACTH- and cortisol secretion by CRH, ACTH and prednisolon. The basal rate of cortisol secretion was examinated in a long term experiment.
Material and methods: Trophoblast cells were prepared from human term placentae by standard trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation step. After adjusting the cell suspension to a defined cell concentration of 1 × 10⁶ cells/ml the cells were cultivated. The addition of CRH, ACTH or prednisolon followed every eight hours. The samples collected 20 or 30 minutes later, also from unstimulated cultures, were assayed for ACTH and cortisol by enzyme-immunometric methods.
Results: The concentration of cortisol shows a rhythmical course in long term cell cultures. The addition of CRH (500 ng/ml, 1 µg/ml) stimulates the concentration of ACTH- and cortisol in a time-depending manner. The addition of ACTH (500 ng/ml-2 µg/ml) stimulates the concentration of cortisol in a time-depending manner. The addition of prednisolon stimulates the concentration of ACTH.
Conclusions: The trophoblast cell shows a rhythmical course in the concentration of cortisol. For the first time a CRH-ACTH-cortisol feedback loop could be demonstrated in cultured trophoblast cells.

Trophoblastzelle - CRH - ACTH - Kortisol - in vitro

Trophoblast cells - CRH - ACTH - cortisol - in vitro

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