Osteoblastäre Differenzierung von humanen adulten mesenchymalen Stammzellen durch transgenes BMP-2 in Abwesenheit von Dexamethason

Zusammenfassung

Studienziel: Mesenchymale Stammzellen (MSC) verschiedener Spezies scheinen unterschiedlich auf osteogene Stimuli zu reagieren. Es wird beschrieben, dass MSC humanen Ursprungs nach Behandlung mit BMP-2 nicht osteoblastär differenzieren, sondern dass es hierzu der Wirkung von Dexamethason bedarf. In vorliegender Studie wurde die Wirkung des adenoviralen Transfers des BMP-2-Gens auf Genotyp und Phänotyp von hMSC in Abwesenheit von Dexamethason untersucht. Methode: hMSC wurden mit adenoviralen Vektoren für BMP-2 transduziert und in Abwesenheit von Dexamethason oder anderen osteogenen Supplementen in Kultur gehalten. Änderungen in der Expression von Knochenmarkergenen Runx2, Osterix und Typ-I-Kollagen wurden mittels quantitativer PCR bestimmt. Als phänotypischer Marker wurde die alkalische Phosphataseaktivität untersucht. Die statistische Auswertung erfolgte mittels Varianzanalyse und Post-hoc-Tests (p < 0,05). Ergebnisse: Der adenovirale Transfer des BMP-2-Gens in hMSC und die nachfolgende Produktion von transgenem BMP-2 führte ohne Zugabe osteogener Supplemente zu einem Expressionsanstieg der genotypischen Marker und zu einer Zunahme der alkalischen Phosphataseaktivität als Ausdruck osteoblastärer Differenzierung. Schlussfolgerung: Unsere Beobachtungen sind bemerkenswert als möglicher Hinweis auf eine Überlegenheit transgener, endogen produzierter Proteine gegenüber exogen zugeführten rekombinanten Proteinen.

Abstract

Aim: Mesenchymal stem cells (MSC) of various species appear to require different cues to differentiate towards the osteoblastic lineage. For MSC of human origin, recombinant hBMP-2 is reported to be not sufficient but dexamethasone seems to be essential. The aim of this study was to analyse changes in genotype and phenotype of hMSC after adenoviral transfer of the BMP-2 gene in the absence of dexamethasone. Methods: We employed hMSC and analysed changes in expression of the Runx2, Osterix and type I collagen gene by quantitative PCR after adenoviral transfer of the human BMP-2 gene in the absence of dexamethasone. As a phenotypic marker alkaline phosphatase activity was assessed. ANOVA and post hoc statistical analyses were used to determine differences among data (p < 0.05). Results: Transfer of the hBMP-2 gene and consecutive production of transgenic BMP-2 up-regulated bone marker gene expression and increased alkaline phosphatase activity and thus promoted an enhanced lineage progression to the osteoblast phenotype without the addition of dexamethasone. Conclusion: These findings are noteworthy in the light of a possible superiority of endogenous transgenic proteins compared to exogenous recombinant proteins.

humane mesenchymale Stammzellen - adenovirale Transduktion - BMP-2 - osteoblastäre Differenzierung - quantitative real-time PCR

Human mesenchymal stem cells - adenoviral transduction - BMP-2 - osteoblastic differentiation - quantitative real-time PCR

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