Liposomal vermittelte Transfektion von Thrombomodulin in vaskuläre glatte Muskelzellen - ein Gentherapie-Konzept zur Inhibition der Rezidiv-Stenose?

Zusammenfassung

Ziel: Thrombomodulin (TM), ein membranständiger Rezeptor, ist bei Endothelzellen für seine antikoagulative Wirkung bekannt. Zudem gibt es Hinweise für wachstumsregulatorische Effekte des Oberflächenproteins. Ziel dieses in vitro Projektes war es, stabile Zellreihen glatter vaskulärer Myozyten mit TM-Überexpression durch Transfektion zu erhalten, um die wachstumsmodulierenden Eigenschaften von TM und seine mögliche Rolle im Rahmen der Restenoseausbildung näher zu charakterisieren. Methode: In aortale glatte Muskelzellen der Ratte wurde die cDNA von Mäuse-TM oder einer seiner drei Mutanten (M1, 2, 4) mit einer liposomenvermittelten Transfektionsmethode eingebracht. Mittels RT-PCR wurde die murine TM-mRNA-Expression in den selektierten Klonen nachgewiesen. Das Zellwachstum wurde anhand von Proliferationskinetiken untersucht. Die Quantifizierung der TM-Gesamtmenge erfolgte mit Western Blots. Ergebnisse: Die RT-PCR wies in 44 % erfolgreiche Transfektionen mit Mäuse-TM nach. Die Klone mit TM, M1 oder M2 zeigten im Mittel eine Wachstumsinhibition, M4 dagegen zur Kontrolle eine gesteigerte Proliferation. Die Gegenüberstellung der TM-Gesamtmenge zum Wachstumsverhalten der einzelnen Klone ergab eine negative Korrelation zwischen Proliferation und TM-Expression (Korrelationskoeffizienten TM -0,87, M1 -0,59). Schlussfolgerungen: Dieses Transfektionsmodell ermöglicht die Reproduktion stabiler Zellreihen vaskulärer glatter Myozyten mit TM-Überexpression in vitro und ist damit Grundlage für detaillierte Untersuchungen der Mechanismen der Wachstumsregulation durch TM.

Abstract

Purpose: Thrombomodulin (TM), an integral endothelial receptor, is known for its anticoagulant functions. Moreover, there is evidence of growth-modulating effects of this cell surface ­protein. The aim of our study was to establish by in vitro transfection a stable cell line of vascular smooth muscle cells with overexpression of TM for further investigations concerning the influence of TM on cellular proliferation and its potential role during the formation of restenosis. Methods: Aortic smooth muscle cells of the rat were transfected with cDNA of mouse TM or one of its three mutants (M1, M2, M4) by a liposome-mediated technique. The expression of mouse TM mRNA in the selected clones was proven with the help of RT-PCR. Changes of cell proliferation were determined by proliferation kinetics over 24 days. The quantification of the total protein TM was made by Western blots. Results: In 44 of 100 cases the RT-PCR confirmed a successful transfection of mouse-TM. The clones with transfected TM, M1 or M2 showed an inhibited cell growth, whereas M4 demonstrated an increased proliferation compared with controls. The comparison of amounts of total TM with cell growth of individual clones resulted in a negative correlation between proliferation and TM-expression (coefficient of correlation for TM -0.87, for M1 -0.59). Conclusions: It is possible to reproduce stable cell-lines of vascular smooth muscle cells with overexpression of TM by the presented model of in vitro transfection. Thus, a basis exists for detailed examinations of growth-regulating mechanisms by TM.

Key words

Thrombomodulin - proliferation - vascular smooth muscle cells - transfection - restenosis

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