Osteogenic ability of reversibly immortalized valve mesenchymal cells induced by BMP9 in mice

Abstract:

Objective To establish a reversibly immortalized aortic valve mesenchymal cell line with rapid proliferation.Methods Valve tissue was clamped with spring scissors under stereomicroscope，and the valve tissue was digested with collagenase and affected with immortalized retrovirus. The cell proliferation ability was detected by crystal violet，CCK-8 and flow cytometry. The osteogenic ability of the cells was detected by ALP staining and alizarin red calcium salt deposition assay. The osteogenic markers were detected by q-PCR and Western blot.Results By light microscopy and immunofluorescence identification，we confirmed that the mesenchymal cells were successfully obtained. After affected with immortalized virus，light microscopy and immunofluorescence confirmed that there was no phenotypic change in valve mesenchymal cells，and a large amount of SV40T could be detected in immortalized cells（P<0.001）. The results of crystal violet staining，CCK-8（P<0.001） and flow cytometry showed that the proliferation rate of valve mesenchymal cells was faster after immortalization. After addition of osteogenic inducible factors BMP9，immortalized valve mesenchymal cells showed better osteogenic ability，and the most of osteogenic markers were significantly increased at the molecular level. There was no significant change in the morphology of valve mesenchymal cells after deimmortalization，and q-PCR results showed that SV40T was significantly reduced（P<0.001）. Cell proliferation was significantly decreased， and the cell cycle was arrested in the G₀/G₁ phase. Deimmortalization valve mesenchymal cells induced by BMP9 also had osteogenic differentiation ability，and most osteogenic markers were significantly increased.Conclusion We have successfully established reversibly immortalized valve mesenchymal cell lines，which can be induced by BMP9 for osteogenic differentiation and can be used for subsequent studies of calcified aortic valve disease.