Primary culture of mouse vascular endothelial cells and establishment of a senescence model

Objective To establish a straightforward and efficient method for the primary isolation and cultivation of mouse aortic endothelial cells(ECs), and to induce a cellular senescence model.

Methods The thoracic and abdominal aortas of 8-week-old C57BL/6 mice were harvested under aseptic conditions and enzymatically digested with collagenase.The ECs were collected and cultured.The cell growth condition was continuously monitored using an inverted microscope, and the expression levels of CD31 and CD105 (the specific markers of ECs) were assessed by flow cytometry.After stimulating ECs with 200 μmol/L H₂O₂ for 24 h, the expression levels of cellular senescence markers p53 and p21 were measured by Western blotting and qRT-PCR, and SA-β-Gal staining were performed.

Results In the early stages of the cell culture, scattered, irregular, spindle-shaped adherent growth was observed under the inverted microscope.These cells were gradually divided and proliferated, forming a network-like structure.After the first passage, the cells exhibited the typical "paving stone" morphology.Flow cytometry results revealed that 95.8% cells expressed with CD31 and CD105 synchronously.SA-β-Gal staining results demonstrated that the proportion of positive cells in the H₂O₂-treated group was significantly higher than that in the control group(P < 0.01).Furthermore, the results of Western blotting and qRT-PCR experiments proved that the expression levels of p53 and p21 were both elevated in the H₂O₂-treated group compared with the control group, and the differences were statistically significant (P < 0.05 to P < 0.01).

Conclusions A simple primary culture system for mouse aortic ECs and a stress-induced premature senescence model are successfully established, which provide a reliable technical support for the study of ECs biology in vitro.