[A novel extraction method and identification of primary mouse pulmonary artery smooth muscle cells].

Abstract

Objective To establish an efficient and rapid primary culture protocol for mouse pulmonary artery smooth muscle cell (PASMC). Methods PASMC were isolated from mice using a novel dual-enzyme digestion method. Cell morphology was observed under an inverted phase-contrast microscope, and cell type and purity were identified by immunofluorescence staining for α-smooth muscle actin (α-SMA). Cell growth after passage was assessed using the CCK-8 assay. The migratory response of PASMC to platelet-derived growth factor-BB (PDGF-BB) was evaluated by cell scratch assay. Changes of protein expression in primary PASMC after stimulation of transforming growth factor β1 (TGF-β1) were detected by Western blot. Results Under light microscopy, PASMC showed a long spindle-shaped morphology with a positive rate reaching as high as 98%. The growth curve of passaged cells approximated an "S" shape. Primary cultured PASMC showed a significant migratory response to stimulation of PDGF-BB. TGF-β1 stimulation significantly increased the expression of phospho-Smad2 (p-Smad2) in PASMC. Conclusion The novel dual-enzyme digestion method significantly improves the isolation efficiency and viability of primary PASMC, providing a sufficient and phenotypically stable source of high-quality cells for in vitro studies.