Isolation, culture and identification of vascular smooth muscle cells with synthetic phenotype

Abstract

Objective To investigate a method to obtain vascular smooth muscle cells (VSMCs),and isolate,culture and identify the VSMCs with highly synthetic phenotype.Methods The primary and subculture of VSMCs from thoracic aorta of SD rats was done by modified collage Ⅱ enzymatic dispersion and trypsin digestion,respectively.The third passage VSMCs were treated with platelet-derived growth factorBB (PDGF-BB).The cell viability and migration were detected by methyl thiazol tetrazolium (MTT) assay and wound-healing assay,respectively.The VSMCs were observed using inverted microscope and crystal violet staining for morphological observation.Immunofluorescence staining and immunocytochemical staining were used to examine contractile marker α-smooth muscle actin (SMA) and synthetic markers nonmuscle MHC isoform-B (SMemb),Tropomyosin-4 (TPM-4) for VSMCs identification and quantitative analysis.Results The post-confluent cells in the control group exhibited the hill and valley growth pattern typical of cultured VSMCs.More than 95％ of cells were positively stained by both stains for SMA.VSMCs under PDGF-BB treatment underwent a significant morphological alteration,from spindle to polygonal shape (P ＜0.05).The viability and mobility of VSMCs was increased by 11.48％ and 1.92 times,respectively (P ＜0.05).The expression of both SMemb and TPM-4 proteins was increased,and that of SMA protein decreased,significantly (P ＜ 0.05).Conclusion PDGF-BB treatment could lead to a significant phenotype modulation (highly synthetic VSMCs) in accompanied with enhancement of viability and mobility,highly increased synthetic markers while decreased contractile marker,significantly. Key words: Vascular smooth muscle cells; Cell phenotype; Platelet-derived growth factor; Cell culture