Primary culture and in vitro calcification model establishment of human aortic valve interstitial cells

Abstract

Objective To primary culture human aortic valvular interstitial cells(hVlCs).establish their in vitro calcification model,and to induce hVICs differentiation to osteogenesis and to observe the phenotype changes.Methods hVICs were digested from native valves and used for experiments after 3-7 passages.The cells were cultured in osteogenic media or in normal media.One week later the calcified nodules were stained and measured by von Kossa.The activity of alkaline phosphatase(ALP) was examined by spectrophotometer,immunofluorescence staining was used to detect the phenotype protein of hVICs,and real-time PCR and Western blotting analysis were used to examine the osteogenesis associated factors to assess the calcification model of hVICs.Results The calcified nodules were found 7 days after osteogenic induction.The calcified nodules in the experimental group were significantly more than that in the control group(51.20±14.31/well vs 3.60±1.82/ well,P0.05).The activity of ALP was significantly increased after osteogenic induction compared with the control group (increased by about 4 folds,P0.05),with increased contractile phenotypeα-smooth muscle actin(α-SMA).Real-time PCR and Western blotting results indicated that the expressions of Run×2,osteocalcin,and osteopontin in the experiment group were significantly higher than those in the control group(P0.05),so was the expression of phosphorylated Smad1/5/8(P0.05). Conclusion We have successfully established the in vitro calcification model of hVICs,with hVICs in an activated state;and the phenotype shifts to contraction and ossification,which provide a reliable cell model for the future study.