Taurine Inhibits Osteoblastic Differentiation Of Aortic Valve Interstitial Cells

Chapter One:Isolation, culture and identification of aortic valve interstitial cells.Objective:To isolate and cultivate aortic valve interstitial cells, and prove that aortic valve interstitial cells express taurine transporter and are not contamintated by other cells.Methods:Enzymatic method was obtained to dissolve extracellular matrix of the aortic valves from patients with acute dissection(type A). Cell phenotypic identification was achieved by immunochemistry staining of taurine transporter, SM a-actin, vimentin, CD31, SM-myosin and desmin.Results:Isolated cells proliferated well, expressed taurine transporter, SM a-actin and vimentin, but no CD31, SM-myosin or desmin.Conclusion:Isolated aortic valve interstitial cells grew well in vitro, expressed specific proteins and were not contamintated by endothelial cells or muscle cells. Chapter two:The establishment of osteoblastic differentiation model of aortic valve interstitial cells. Objective:To establish the osteoblastic differentiation model of aortic valve interstitial cells by pro-calcific medium.Methods:Pro-calcific medium (containing β-glycerophosphate disodium, dexamethasone and ascorbic acid) was used to promote the osteoblastic differentiation of cultured cells. This cell model was evaluated by alkaline phosphatase activity/staining, calcified nodules(von Kossa staining) and the expression of alkaline phosphatase and Cbfal by western blotting.Results:On the21st day of intervention, the cell model was achieved by the positive staining of alkaline phosphatase and von Kossa and more expression of alkaline phosphatase and Cbfal compared with blank control.Conclusion:Pro-calcific medium could promote the osteoblastic differentiation of cultured cells in the form of expressing alkaline nhosbhatase and Cbfal and forming calcified nodules. Chapter three:Taurine suppresses the osteoblastic differentiation of aortic valve interstitial cells via the ERK pathway Objective:To prove that taurine can attenuate the osteoblastic differentiation of aortic valve interstitial cells, and explore the role of taurine transporter and ERK pathway.Methods:Different concentrations of taurine (1mM,3mM,5mM,10mM,20mM) or taurine(10mM) plus the ERK pathway blocker PD98059were added into the pro-calcific medium. The expression of Cbfα1was detected after24h of intervention, and the activity/expression of alkaline phosphatase was detected on the14th and21st day of intervention. The activation level of ERK pathway was examed on different time point (5min,10min,30min,60min,120min) after the addition of taurine into the medium. And the blockade effect of β-alanine and PD98059on the activation of ERK pathway by taurine(lOmM) was detected after20min of intervention.Results:Taurine attenuated the expression of Cbfα1and alkaline phosphatase on a dose dependent manner (reaching the maximum effect at10mM) which could be attenuated by PD98059. Taurine could activate the ERK pathway whose effect could be abolished by β-alanine.Conclusion:Taurine attenuated the osteoblastic differentiation of aortic valve interstitial cells via taurine transporter and ERK pathway.