Experimental study of influence on 5-azacytine induced cardiomyogenic differentiation of human bone marrow mesenchymal stem cell in vitro by ultrasonic exposure combined with microbubbles

Abstract

Objective To explore and establish the possible method of low-intensity pulsed ultrasound(LIPUS) irradiation directly combined with SonoVue microbubbles could enhance 5-azacytine (Aza) induced cardiomyogenic differentiation of human bone marrow mesenchymal stem cellin(MSCs)vitro,initially explore the appropriate irradiation condition,and to provide experimental basic data for ultrasound-mediated microbubble destruction application in MSCs transplantation for acute myocardial infarction.Methods (1) In order to screen out the appropriate ultrasound irradiation intensity and time without conspicuous harm to MSCs,stem cells were exposed to different ultrasound intensity irradiation (0.11,0.33,0.55,0.77,0.99 W/cm2) and time (0,30,60,90 s) and then cell proliferation determined by methyl thiazolyl tetrazolium(MTT).In order to screen out the appropriate microbubble-to-cell ratio without conspicuous harm to MSCs,stem cells of different microbubble-to-cell ratios (0,10,50,100,150) were exposed above the screened ultrasound irradiation intensity and time and then cell proliferation determined by MTT.(2) MSCs were divided into four groups and induced cardiomyogenic differentiation by different inducing systems in vitro as follows:no-treatment group,5-Aza inducing group(10 μmol/L),microbubble and ultrasound group,5-Aza inducing (10 μmol/L) with microbubble and ultrasound group,followed by four weeks of further culture.The morphological changes were studied daily under phase-contrast microscope,then the expression of cardiac troponin-T (cTnT) was detected by immunocytochemistry method to analyze and compare differentiation rate of cardiomyocyte-like cells in each group.Results (1)The direct ultrasound irradiation condition of no conspicuous harm to MSCs,but with destruction was intensity of 0.55 W/cm2,exposure time of 30 s and microbubble-to-cell ratio of 50.(2) This study confirmed the feasibility of direct ultrasound exposure of LIPUS on MSCs in vitro.(3) At 28 days after induction,the stem cells of 5-Aza inducing group and 5-Aza inducing with microbubble and ultrasound group expressed cTnT and the positive rate of the latter was higher than the former by immunocytochemistry (13.69％ vs 34.13％,P ＜0.05).Conclusions The appropriate ultrasound direct irradiation condition was intensity of 0.55 W/cm2,exposure time of 30 s and microbubble-to-cell ratio of 50.Appropriate conditions of LIPUS could enhance 5-Aza induced cardiomyogenic differentiation of MSCs in vitro. Key words: Ultrasonography; Microbubbles ; Azacitidine; Mesenchymal stem cell ; Cell differentiation