超声辐照微泡介导5-氮杂胞苷诱导人骨髓间充质干细胞心肌样分化的实验研究

摘要

目的 探索建立低声强脉冲超声联合SonoVue微泡培养孔内直接辐照增强5-氮杂胞苷(5-Aza)体外诱导入骨髓间质干细胞(MSCs)心肌样分化的方法,摸索超声辐照条件,为超声靶向击破微泡技术在MSCs移植治疗急性心肌梗死中的应用提供实验数据.方法 ①以不同的超声辐照强度(0.11、0.33、0.55、0.77、0.99 W/cm2)与不同的辐照时间(0、30、60、90 s)组合于六孔板培养孔内直接超声辐照MSCs,用四甲基偶氮唑蓝(MTT)比色法筛选出对其活性无明显抑制的声强-时间组;将筛选出的超声辐照强度-时间联合不同微泡数/干细胞数比例(0、10、50、100、150)对应的造影剂剂量于培养孔内直接超声辐照MSCs,用MTT筛选出对其无明显抑制的微泡数/干细胞数比例;②将MSCs随机分为4组,各自采用如下体外心肌化诱导方式:对照组、5-Aza(10μmol/L)组、超声辐照微泡组、5-Aza(10 μmol/L)+超声辐照微泡组,诱导后培养4周,倒置相差显微镜逐日观察细胞形态学变化,并于诱导后28 d免疫细胞化学染色法检测心肌特异肌钙蛋白T(cTnT)表达,计算并比较各组的心肌样细胞分化率.结果 ①无明显抑制MSCs且具有一定微泡击破效应的培养孔内直接超声辐照条件为辐照强度0.55 W/cm2,辐照时间为30 s,微泡数/干细胞数比值为50;②各组MSCs诱导后免疫细胞化学染色法检测发现5-Aza组及5-Aza+超声辐照微泡组可见cTnT表达阳性,且后组阳性率显著高于前组(13.69％对34.13％,P＜0.05).结论 声强0.55 W/cm2、辐照时间30 s、微泡数/干细胞数比50是适当的直接孔内超声辐照条件;在适当条件下的低声强脉冲超声辐照能够增强5-Aza体外诱导MSCs心肌样分化效果.%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.