骨髓间充质干细胞向心肌细胞诱导过程中脉动电流刺激对肌细胞增强因子2C基因表达的影响  

Effect of pulse electric stimulation on myocyte enhancer factor 2C gene expression during differentiation of bone marrow mesenchymal stem cells into cardiomyocytes

作　　者：伍长学[1] 杨思远[2] 马建旸[2] 张尔永[2] 安琪[2] 赁可[2] 石应康[2] 

机构地区：[1]泸州医学院附属医院胸外科,四川省泸州市646000 [2]四川大学华西医院胸外科,四川省成都市610041

出　　处：《中国组织工程研究与临床康复》2008年第43期8408-8412,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：重大基础("九七三")前期研究专项(2005CCA03600);国家自然科学基金(30500497);四川省科技厅攻关/重点项目(05SG022-020)~~

摘　　要：背景:一些研究发现电刺激可以改变成熟心肌细胞的分布和电生理特性,其是否对骨髓间充质干细胞向心肌细胞分化的过程产生影响目前还不清楚。目的:在诱导骨髓间充质干细胞向心肌细胞分化过程中,观察低压脉动电刺激对肌细胞增强因子2C基因表达的影响。设计、时间及地点:对比观察电刺激细胞学体外实验,于2005-09/2007-03在四川大学老年病研究室完成。材料:清洁级4周龄雄性SD大鼠5只,由四川大学华西医学中心动物实验室中心提供。诱导剂5-氮胞苷为Sigma产品。使用一次性100mL培养瓶、镍钛合金金属丝、心脏起搏器和连接导线设计制作电场。方法:密度梯度离心+贴壁法体外分离培养大鼠骨髓间充质干细胞,传至第3代按1×107L-1接种到电场内,细胞爬满80%后,加入终浓度为10μmol/L5-氮胞苷进行诱导。分别于诱导第1,2,3,4周收集细胞,分为2组:刺激组施加电场刺激,刺激参数选择1.5V/cm电压,2ms方波,频率1Hz,刺激2周;对照组单纯以5-氮胞苷诱导。主要观察指标:免疫细胞化学法和westernblot技术检测肌钙蛋白I表达,RT-PCR法分析肌细胞增强因子2C基因的表达。结果:诱导后细胞停止生长,并倾向集落化,诱导第2周两组均开始出现肌钙蛋白I阳性细胞,第3、4周明显增强,诱导各时间点刺激组肌钙蛋白I表达量均显著高于对照组(P<0.05)。诱导第1周两组均开始表达肌细胞增强因子2C基因,第2周明显增强,第3周达高峰,持续到第4周,诱导各时间点刺激组肌细胞增强因子2C基因的表达量均显著高于对照组(P<0.05)。结论:肌细胞增强因子2C基因可能参与调控心肌细胞的形成过程,而电场刺激通过上调肌细胞增强因子2C基因的表达,进而促进肌钙蛋白I的表达和心肌细胞的形成。BACKGROUND： Electric stimulation can change distribution of mature cardiomyocytes and electrophysiological characteristics. It is unclear whether electric stimulation has effects on differentiation of bone marrow mesenchymal stem cells （BMSCs） into cardiomyocytes. OBJECTIVE： To investigate the influence of pulse electric stimulation on the expression of the transcriptive factors, myocyte enhancer factor （MEF） 2C mRNA, in the course of differentiation of BMSCs into cardiomyocytes. DESIGN, TIME AND SETTING： The controlled electric stimulation cytology in vitro experiment was performed at the Research Room of Senile Disease, Sichuan University from September 2005 to March 2007. MATERIALS： Five clean male Sprague Dawley rats aged 4 weeks were obtained from Animal Laboratory Center, West China Medical Center, Sichuan University. 5-azacitidine was purchased from Sigma, USA. 100 mL culture flask, nitinol wire, cardiac pacemaker and connecting lead were used to make electric field. METHODS： Rat BMSCs were isolated and cultured in vitro by density gradient centrifugation ＋ adherence method. The third passage of BMSCs were incubated in electric field at a density of 1 × 10^7 L^-1. When 80% cells were confluent, BMSCs were induced with 10 μmol/L 5-azacytidine. BMSCs were collected at 1, 2, 3 and 4 weeks and divided into 2 groups. BMSCs in the stimulation group received1.5 V/cm pulse electric stimulation, 2 ms square wave, a frequency of 1 Hz for 2 weeks. BMSCs in the control group were treated with 5-azacytidine. MAIN OUTCOME MEASURES： Troponin I was measured by immunocytochemistry and Western Blot. MEF2C gene were analyzed by reverse transcfiption-polymerase chain reaction （RT-PCR）. RESULTS： Growth of BMSCs stopped after being induced, trending to colony. Troponin I positive cells expressed at 2 weeks, and increased at 3 and 4 weeks in both groups. Troponin I expression was significantly higher in the stimulation group than in the control group （P 〈 0.05）. MEF2C mRNA began to express at

关 键 词：骨髓间充质干细胞 心肌细胞 电场刺激 肌细胞增强因子2C 

分 类 号：R394.2[医药卫生—医学遗传学]