低压脉动电刺激大鼠骨髓间充质干细胞向心肌细胞的分化:希望验证电学因素在心肌发育过程中作用  

Differentiation of bone marrow mesenchymal stem cells into cardiomyocytes in rats following low-tension pulse electric stimulation:In the hope of verifying effects of electricity factors in development of the myocardium

作　　者：伍长学[1] 马建旸[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]四川大学华西医院心脏外科,四川省成都市610041

出　　处：《中国组织工程研究与临床康复》2009年第40期7859-7864,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：重大基础(973)前期研究专项基金(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:电学微环境是心肌细胞所处微环境的要素之一。在体外干细胞向心肌细胞诱导分化的过程中,模拟心肌微电学环境的脉动性电流刺激是否有助于心肌分化呢?目的:大鼠骨髓间充质干细胞向心肌细胞诱导分化过程中,验证课题提出的低压脉动电刺激对心肌分化过程影响的假设,并分析其分子生物学机制。设计、时间及地点:细胞-基因学实验,于2005-09/2007-03在四川大学老年病研究室完成。材料:4周龄SD大鼠5只,购自四川大学华西医学中心动物实验室中心。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,取传至第3代细胞,设立2组,5-氮胞苷诱导组单纯给予10μmol/L5-氮胞苷诱导;5-氮胞苷诱导+电刺激组在5-氮胞苷诱导基础上,对骨髓间充质干细胞施加电场刺激,刺激参数选择1.5V/cm电压,2ms方波,频率1Hz,刺激时间为2h/d。主要观察指标:分别于诱导第1,2,3,4周收集细胞,western blot技术检测TroponinⅠ,Desmin,Connexin-43蛋白的表达,RT-PCR法分析GATA-4,NKx2.5,MEF2C基因的表达。结果:诱导后细胞停止生长,并倾向集落化。两组均在诱导第2周开始表达TroponinⅠ,Desmin,Connexin-43蛋白,第3,4周表达明显增强。两组均在诱导第1周开始表达GATA-4,NKx2.5,MEF2C基因,并逐渐增强,至第3周达高峰,持续到第4周。在同一时间点,5-氮胞苷诱导+电刺激组蛋白、基因的表达量均显著高于5-氮胞苷诱导组(P<0.01)。结论:GATA-4,NKx2.5和MEF2C基因调控心肌细胞的形成,而低压脉动电场刺激通过上调其表达进而促进大鼠骨髓间充质干细胞向心肌细胞的分化过程。课题结果验证了电学因素在心肌发育过程中具有重要意义的理论假设。BACKGROUND：Electricity microenvironment is one of factors of microenvironment of cardiomyocytes. In the processing of differentiation of stem cells into cardiomyocytes in vitro,whether to simulate pulsing electric stimulation in electricity microenvironment of the myocardium is beneficial for myocardium differentiation？ OBJECTIVE：To verify the influence of low-tension pulse electric stimulation on the course of myocardium differentiation,analyze its molecular biological mechanisms during the differentiation of rat bone marrow mesenchymal stem cells（BMSCs） into cardiomyocytes. DESIGN,TIME AND SETTING：Cell-gene study was performed at the Research Room of Senile Disease,Sichuan University from September 2005 to March 2007. MATERIALS：A total of 5 Sprague Dawley rats aged 4 weeks old were purchased from the Animal Laboratory Center of West China Medical Center of Sichuan University. METHODS：Rat BMSCs were cultured in vitro by density gradient centrifugation and adherence method. At the third passage,BMSCs were divided into 2 groups. BMSCs in the 5-azacytidine group were administered 10 μmol/L 5-azacytidine. BMSCs in the 5-azacytidine ＋ electric stimulation group were treated with 5-azacytidine and electric stimulation,1.5 V/cm voltage,2 ms square wave,1 Hz frequency,2 hours per day. MAIN OUTCOME MEASURES：BMSCs were collected at 1,2,3 and 4 weeks following induction. Western blot was used to determine Troponin I,Desmin,Connexin-43 protein expression. Reverse transcription-polymerase chain reaction（RT-PCR） was utilized to analyze expression of GATA-4,NKx2.5 and MEF2C genes. RESULTS：Growth of BMSCs stopped after being induced,trending to colony. BMSCs were positive for Troponin I,Desmin,Connexin-43 protein at week 2,and became strong at weeks 3 and 4. In both groups,BMSCs were positive for GATA-4,NKx2.5 and MEF2C genes at week 1,and gradually enhanced,peaked at week 3,lasted for week 4. At the same time point,protein and gene expression in the 5-azacytidine ＋ electric stimulation group was si

关 键 词：电场刺激 骨髓间充质干细胞 心肌细胞 转录因子 

分 类 号：R394.2[医药卫生—医学遗传学]