P19-αMHC—EGFP干细胞株的构建及其向心肌细胞诱导分化的初步研究  

Generation of a P19-αMHC-EGFP reporter line and cardiomyocyte differentiation

作　　者：龚杰 张奇军 汪剑 孙凤荣 钱玲梅 孔祥清 杨荣 盛燕辉 曹克将 

机构地区：[1]江苏省心血管病临床医学中心南京医科大学第一附属医院心脏科,210029

出　　处：《中华心血管病杂志》2008年第8期691-694,共4页Chinese Journal of Cardiology

基　　金：国家自然科学基金资助项目（30470709）;江苏省自然科学基金资助项目（BK2004156）

摘　　要：目的构建稳定携带心肌特异报告基因pαMHC—EGFP的P19细胞株并诱导其向心肌细胞分化。方法应用脂质体转染法转染质粒pαMHC—EGFP到P19干细胞，通过G418筛选和有限稀释法得到稳定携带pαMHC—EGFP的细胞克隆P19-αMHC—EGFP。诱导P19一otMHC—EGFP向心肌细胞分化，在诱导的不同阶段观测其超微结构的变化，在诱导分化进程中监测“跳动”的发绿色荧光的心肌细胞出现。同时与未转染pαMHC—EGFP的P19细胞进行心肌细胞分化率及心肌肌钙蛋白I（cTnI）表达量的对比。结果电镜结果提示在诱导的第10天，部分细胞出现心肌细胞样特征。P19-αMHC—EGFP在诱导分化的第7天出现发强绿色荧光并“跳动”的心肌细胞，同时与对照P19细胞在心肌细胞分化率及cTnI表达量上差异无统计学意义（P〉0．05）。结论P19-αMHC-EGFP细胞株在分化为心肌细胞的同时具有强绿色荧光标记，其向心肌细胞分化的效率及cTnI的合成未受影响。这一特征有利于对分化成熟的心肌细胞进行识别和纯化，从而应用于心肌细胞替代治疗。Objective To generate a P19-αMHC-EGFP reporter line and induce cardiomyocyte differentiation of this reporter line. Methods The P19 cells were transfected with pαMHC-EGFP, a P19- αMHC-EGFP reporter line was obtained after G418 selection and limited dilution of recombinant clones. The reporter line was induced to differentiate into cardiomyocytes which would beat and express green fluorescent protein. A comparison of cardiomyocyte differentiation rate and cTnI expression amount between the reporter line and the untransfected P19 cells was also performed. The ultrastructure was observed under transmission electron microscope. Results The uhrastructure characteristics indicated cardiomyocytes-like changes on induction day 10. The beating cardiomyocytes which express GFP appear in the seventh induction day. The cardiomyocyte differentiation rate and cTnI expression amount of P19-αMHC-EGFP reporter line were similar as those in untransfected P19 cells （P 〉 0. 05 ）. Conclusion The P19-αMHC-EGFP reporter line is of great benefit for identifying and purifying cardiomyocytes from undifferentiated P19 cells without influencing the differentiation of P19 cells. This feature makes P19-αMHC-EGFP reporter line a promising cell source for clinical cardiomyocyte replacement therapy.

关 键 词：心肌 干细胞 细胞分化 

分 类 号：R686[医药卫生—骨科学]