CAJ | 학술논문

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-αMHC-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.
목적 구건은정휴대심기특이보고기인pαMHC-EGFP적P19세포주병유도기향심기세포분화.방법 응용지질체전염법전염질립pαMHC-EGFP도P19간세포,통과G418사선화유한희석법득도은정휴대pαMHC-EGFP적세포극륭P19-αMHC-EGFP.유도P19-αMHC-EGFP향심기세포분화,재유도적불동계단관측기초미결구적변화,재유도분화진정중감측"도동"적발록색형광적심기세포출현.동시여미전염pαMHC-EGFP적P19세포진행심기세포분화솔급심기기개단백I(cTnI)표체량적대비.결과 전경결과 제시재유도적제10천,부분세포출현심기세포양특정.P19-αMHC-EGFP재유도분화적제7천출현발강록색형광병"도동"적심기세포,동시여대조P19세포재심기세포분화솔급cTnI표체량상차이무통계학의의(P>0.05).결론 P19-αMHC-EGFP세포주재분화위심기세포적동시구유강록색형광표기,기향심기세포분화적효솔급cTnI적합성미수영향.저일특정유리우대분화성숙적심기세포진행식별화순화,종이응용우심기세포체대치료.
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 ultrastructure 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.