CAJ | 학술논문

研究小鼠胚胎成纤维细胞与新生小鼠心肌细胞共培养、并过表达心肌特异性转录因子对小鼠胚胎成纤维细胞向心肌细胞转分化效率的影响.首先取出生48 h 的新生小鼠的心肌细胞并进行培养,模拟新生状态下的心脏内环境.在这一基础上,将小鼠胚胎成纤维细胞与新生小鼠心肌细胞共培养,并使用相关转录因子过表达的策略,使用质粒载体在小鼠胚胎成纤维细胞中过表达 GATA4,MEF2C,TBX5,HAND,NKX2.5.15 d 后通过鉴定相关mRNA 表达量,免疫荧光观察特定蛋白分布以及流式细胞仪分析,来评估转分化的效率.结果表明,在模拟新生心脏内环境以及过表达心肌相关转录因子的条件下,小鼠胚胎成纤维细胞向心肌细胞转分化的效率得到提高,但是经由转分化得到的心肌细胞的成熟程度依然不足.
연구소서배태성섬유세포여신생소서심기세포공배양、병과표체심기특이성전록인자대소서배태성섬유세포향심기세포전분화효솔적영향.수선취출생48 h 적신생소서적심기세포병진행배양,모의신생상태하적심장내배경.재저일기출상,장소서배태성섬유세포여신생소서심기세포공배양,병사용상관전록인자과표체적책략,사용질립재체재소서배태성섬유세포중과표체 GATA4,MEF2C,TBX5,HAND,NKX2.5.15 d 후통과감정상관mRNA 표체량,면역형광관찰특정단백분포이급류식세포의분석,래평고전분화적효솔.결과표명,재모의신생심장내배경이급과표체심기상관전록인자적조건하,소서배태성섬유세포향심기세포전분화적효솔득도제고,단시경유전분화득도적심기세포적성숙정도의연불족.
To evaluate the effect of natal cardiac microenvironment in cardiomyocytes transdifferentiation,extraction of natal murine cardiomyocytes was performed 48 hours after birth.The mouse embryonic fibroblast(MEF)were co-cultured with murine cardiomyocytes in transwell culturing system and then transfected with a plasmid vector to overexpress 5 key cardiac transcription factors,including GATA4,MEF2C,TBX5,HAND and NKX2.5.On Day 1 5 after transdifferentiation,real-time qPCR,immunofluorescence and flow cytometry were used to evaluate the efficiency of transdifferentiation.Real-time qPCR showed that co-cultured with murine cardiomyocytes,the expression of cardiac specific gene cTnT was upregulated,and the non-cardiac but a muscle specific gene Myh1 1 was downregulated.Immunofluorescence demonstrated the distribution of cardiac specific protein cTnT and α-MHC,but no sarcomere detected.Flow cytometry indicated that co-cultured with murine cardiomyocytes,the cTnT+ cell rate increased much.However,with the extension of culture time,the maturity of these cells was inhibited and showed the backward development.Therefore,microenvironment of natal cardiomyocytes could significantly improve the efficiency and specificity of transdifferentiation.