CAJ | 학술논문

背景：微小 RNA(miRNA)是一类非编码小分子 RNA，miRNA参与调控基因表达，在促进前体细胞增殖与分化中发挥了重要的作用。miRNA是否可促进前体细胞向心肌细胞的分化则鲜见报道。 　　目的：验证转入特定miRNA(miRNA-1、miRNA-499)诱导新生小鼠心脏肌成纤维细胞转分化为心肌样细胞的可行性。 　　方法：体外培养新生小鼠心脏成纤维细胞，常氧培养传代至P2；37℃低氧(体积分数3%O2)培养48 h；免疫荧光染色检测心脏成纤维细胞特异性标记物波形蛋白的表达，免疫荧光染色、流式细胞技术检测心脏成纤维细胞向心脏肌成纤维细胞表型转化的标记物α-平滑肌激动蛋白的表达。实验分为4组：miRNA-1转染组、miRNA-499转染组、miRNA-1/miRNA-499共转染组及空白对照组，采用 miRNA 芯片检测心脏肌成纤维细胞与心肌细胞中微小RNA的表达差异；real-time qPCR方法验证芯片检测结果。 　　结果与结论：心脏肌成纤维细胞中过表达 miR-1、miR-499后，心脏发育不同阶段特异性因子发生不同程度的表达变化，与空白对照组相比，其余3组 GATA4、Tbx5、Mef-2c、α-MHC表达明显增高，Mesp1、Isl1表达均无显著变化。结果表明，特定miRNAs可诱导心脏肌成纤维细胞表达心肌细胞特异性性标志物，具备诱导心脏肌成纤维细胞向心肌细胞直接转分化的潜力。
 　　배경：미소 RNA(miRNA)시일류비편마소분자 RNA，miRNA삼여조공기인표체，재촉진전체세포증식여분화중발휘료중요적작용。miRNA시부가촉진전체세포향심기세포적분화칙선견보도。 　　목적：험증전입특정miRNA(miRNA-1、miRNA-499)유도신생소서심장기성섬유세포전분화위심기양세포적가행성。 　　방법：체외배양신생소서심장성섬유세포，상양배양전대지P2；37℃저양(체적분수3%O2)배양48 h；면역형광염색검측심장성섬유세포특이성표기물파형단백적표체，면역형광염색、류식세포기술검측심장성섬유세포향심장기성섬유세포표형전화적표기물α-평활기격동단백적표체。실험분위4조：miRNA-1전염조、miRNA-499전염조、miRNA-1/miRNA-499공전염조급공백대조조，채용 miRNA 심편검측심장기성섬유세포여심기세포중미소RNA적표체차이；real-time qPCR방법험증심편검측결과。 　　결과여결론：심장기성섬유세포중과표체 miR-1、miR-499후，심장발육불동계단특이성인자발생불동정도적표체변화，여공백대조조상비，기여3조 GATA4、Tbx5、Mef-2c、α-MHC표체명현증고，Mesp1、Isl1표체균무현저변화。결과표명，특정miRNAs가유도심장기성섬유세포표체심기세포특이성성표지물，구비유도심장기성섬유세포향심기세포직접전분화적잠력。
BACKGROUND:MicroRNAs are a class of smal non-coding RNA molecules. MicroRNA is involved in the regulation of gene expression, and plays an important role in promoting the proliferation and differentiation of precursor cells. Whether microRNAs can promote progenitor cells to differentiate into myocardial cells has been rarely reported. OBJECTIVE:To investigate the feasibility of direct reprogramming of neonatal mouse cardiac myofibroblasts to cardiomyocyte-like cells by specific microRNAs (microRNA-1 and microRNA-499). METHODS:Cardiac fibroblasts isolated from neonatal mice were cultured in vitro at 37 ℃ under normal oxygen, cells were used at the second passage for the fol owing experiments, and then cultured at 37 ℃ in 3%O2 for 48 hours. Immunofluorescent staining was used to detect expression of vimentin which is a specific marker for Cardiac fibroblasts. Immunofluorescent staining and flow cytometry assay were employed to detect expression ofα-smooth muscle actin which is a specific marker for cardiac myofibroblasts. There were four groups in the experiment:microRNA-1 group, microRNA-499 group, microRNA-1/microRNA-499 co-tranfection group, and blank control group. MicroRNA profile between cardiac myofibroblasts and myocardial cells was determined by microarray analysis via the miRCURYTM Array microarray kit. MicroRNA microarray results were validated by real-time quantitative PCR. RESULTS AND CONCLUSION:After overexpression of microRNA-1 and microRNA-499 in cardiac myofibroblasts, specific factors of heart development at different stages had different expressions;compared with the blank control group, the expression of GATA4, Tbx5, Mef-2c,α-MHC increased significantly in the remaining three groups, but Mesp1, Isl1 expression was not significantly changed). The results provide strong evidence for the capacity of microRNAs to induce expression of cardiomyocyte markers in cardiac myofibroblasts and demonstrate the potential of specific microRNAs that can direct reprogram cardiac myofibroblasts to cardiomyocytes in vitro.