目的：采用非特异性转录因子介导的谱系重编程系统，将小鼠心脏成纤维细胞（ cardiac fibroblasts ， CFs）直接转分化为心脏祖细胞（ cardiac progenitor cells ，CPCs）。方法取昆明小鼠乳鼠心肌组织，差速贴壁法分离CFs。免疫荧光染色、流式细胞术分别检测CFs性状及纯度，以携带含小鼠tet－on系统的4种重编程因子Oct4、Sox2、Klf4、c－myc（OSKC）慢病毒等比例感染第3代（P3）CFs。 A组：感染含小鼠tet－on系统的OSKC慢病毒，重编程培养基中不加入白血病抑制因子（LIF），加入JAK inhabit 1（JI1），以抑制向iPSCs方向的转分化。 B组：对照组感染空载体病毒。 C组：感染含小鼠tet－on系统的OSKC慢病毒，重编程培养基中加入LIF，不加入JI1，不抑制向iPSCs方向的转分化。在特定时间点为感染的细胞更换不同培养条件，观察细胞形态及生物特性的变化：①实时荧光定量PCR（Real time－PCR）法检测重编程各个时间点CPCs特异性转录因子Mesp1、Nkx2．5、Isl1、GATA4及胚胎干细胞多能性因子Oct4、Sox2、c－myc、Klf4、Naong的表达；②CPCs特异性标记Nkx2．5、Isl1免疫荧光染色鉴定；③CPCs自分化为搏动心肌细胞的检测；④心肌细胞特异性标记cTnI免疫荧光染色鉴定CPCs向心肌细胞方向的分化潜能。结果①形态学观察，病毒感染11～14天，可见部分CFs形态发生改变，形成CPCs样克隆；②CPCs克隆Nkx2．5、Isl1免疫荧光染色阳性；③CPCs可自分化为搏动的细胞团，其中的细胞cTnI免疫荧光染色阳性；④Real－time RCR检测结果显示，重编程第4天，CPCs早期特异性转录因子Mesp1即开始表达，而iPSCs多能性因子Naong在重编程第9天才开始表达。结论心脏成纤维细胞可以直接转分化为CPCs。%Objective To investigate the direct conversion of mouse cardiac fibroblasts ( CFs) into cardiac progeni-tor cells ( CPCs) using non-specific transcription factors based lineage reprogramming strategy .Methods Primary CFs were isolated from the myocardial tissues of neonatal Kunming mice through the preplate technique .The obtained cells were identified by immunofluorescence staining and flow cytometry .Then, the third generation of CFs was transfected with a mixture medium containing equal parts of Oct -3/4, Sox2, Klf4 and c-Myc ( OSKC) lentiviruses.The resultant cells were divided into three groups:Group A (exposed to JAK inhabit 1 (JI1) alone), Group B (transfected with empty vec-tors) and Group C (exposed to leukemia inhibitory factor (LIF) alone).Then, the quantities of CPC-specific transcrip-tion factors (Mesp1, Nkx2.5, Isl1, and GATA4)and pluripotent transcription factors (Oct4, Sox2, c-myc, Klf4, and Naong) were measured by Real time -PCR at each time points.The CPC-specific transcription factors (Nkx2.5 and Isl1) were identified by immunofluorescence .The direct conversion of CPCs into beating cardiomyocytes was examined .The dif-ferential capacities of CPCs into cardia myocytes were determined by immunofluorescence staining for cardiac -specific tro-poninI(cTnI).Results 〗MorphologicalchangesappearedinpartsofCFs,inadditiontotheformationofCPC-likecol-onies after 10-12 days of viral transfection .CPC colonies showed positive immunofluorescence staining for Nkx 2.5 and Isl1.CPCs could differentiate into beating cardiomyocytes and display positive immunofluorescence staining for cTnI .Ac-cording to Real-time PCR results, the transcription factor Mesp1 and the pluripotency factor NANOG were expressed on Day 4 and Day 9 of lineage reprogramming , respectively .Conclusion CFs can be directly differentiate into CPCs .
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