低氧/HIF对小鼠成纤维细胞直接重编程为心肌细胞的作用及机制的研究

The development by the Yamanaka group of a method to reprogram somatic cells into induced pluripotent stem cells (iPS) by over expression of pluripotency factors hold considerable promises for the development of stem cell therapies. Here, we propose to directly induce fibroblast differentiate into myocardial cells through the same or similar mechanisms for fibroblast reprogramming. Our working hypothesis is that fibroblast can be induced and differentiated into myocardial cell through manipulated cellular microenvironment. The resultant fibroblast cells with myocardial cell characteristics could become important myocardial cell source used for myocardial cells transplantation..To test our hypothesis, first, we will establish transgenic mice harburing αMHC promoter driven EGFP-IRES-Puromycin (αMHC-EGFP) as report gene. This biomarker system will eusure only differenatiated cell with myocardial characteristics cells positively expressed the green fluorescent protein (GFP). Secondly, Myocardial specific transcriptional factors, Gata4、Mef 2c and Tbx5, Combining traditional Chinese medicine monomer ICA, will be introducd into fibroblast, we expect directly reprogramming of fibroblasts into functional myocardial cells will occur. To enhance efficiency of reprogramming, we will modify O2 concentration to optimize ideal O2 concentration for reprogramming and correlate low O2 concentration with dynamic expression profile such as phenotype, functional genomics, and epigenetics during the transition from fibroblast into myocardial cells in the process of reprogramming. Then, to functional observation of the process of reprogramming in living cell system and explore the mechanisms, we will e employ EYFP-HIF1α labeled mouse fibroblast cells to perform living cell observation and explore the modification of HIF-1Α expression along with epigenetic variation during the process of fibroblast cells reprogramming. This would be benefitial to increase our knowedlge to understand the mechanisms how HIF-1Α expression involves fibroblast cell dedifferentiation and reprogramming, enventually complete erasure of all functionally important epigenetic marks associated with fibroblast differentiation. The ability to produce large number of myocardial cell cells with myocardial cell characteristics is likely to have many translational applications.

直接诱导成纤维细胞重编程为心肌细胞有望为心肌细胞移植提供新的细胞来源。细胞微环境不仅支持细胞发育，也可以通过影响细胞的极化来调节改变信号转导通路从而指导细胞的分化方向。本研究拟构建αMHC启动子驱动的EGFP标记转基因小鼠，以确保只有定向分化的心肌细胞才能表达GFP。通过特定转录因子（Gata4、Mef2c和Tbx5）和中药单体淫羊藿苷直接重编程成纤维细胞为GFP+的心肌细胞。通过调节细胞培养过程中不同氧气浓度，检测相应条件下诱导的成纤维细胞表型、功能基因组和表观遗传学改变来评估其对重编程的作用，以期探讨诱导成纤维细胞定向分化为心肌细胞的作用机制。利用EYFP-HIF1α标记的小鼠成纤维细胞系，活体动态观察HIF-1α表达与定向诱导分化过程中成纤维细胞表观遗传学变化，获得HIF-1α表达与成纤维细胞重编程相关性的内在关系及可能的信号转导通路，并研究低氧或低HIF活性对直接重编程的作用机制。

本课题成功获得Myh6-mcherry转基因小鼠，小鼠成熟心肌表达内源性红色荧光。通过特定的转录因子（Gata4、Mef2c和Tbx5）直接重编程成纤维细胞为心肌细胞，但其重编程效率低，重编程心肌细胞不能自发跳动。通过低氧预处理成纤维细胞能明显提高成纤维细胞直接重编程为心肌细胞的效率，表现为低氧预处理后直接重编程的心肌细胞在表型、表观遗传学等方面更接近成熟心肌细胞。其中HIF-1α、Oct4、Uhrf1参与低氧促进成纤维细胞直接重编程的过程。低氧促进重编程过程中机制研究发现，通过模式识别和生物信息学数据分析，从糖代谢众多物质中筛选出标志性的代谢物，确立分子靶点，构建分子网络联系。低氧本身可影响细胞的代谢由有氧代谢模式转变为糖酵解模式，并有利于重编程的发生；低氧可以改变成纤维细胞相应的表观遗传学修饰促进有重编程发生。另外，本课题还利用Myh6-mcherry转基因小鼠只有成熟的心肌细胞才能表达红色荧光蛋白的特性，研究了小鼠胚胎及生后心脏的发育过程，并对生后1天以及胚胎11.5天小鼠心脏表面及内腔进行三维模型重建。观察了小鼠心脏发育成熟过程中表观遗传调节因子Meis1的时空表达规律，以期对心脏的发育、再生和细胞移植进行更深刻的理解。目前正在通过整合细胞重编程过程中的基因组、转录组、表观基因组、蛋白质组等高通量多维组学数据，在融合TF-gene/miRNA及miRNA-gene /TF靶向关系的基础上，构建包含转录调控信息及转录后调控信息的、考虑表观遗传对基因调控影响的、重编程过程驱动的加权基因调控网络，解析重编程过程中的分子事件。期间发表论文7篇，其中SCI收录2篇。出版专著1部。获得国家发明专利1项，著作权专利1项。本研究取得的研究成果，揭示了低氧对心肌细胞直接重编程的机制，为提高临床细胞移植的安全性和效率打下基础。