人牙源性TF-iPS细胞miRNAs谱系特征及其促进牙髓再生的研究

Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells similar to embryonic stem cells (ESCs) in cell morphology and function, but avoid the ethical and legal problems that ESCs have. iPSCs can be applied to many aspects, such as regenerative medicine, disease model, drugs screening, individual therapy, tissue and organ repair, et al. Our previous research has proved that human stem cells from apical papilla (SCAP) can be successfully reprogrammed into iPSCs by a single lentivirus cassette hSTEMCCA-LoxP, and transgene-free iPSCs （TF-iPSCs）were generated after plasmid transfection and puromycin selection. But the role of miRNAs in the process remains unkonwn. In this study, hSTEMCCA reprogramming kit (a single polycistronic lentivirus vector, composed of 4 transcription factors: Oct4/Sox2/c-Myc/Klf4) will be utilized to induce human DPSC, SCAP and gingival fibroblasts (GF) derived from young third molars to get iPSCs. Then Cre/LoxP recombination enzyme and puromycin will be applied to excise the 4 exogenous transgenes to generate TF-iPSCs. microRNA microarray will be used to analyze the microRNAs profiling of 3 iPSCs before and after excision and validation of the microarray results will be carried out by real-time quantitative RT-PCR (qRT-PCR). Then generated TF-iPSCs will be transplanted into a root canal with stromal cell-derived factor-1 (SDF-1) and TE collagen scaffold after pulpectomy in mature teeth with complete apical closure in dogs, to find out if the root canal will be filled with regenerative pulp-like tissue, followed by new dentin formation along the dentinal wall. Our research will provide a foundation for the research of the role of miRNAs in the induction of human TF-iPSCs and has theoretical significance and application value in the regenerative treatment of pulpitis.

iPS细胞与胚胎干细胞形态和功能相似，却避开了其面临的伦理问题，可用于再生治疗、药物筛选等方面。我们的前期研究证实人SCAP可被hSTEMCCA-loxP诱导为iPSCs，经质粒转染、嘌呤霉素筛选后得到TF-iPSCs，但诱导过程中miRNAs的作用机制不明。本课题采用hSTEMCCA多顺反子慢病毒重编程试剂盒诱导人DPSC、SCAP、GF获得iPSCs，Cre/LoxP切除、嘌呤霉素筛选后得到TF-iPSCs，miRNAs芯片筛选人TF-iPSCs切除前后miRNAs差异表达谱，为miRNAs在iPSCs诱导中作用机制研究提供实验基础；建立家犬恒牙牙髓炎模型，行牙髓摘除术后，将人TF-iPSCs、基质细胞衍生因子-1、TE胶原植入空根管内，观察是否可以诱导牙髓样组织再生，研究结果对于人牙源性TF-iPS细胞诱导过程中miRNAs的调控作用机制、牙髓再生等具有理论意义和应用价值。

诱导性多潜能干细胞（Induced pluripotent stem cells，iPSCs）与胚胎干细胞形态和功能相似，却避开其面临的伦理和法律问题，在再生医学、组织工程等方面有广泛的应用前景。我们的前期研究采用hSTEMCCA-loxP系统将人SCAP 诱导为iPSCs，但仍有转录基因残留，诱导过程中miRNAs 的调控机制也未进行研究。. 本课题采用Sendai Virus重编程系统将人牙髓干细胞（Dental pulp stem cells，DPSCs）和根尖乳头干细胞（Stem cells from apical papilla，SCAP）诱导为iPSCs，经多潜能标记物染色、核型与特异性标记物分析、畸胎瘤实验、体内外分化等证实为iPSCs，RT-PCR结果证实无外源性病毒或转录基因序列的表达，建立一种高效率的人牙源性iPSCs重编程方法；研究一种组分明确的心肌分化培养基CDM3，可将人牙源性iPSCs分化为高纯度心肌细胞，建立一种高效稳定的iPSCs-心肌细胞分化体系；对比研究人牙源性iPSCs在3种底物的生长情况，发现重组人玻连蛋白制备简便、成分明确、重编程时间短、效率高，是人牙源性iPSCs理想的支持底物。miRNAs探针分析筛选人iPSCs重编程前后miRNAs表达，结果筛选到3个高表达miRNAs：miR-19a-3p、miR-92b-3p、miR-130b-3p，GO分析显示这些miRNAs通过不同通路参与了细胞生物过程、代谢调节与刺激反应，KEGG生物学功能分析显示miR-19a-3p与15个通道相关，miR-92b-3p参与肌动蛋白细胞骨架和多巴胺能神经突触的调节，miR-130b-3p参与干细胞多潜能性调节信号通路，预示这些高表达miRNA可靶向作用于一些重要通路中的某些成分，对iPSCs的重编程起到调控作用。动物实验研究将人iPSCs + Puramatrix置于离体牙根管内，植入裸鼠皮下，8周后发现根管内充满牙髓样组织，可见明显血管、纤维组织等结构，最外层为成牙本质样细胞及再生牙本质样结构，免疫组化染色结果显示CD105（+）、DSP（+），说明有新生血管和牙本质样组织形成。. 我们的研究结果对于人牙源性iPSCs的重编程、诱导过程中miRNAs 的调控作用机制、牙髓炎的生物再生治疗等研究具有理论意义和应用价值。