CD66a影响诱导多能干细胞形成的机制研究

诱导多能干细胞(iPSCs) 是体外研究疾病发病机制和药物筛选的良好模型，但如何提高其形成效率及阐明细胞重编程机制仍是未解决的问题。申请人前期研究发现在小鼠和人iPSCs 形成过程中癌胚抗原粘附分子1（又名CD66a）的表达量会在早期迅速上调，且发现早期流式分选富集的CD66a阳性的细胞更易形成完全重编程的iPS克隆，该技术大幅度提升了iPS的诱导效率和克隆质量。但CD66a影响iPSCs形成的机制尚未明确，鉴于CD66a阳性细胞组具有极强的间质细胞上皮化（MET）特性以及粘附分子被报道具有细胞胞内信号的调控能力，我们推测CD66a可能直接参与胞内信号通路调控影响iPSCs形成。本课题拟在细胞重编程过程中干扰和过表达CD66a蛋白的水平，并利用结合高通量测序、免疫印迹、免疫共沉淀等方法探讨CD66a是否作为信号分子直接参与胞内信号通路的调控，以期阐明CD66a影响iPSCs形成的分子机理。

The creation of induced pluripotent stem cells (iPS cells) facilitate the generation of human patient-specific stem cell lines for modeling disease processes in vitro, providing a useful tool in studies of developmental biology, drug discovery and regenerative medicine. Although great progress has been made in the iPS cell field, reprogramming efficiency is low and the reprogramming mechanism remains unclear. The applicant has found that Carcinoembryonic antigen adhesion molecule 1（CD66a）was rapidly increased at the early stage when miPS and hiPS was forming ,and found that the CD66a positive cells sorted by flow cytometry can always converted to fully reprogrammed iPS clones.This technology can robustly promote the reprogramming efficiency and the quality of the clones.But the mechanism of CD66a effecting the cell reprogramming efficiency is still unclear.In view of the CD66a positive cells have strong feature to undergoing the MET pathway and other cadherin moleculars have the ability to regulate the cells’ signal,we speculate that the CD66a maybe directly involved in intracellular signaling pathways and regulate the formation of iPSCs.In this study, we will upregulate and downregulate the CD66a’s level during the iPS reprogramming process and observe the reprogramming efficiency. High-throughput sequencing, Western blot and co-immunoprecipitation will be performed to find out whether CD66a effecting the cell reprogramming by playing a direct role in a signaling pathway as a signaling molecule. We aim to found out the function of CD66a play on cell reprogramming and the mechanism of cell reprogramming.