干细胞Marker 示踪的涡虫转基因模型建立及活体水平重编程研究

Stem cells are undeveloped cells capable of proliferation, self renewal, conversion to differentiated cells, and regenerating tissues. There are three main types of stem cells, embryonic stem cell, adult stem cells and induced pluripotent stem cells (iPSCs). iPSCs are a type of pluripotent stem cell that can be generated directly from adult cells, which hold great promise in the field of regenerative medicine. Because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic, and liver cells), they represent a single source of cells that could be used to replace those lost to damage or disease.. Cellular reprogramming has been achieved under carefully controlled in vitroculture conditions. It is mainly strategy used to induced in vitro, transplantation into the body, there is a problems of survival, differentiation and immunity of the transplant, limiting its application. In vivo induction of iPSC is a potential way to solve the above problem. But, in vivo studies of iPSCs are not easy in mammals due to the difficulty of developmental biology studiesin vivousing vertebrate models. For this reason, most of the knowledge on iPSC biology results from in vitro studies. Non-mammalian model systems can be alternatively used to assessin vivo the effect of substances on these cells.. Planarians are an ideal animal model for in vivo analysis of iPSCs. Their extraordinary ability to regenerate, as well as to sustain continuous homeostatic turnover of all differentiated cell types, relies on the presence of stem cells that share characteristics with the stem cells of higher organisms, including humans. Based on the previous work of planarians transgenic and reprogramming technique, the project intends to planarians as model organisms, to construct these transgenic planarians which multiple stem cell marker co-expression with different fluorescent protein using the piggyBac and CRISPR/Cas9 genome editing technology, tracing of stem cell stages of proliferation and differentiation in vivo, to explore the simple and effective method for inducing stem cell in vivo of planarian, provides new ideas and useful information to the field of regenerative medicine base on stem cells.

诱导多能干细胞（iPSC）因其获得方便、较少伦理问题等成为干细胞研究的热点，目前主要采用的是体外诱导、体内移植的研究思路，但因在移植时存在存活率、免疫原性、定向分化困难等问题，限制了其应用。iPSC在活体水平的诱导是解决上述问题的一种新思路，并有人进行了有益的探索，表明具有很好的可行性；但他们都是利用哺乳动物作为研究对象，操作不便、观察不易，得到的主要是间接结果。本研究拟以涡虫为模式生物，利用前期工作中建立的涡虫转基因和重编程技术，通过piggyBac转座子和CRISPR/Cas9基因组编辑技术构建多种干细胞Marker与不同荧光蛋白共表达的转基因涡虫，在活体水平对干细胞增殖与分化的不同阶段进行示踪，可以实时观察的细胞重编程过程；探索和优化涡虫活体水平的体细胞重编程方法，利用单细胞技术研究涡虫体细胞重编程过程中基因表达调控机制；为基于干细胞的再生医学研究提供新的研究思路和有用信息。

在本项目中我们通过直接注射mRNA 的方法实现了涡虫体内的转基因；克隆并构建了管家基因gapdh 和actin 的启动子荧光报告载体；建立并利用原核表达的融合穿膜肽的CAS9蛋白（NLS-CPP-Cas9）和体外转录的marker基因sgRNA，以及靶基因的同源DNA donor（PCNA-P2A-GFP、PIWI-P2A-RFP DNA donor）这一基因编辑系统，尝试对涡虫进行基因敲入，实现了少量细胞的编辑。结合生物信息学分析，从本实验室前期的再生差异蛋白组、再生差异转录组测序结果以及参考文献中筛选可能对涡虫细胞自我更新和再生具有调控作用的基因。对涡虫中这些候选基因进行进一步的RNAi筛选，发现其中Djrfc、DjArid1、DjArid2、DjERas、EXPORTIN1、Cdc42等一些涡虫干细胞调控因子被干扰下调后，涡虫无法再生，也无法维持平衡态，我们对这些基因进行了进一步的功能和调控机制研究，为干细胞在体研究、再生医学和创伤修复研究提供新的研究思路和有用信息。