RSPO1/Wnt通路在小鼠胰腺祖细胞增殖和分化中的作用及机制研究

我们发现源于小鼠胰腺的CD133+Sox9+细胞具备胰腺祖细胞（PPCs）的生物学特性，但该类细胞增殖、分化困难。经典Wnt通路与胰腺发育相关，我们推测其可能会对PPCs的增殖和分化起调控作用。本项目拟通过qPCR、免疫组化等技术手段研究RSPO1及其受体在小鼠胰腺中的表达及其与 PPCs的共定位关系，为其体外功能提供依据；采用二次克隆实验、免疫组化及激光共聚焦等检测手段，以细胞数目、胰腺分化相关基因表达水平变化为指标，在体内、外水平研究RSPO1/Wnt通路对PPCs增殖和分化的作用；采用启动子活体荧光标记、RNA干扰及二代测序等方法，以细胞分裂标志、PPCs分化相关基因及Wnt通路下游因子表达水平变化为指标，揭示RSPO1/Wnt通路对PPCs增殖和分化作用的分子机制。本研究将有助于构建Wnt通路调控PPCs增殖和分化的理论基础，为开发信号转导相关药物提供科学依据。

Our previous research showed that CD133+Sox9+ cells derived from mouse pancreas exhibited biological properties of PPCs. However, in vitro amplification of the cells is difficult due to low abundance of these types of cells. Because of the close relationship of canonical Win pathway to pancreas development, we speculate that it mayregulate the proliferation and differentiation of PPCs. This project is aimed at investigating the expression and co-localization with PPCs of Wnt agonist-RSPO1 and its receptor as well as downstream regulatorsin adult mouse pancreatic tissues to provide evidence for its in vitro function utilizing techniques such as Real-time PCR and Immunohistochemistry. By using re-plating assay, BrdU incorporation, Western Blot, IHC double staining and Confocal laser scanning microscopewere to measure cell cycle-related proteins, transcription factors associated with PPCs differentiation and alterations of Wnt pathway relevant downstream genes, and thereby to elucidate the effect of RSPO1/Wnt pathway on PPCs proliferation and differentiation and the molecular mechanism of the role of RSPO1 in that process. This research has the potential of providing seeding cells for diabetes cell replacement therapy and is essential to lay the theoretical foundation of the regulation of Wnt pathway in PPCs proliferation and differentiation, and provide scientific basis for developing drugs targeting signal transduction pathways.