重组Pax4和MafA蛋白质在小鼠糖尿病治疗中的作用及机制研究

Differentiation and regeneration of pancreatic β cells has become the hotspot of biomedical research. Viral vectors, a tool commonly used in inducing differentiation, raise the potential for unexpected genetic modifications. Other studies and our previous data show that protein transduction provide a possible way to aviod introducing exogenous genetic modification, may be a promising way to induce pancreatic β-cells.In our study, we will use recombinant Pax4-, MafA-mCherry, which possesses protein transduction domain (PTD) that facilitates its entry into cells, to activate endogenous insulin synthesis in stretozotocin (STZ)-induced diabetes mice. Firstly, we will test the permeability of the recombinant proteins in vitro. The dynamic distribution of the recombinant proteins in mice will be observed by small animal imaging system. Nextly, we will perform an evaluation to confirm the adverse effects of administration of the recombinant proteins. Then the effects of recombinant proteins on blood glucose levels in diabetic mice will be confirmed. Finally, we will demonstrate that recombinant Pax4, MafA-mCherry treatment could promote β-cell regeneration and liver and/or jejunum cell reprogramming, leading to restoration of normoglycemia in diabetic mice. To explore recombinant proteins mediated normoglycemia resulted from islet β-cell regeneration, paraffin blocks of pancreas will be tested for immunofluorescence, and pancreas insulin levels will be measured using an ultrasensitive mouse insulin ELISA kit. To assess whether in vivo intraperiteonal recombinant Pax4 and MafA delivery affects the liver and intestine, we will examine the expression of pancreatic genes by RT-PCR and real-time PCR, tissue insulin level will be meatured, and paraffin blocks of liver, jejunum, spleen and pancreas will be immunostained with anti-insulin antibody. This novel PTD-based protein therapy offers a promising way to treat patients with diabetes.

胰岛β细胞的分化再生是生物医学领域的研究热点，病毒载体作为诱导分化的常用工具存在潜在致瘤性。文献和我们的前期研究表明，蛋白质转导避免了外源基因的整合风险，可能成为诱导产生胰岛β细胞的有效手段。本项目拟采用自身带有蛋白质转导结构域(PTD)的重组Pax4-，MafA-mCherry蛋白质活体转导诱导体内产生胰岛素分泌细胞。我们将首先在体外确定重组Pax4-，MafA-mCherry的转导功能；利用小动物成像系统研究重组蛋白质在小鼠体内的动态分布，观察重组蛋白质活体转导是否存在毒副反应；揭示重组Pax4-,MafA-mCherry多次重复转导对1型糖尿病模型小鼠高血糖症状的改善作用；检测胰岛特异基因的转录表达，确定血清及组织中的胰岛素水平，系统的从胰岛β细胞再生及非胰腺组织细胞转分化两方面阐明重组蛋白质降血糖的作用机制。本课题将为开辟新的糖尿病治疗途径提供理论和实验依据。

胰岛β细胞的分化再生是生物医学领域的研究热点，病毒载体作为诱导分化的常用工具存在潜在致瘤性。本项目采用蛋白质转导结构域(PTD)实现重组Pax4，MafA蛋白质活体转导，诱导体内产生胰岛素分泌细胞。研究首先在体外确定重组蛋白质具有转导功能，细胞生存试验证明蛋白质转导细胞毒性较小；小动物成像系统观察注射重组蛋白质主要分布在小鼠肝脏和小肠组织，其他器官分布较少。建立小鼠糖尿病模型，重组Pax4、MafA通过小鼠尾静脉多次重复输注。MafA输注小鼠高血糖症状明显改善，血清中胰岛素水平升高。结果证实MafA输注小鼠胰腺内未发生明显的胰岛增殖、肝脏中未检测到胰岛素分泌细胞，而在小肠组织中观察到胰岛素表达细胞。本课题为开辟糖尿病治疗新途径提供了理论依据。