利用斑马鱼在体荧光成像研究胰岛beta细胞的分泌和成熟机制

Pancreatic beta cells are very important in the pathogenesis of Diabetes. To study the mechanisms that control beta cell function, differentiation and maturation will provide a theory basis for developing new therapy for Diabetes. However currently, there does not exist an in-vivo model to observe time-lapse changes of beta cell function during development,thereby to further study the differentiation and maturation of beta cells. So people could not unravel how insulin positive cells differentiate to mature functional beta cells and what signals and mechanisms control this procedure. We will establish a transgenic zebrafish in which the insulin secretory granules of beta cells are fluorescently labelled, to directly observe insulin secretory granule exocytosis in response to glucose stimulation by time-lapse in vivo imaging. Furthermore,this system will be used to study when and how the beta cells become responsive to glucose stimulation in vivo.This will be the first imaging study of insulin secretory granule exocytosis within a living organism. The signals and mechanisms of controlling beta cell maturation revealed by this study will help to improve the protocals of inducing embryonic stem cells to differentiate into mature beta cells in vitro.

胰岛beta细胞在糖尿病发病过程中十分重要。研究beta细胞的功能及其分化、成熟的机制会对糖尿病治疗提供理论依据。目前人们无从了解机体内胰岛素阳性的细胞如何分化成为成熟的beta细胞，是什么信号和机制起了决定作用。主要原因是缺乏研究的手段：一个在体的模型可以实时观测发育过程中beta细胞的功能变化，进而研究其分化、成熟的过程。我们将建立荧光特异性标记beta细胞胰岛素分泌囊泡的转基因斑马鱼，建立在体实时成像新方法直接观测葡萄糖刺激下胰岛素分泌囊泡的释放,并以此作为beta细胞成熟的判定标准，研究beta细胞在体分化和成熟的机制。此研究所揭示的调控beta细胞分化和成熟的信号和机制将指导改进体外诱导胚胎干细胞分化成胰岛beta细胞的方法。该研究也将为加强糖尿病病人体内自身的beta细胞的功能和再生的治疗策略提供新的思路。

体外诱导干细胞分化为分泌胰岛素的细胞，为治疗糖尿病提供beta细胞替代疗法一直是业界的研究热点。但从干细胞分化成的分泌胰岛素的细胞与内源的beta细胞相比，对葡萄糖的响应能力比较差。为了探寻促进beta细胞功能成熟的因子，我们利用本实验室自主研发的双光子三轴扫描光片显微镜，在单细胞水平在体实时观测斑马鱼胰岛beta细胞功能，并研究在体胰岛beta细胞成熟的机制。我们的研究揭示了胚胎期胰岛微循环系统的建立精确调控了逐渐升高的葡萄糖进入胰岛外围和内部的beta细胞内，而合适的葡萄糖浓度通过激活calcineurin/NFAT信号通路导致胰岛beta细胞的功能成熟。在缺乏微循环系统的条件下，体外培养小鼠新生胰岛beta细胞无法实现胰岛外围和内部的beta细胞受到相同最适浓度的葡萄糖的刺激而获得最大程度的成熟。而有趣的是，这个缺陷可以通过在培养基中加入calcineurin的激活剂而得到纠正。我们的发现将为体外诱导人胚胎干细胞分化为功能完全成熟的beta细胞提供一个新的策略。