人诱导多能干细胞来源功能性胰岛β细胞的体外定向分化方法的建立  

In vitro Directed Differentiation of Functional Pancreatic β Cells from Human Induced Pluripotent Stem Cells

作　　者：陈馨雅 陈龙 王玉姣 薛群航 冯志伟 刘志贞[1] 周冰蕊 解军[1] CHEN Xin-Ya;CHEN Long;WANG Yu-Jiao;XUE Qun-Hang;FENG Zhi-Wei;LIU Zhi-Zhen;ZHOU Bing-Rui;XIE Jun(Department of Biochemistry and Molecular Biology,Shanxi Key Laboratory of Birth Defect and Cell Regeneration,MOE Key Laboratory of Coal Environmental Pathogenicity and Prevention,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学生物化学与分子生物学教研室出生缺陷与细胞再生山西省重点实验室,煤炭环境致病与预防教育部重点实验室,太原030001

出　　处：《中国生物化学与分子生物学报》2024年第5期664-673,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.82100821);中央引导地方科技发展资金项目(No.YDZJSX2021b008);山西省基础研究计划(No.20210302124406)资助。

摘　　要：1型糖尿病是由胰岛β细胞功能受损、胰岛素分泌不足所致,目前,主要通过外源性胰岛素补充来治疗,但外源性胰岛素无法精准调控血糖,严重低血糖可危及生命。胰岛移植是一种替代疗法,但面临器官供体不足和异种来源胰岛β细胞存在人畜共患病交叉感染风险的问题。因此,获得足量且安全的胰岛β细胞是1型糖尿病细胞治疗面临的难题。本研究旨在通过人诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)在体外向胰岛β细胞分化,提供一种潜在的1型糖尿病治疗新策略。为实现这一目标,我们采用了结合2D和3D培养系统的分化策略,模拟胰岛β细胞的体内发育环境,并使用多种生长因子调节在胰腺发育和β细胞分化中发挥重要作用的关键信号包括Notch信号通路(Notch signaling pathway)、Wnt信号通路(Wnt signaling pathway)、TGF-β/Smad信号通路(TGF-β/Smad signaling pathway)等,在体外将hiPSC定向诱导分化至胰岛β细胞。结果显示,在2D、3D结合的培养条件下,分化过程中定型内胚层细胞,胰腺祖细胞,胰腺内分泌细胞及胰岛β细胞阶段的特异性基因的表达显著提高(P<0.05),并在胰岛素含量及葡萄糖刺激后表现出显著增强的胰岛素分泌能力(P<0.05)。总之,本研究成功建立了一种从hiPSC到功能性胰岛β细胞的分化策略,为1型糖尿病提供了一种新的细胞治疗途径。这种方法不仅为研究胰岛β细胞的发育生物学提供了新的工具,也为临床应用提供了一种潜在的胰岛β细胞来源,有望解决现有治疗方法的局限性。Type 1 diabetes is caused by impaired function of pancreaticβ-cells and insufficient insulin secretion.Currently,it is primarily managed with exogenous insulin supplementation;however,exogenous insulin cannot precisely regulate blood glucose levels,and severe hypoglycemia can be life-threatening.Islet transplantation serves as an alternative therapy,but faces challenges such as a shortage of organ donors and the risk of cross-species infections from xenogeneic sources of isletβ-cells.Thus,obtaining a sufficient and safe supply of isletβ-cells remains a significant challenge in cell therapy for type 1 diabetes.This study aims to differentiate human induced pluripotent stem cells(hiPSCs)into isletβ-cells in vitro,offering a potential new strategy for treating type 1 diabetes.To achieve this,we utilized a differentiation strategy that combines 2D and 3D culture systems to simulate the in vivo developmental environment of isletβ-cells and employed various growth factors to regulate key signaling pathways crucial in pancreatic development andβ-cell differentiation,including the Notch,Wnt,and TGF-β/Smad signaling pathways.Our results show that under combined 2D and 3D culture conditions,the expression of specific genes at the stages of definitive endoderm,pancreatic progenitors,pancreatic endocrine cells,and isletβ-cells significantly increased(P<0.05).And there was a marked enhancement in insulin content and secretion following glucose stimulation(P<0.05).In summary,this study successfully established a differentiation strategy from hiPSCs to functional isletβ-cells,providing a new cell therapy approach for type 1 diabetes.This method not only offers new tools for studying the developmental biology of isletβ-cells,but also provides a potential source of isletβ-cells for clinical applications,potentially overcoming the limitations of current treatment methods.

关 键 词：人诱导多能干细胞 体外诱导分化 胰岛Β细胞 胰岛素 

分 类 号：Q254[生物学—细胞生物学]