MAFA-PDX1过表达慢病毒感染人脐带间充质干细胞向胰岛素分泌细胞的分化  

Differentiation of insulin-producing cells from human umbilical cord mesenchymal stem cells infected by MAFA-PDX1 overexpressed lentivirus

作　　者：邱晓燕[1] 李碧欣 黎敬弟 范垂钦 马廉[1,2,3] 王鸿武 Qiu Xiaoyan;Li Bixin;Li Jingdi;Fan Chuiqin;Ma Lian;Wang Hongwu(Department of Pediatrics,The Second Affiliated Hospital of Shantou University Medical College,Shantou 515041,Guangdong Province,China;Department of Pediatrics Research Institute,Shenzhen Children’s Hospital,Shenzhen 518038,Guangdong Province,China;The Third Affiliated Hospital of Guangzhou Medical University(Women’s and Children’s Hospital of Guangzhou Medical University),Guangzhou 512000,Guangdong Province,China;Stem Cell Research Center of Shantou University Medical College,Shantou 515041,Guangdong Province,China)

机构地区：[1]汕头大学医学院第二附属医院儿科,广东省汕头市515041 [2]深圳市儿童医院儿科研究所,广东省深圳市518038 [3]广州医科大学第三附属医院(广州医科大学妇女儿童医院),广东省广州市512000 [4]汕头大学医学院干细胞研究中心,广东省汕头市515041

出　　处：《中国组织工程研究》2024年第7期1000-1006,共7页Chinese Journal of Tissue Engineering Research

基　　金：2020年广东省科技计划大专项:基于脐带间充质干细胞来源胰岛素分泌细胞治疗儿童1型糖尿病的转化医学研究,汕府科【2020】53-112,项目负责人:王鸿武。

摘　　要：背景:干细胞来源胰岛β细胞移植被认为是治疗1型糖尿病的有效手段。人脐带间充质干细胞是理想的细胞来源,但其向胰岛β细胞分化的效率不高。目的:研究MAFA、PDX1修饰促进人脐带间充质干细胞向胰岛素分泌细胞分化的潜能。方法:构建MAFA-PDX1过表达慢病毒载体,用细胞形态学、RT-qPCR法、双硫腙染色比较3种方案[方案A:单纯慢病毒组;方案B:先药物(尼克酰胺、β-巯基乙醇)诱导再加慢病毒组;方案C:慢病毒和药物诱导同时进行组]诱导人脐带间充质干细胞分化为胰岛素分泌细胞的效率和潜能。结果与结论:(1)细胞形态学改变:经3种方案诱导后细胞形态均发生了改变,方案B在诱导第11天细胞聚集生长最为明显,出现聚集生长的胰岛样细胞团;(2)RT-qPCR检测胰岛相关基因的表达差异:同一诱导时间点3种方案横向比较,在诱导第5天,方案C的MAFA和PDX1基因表达量最高,方案B的GCG基因表达量最高;在诱导第11天,方案B的MAFA、PDX1基因表达量以及INS和GLUT2基因表达量最高;(3)双硫腙染色鉴定锌离子:3种方案诱导第11天部分细胞被双硫腙染成棕红色,其中方案B中部分小岛状细胞被染成棕红色,颜色较深(阳性表达);(4)结果表明,MAFA和PDX1共过表达可促进人脐带间充质干细胞向胰岛素分泌细胞分化;MAFA-PDX1基因修饰联合药物诱导方案优于单纯基因修饰方案。BACKGROUND:Transplantation of stem cell-derived isletβcells has been considered effective for the treatment of type 1 diabetes.Human umbilical cord mesenchymal stem cell is an ideal cellular source,but with a low differentiation efficiency to isletβcells.OBJECTIVE:To explore the possibility of human umbilical cord mesenchymal stem cells modified by MAFA and PDX1 to differentiate into insulin-producing cells.METHODS:MAFA-PDX1 lentivirus expression vectors were constructed.The efficiency and potentiality of human umbilical cord mesenchymal stem cells differentiated into insulin-producing cells with three methods were compared by cell morphology,RT-qPCR,and dithizone staining[protocol A:Simple lentivirus group;protocol B:Drug(nicotinamideβ-mercaptoethanol)induction followed by lentivirus group;protocol C:lentivirus and drug induction group].RESULTS AND CONCLUSION:(1)Morphological change of cells:Cell morphology was all altered after the induction of three protocols.At day 11,human umbilical cord mesenchymal stem cells induced by protocol B showed the most cell clusters among the three protocols,appearing aggregated islet-like cell clusters.(2)Islet-related gene expression detected by RT-qPCR:Horizontal comparison of the three protocols at the same induction time point showed that the expression levels of MAFA and PDX1 genes were the highest in protocol C on day 5 of induction,and those in protocol B were the highest on day 11 of induction.Human umbilical cord mesenchymal stem cells induced by protocol B had the greatest expression of GCG gene at day 5,INS and GLUT2 genes at day 11.(3)Dithizone staining to identify zinc ions:parts of the post-induced cells were stained brownish red by dithizone on day 11.The partial small island cells were stained brownish red with a darker color(positive expression)in protocol B.(4)It is concluded that the overexpression of MAFA and PDX1 can promote the differentiation of human umbilical cord mesenchymal stem cells into insulin-producing cells.The combination of MAFA-PDX1 gene mo

关 键 词：脐带间充质干细胞 MAFA PDX1 过表达慢病毒载体 胰岛素分泌细胞 糖尿病 诱导分化 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R394.2