小鼠脂肪间充质干细胞向胰岛素分泌细胞的诱导分化研究  

Studies on insulin-producing cells induced from mouse adipose mesenchymal stem cells

作　　者：赵天闯[1] 安星兰[1] 刘阳[2] 唐博[1] 张学明[1] 李子义[1,3] 

机构地区：[1]吉林大学动物科学学院人类疾病动物模型国家地方联合工程实验室,吉林长春130062 [2]西北农林科技大学动物医学院,陕西杨凌712100 [3]吉林大学第一医院转化医学研究院,吉林长春130061

出　　处：《中国兽医学报》2015年第5期755-764,共10页Chinese Journal of Veterinary Science

基　　金："973"国家重点基础研究发展计划资助项目(2011CBA01003)

摘　　要：为了通过特定转录因子将小鼠脂肪间充质干细胞(mADSCs)定向诱导分化为胰岛素分泌细胞(IPCs)。本研究分别构建Pdx1(胰十二指肠同源盒基因1)、MafA(V-maf肌肉腱膜纤维肉瘤癌基因同源物A)、NeuroD1(神经分化因子1)3种基因的慢病毒过表达载体,使用293T细胞对3种因子进行慢病毒包装,将3种慢病毒过表达载体以单因子侵染、双因子侵染、三因子联合侵染的方式对mADSCs进行定向分化诱导,于诱导分化第15天对不同方式诱导的IPCs进行检测鉴定,并对不同方式诱导组的IPCs进行高糖刺激,刺激后30~120min检测培养基中含糖量的变化。结果显示,构建的慢病毒过表达载体pHBLV-CMV-IRES-ZsGreen-Pdx1、pHBLV-CMV-PGK-RFPMafA、pHBLV-CMV-PGK-RFP-NeuroD1所含目的片段基因序列与小鼠全基因编码序列完全一致,三种基因慢病毒过表达载体构建成功;诱导分化第15天,三因子联合诱导组所形成的IPCs克隆双硫腙(DTZ)染色呈阳性,并可表达胰岛素生物合成及分泌相关基因;在高糖刺激条件下,三因子联合诱导组糖分解速度、分解量远优于单因子或双因子诱导组。结果表明,Pdx1、MafA、NeuroD1 3种因子联合作用,可以将小鼠脂肪间充质干细胞定向诱导分化为胰岛素分泌细胞,并可在高糖刺激下,有效发挥降糖作用。To gain insulin-producing cells(IPCs)induced from mice adipose mesenchymal stem cells(mADSCs)through specific transcription factors.Lentiviral overexpression vectors harboring Pdx1(pancreatic and duodenal homeobox-1),MafA(V-maf musculoaponeurotic fibrosarcoma oncogene homolog A)and NeuroD1(neural differentiation factor-1)were constructed,respectively,packing of lentivirus of Pdx1,MafAand NeuroD1 using 293Tcells;mADSCs were induced with different combinations of Pdx1,MafA and NeuroD1genes;at 15 dafter transfection,IPCs induced by different ways were tested.The concentration of glucose were detected during 0-120 min after stimulation.Expressions of Pdx1,MafA,NeuroD1,Ngn3 and Insulin2as specific marker genes were investigated at mRNA level.Lentiviral overexpression vectors,pHBLV-CMVIRES-ZsGreen-Pdx1,pHBLV-CMV-PGK-RFP-MafAand pHBLV-CMV-PGK-RFP-NeuroD1,in which their purpose fragment gene sequences contained exactly the same as that of coding sequence of all the genes in mice,indicating that three genes lentiviral overexpression vectors were successful constructed;At 15 dafter transduction,IPCs clones showed positive staining of DTZ and expressed insulin biosynthesis and secretion related genes;stimulate IPCs with high glucose DMEM mediusm,IPCs induced with a combination of 3factors showed the most effective regulation and the lowest glucose concentration compared with other groups.The combination of Pdx1,MafAand NeuroD1 genes facilitates mADSCs differentiation into insulin-producing cells and shows a potential as a surrogateβ-cells for the treatment of diabetes.

关 键 词：脂肪间充质干细胞(ADSCs) 胰岛素分泌细胞(IPCs) 慢病毒过表达载体 诱导分化 小鼠 

分 类 号：R587.1[医药卫生—内分泌]