大鼠胰腺导管干细胞分化形成类胰岛的改良诱导方法  

Modified induction method for differentiation of rat pancreatic ductal stem cells to form islet-like cells

作　　者：张文琦 杨洁 吴江 郎贯存 效梅[1] ZHANG Wen-qi;YANG Jie;WU Jiang;LANG Guan-cun;XIAO Mei(Department of Veterinary Medicine,College of Coastal Agricultural Sciences,Guangdong Ocean University,Guangdong Zhanjiang 524088,China)

机构地区：[1]广东海洋大学滨海农业学院动物医学系,广东湛江524088

出　　处：《解剖学报》2022年第3期387-395,共9页Acta Anatomica Sinica

基　　金：广东省自然科学基金(10152408801000023);教育部留学基金(教外司留[2010]1561);国家级大学生创新创业训练计划项目(201810566015)。

摘　　要：目的对大鼠胰腺导管干细胞(rPDSCs)分化形成类胰岛的诱导方法进行改良。方法在基础培养液DMEM/F12+10%FBS+1%青霉素/1%链霉素中,分别添加2、4、6和8μmol/L全反式维A酸(ATRA),体外诱导rPDSCs分化形成类胰岛,筛选ATRA最适诱导浓度。以ATRA最适诱导浓度为基础,再分别采用基质胶(matrigel)培养,悬浮培养或悬滴培养方式体外诱导rPDSCs分化形成类胰岛,筛选最适诱导培养方式。采用细胞形态学,双硫腙(DTZ)染色,细胞免疫荧光染色,Real-time PCR和ELISA方法对诱导类胰岛进行检测。结果与对照组相比,在基础培养液中,添加6μmol/L ATRA及采用基质胶培养方式诱导效果最好。诱导28 d,细胞富集分化形成胰岛样球形细胞团;DTZ染色呈阳性;在基因和蛋白水平上分别表达胰岛素(insulin)和胰腺十二指肠同源框1(Pdx1);葡萄糖刺激,释放胰岛素和C肽,且具有葡萄糖浓度依赖性。结论在基质胶培养方式下,采用6μmol/L ATRA+DMEM/F12+10%FBS+1%青霉素/1%链霉素可以成功诱导rPDSCs体外分化形成类胰岛。Objective To establish a modified induction method for differentiation of rat pancreatic ductal stem cells(rPDSCs)to form islet-like cells.Methods All-trans retinic acid(ATRA)was added at 2,4,6 and 8μmol/L in the basal culture medium DMEM/F12+10%FBS+1%penicillin/1%streptomycin to induce the differentiation of rPDSCs to form islet-like cells in vitro,and the optimal induction concentration of ATRA was screened.Based on the optimal ATRA induction concentration,rPDSCs were then induced to form islet-like cells in vitro by matrigel culture,suspension culture or hanging drop culture,respectively,to screen the optimal induction culture method.Cell morphology,dithizone(DTZ)staining,cell immunofluorescence staining,Real-time PCR and ELISA were used to detect the induced islet-like cells.Results Compared with the control group,6μmol/L ATRA and matrigel culture were the best in the basic culture medium.After 28 days of induction,the cells enriched and differentiated to form islet-like spherical cell clusters;DTZ staining was positive;Pancreatic duodenal homeobox-1(Pdx1)and insulin were expressed at gene and protein levels,respectively;Glucose stimulation,release insulin and C-peptide,showed glucose concentration dependent.Conclusion The in vitro differentiation of rPDSCs to form islet-like cells could be successfully induced by using 6μmol/L ATRA+DMEM/F12+10%FBS+1%double antibody under matrigel culture method in the present study.

关 键 词：胰腺导管干细胞 全反式维A酸 胰岛 实时定量聚合酶链反应 大鼠 

分 类 号：Q813.1[生物学—生物工程]