大鼠胰腺导管干细胞分化形成类胰岛的改良诱导方法

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8 μmol/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6 μmol/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6 μmol/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。

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

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 8 μmol/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, 6 μmol/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 6 μmol/L ATRA+DMEM/F12+10% FBS+1% double antibody under matrigel culture method in the present study.