解剖学报 ›› 2022, Vol. 53 ›› Issue (3): 387-395.doi: 10.16098/j.issn.0529-1356.2022.03.018

• 技术方法 • 上一篇    下一篇

大鼠胰腺导管干细胞分化形成类胰岛的改良诱导方法

张文琦 杨洁 吴江 郎贯存 效梅*   

  1. 广东海洋大学滨海农业学院动物医学系,广东 湛江 524088
  • 收稿日期:2021-10-14 修回日期:2021-12-08 出版日期:2022-06-06 发布日期:2019-06-06
  • 通讯作者: 效梅 E-mail:xiao0812@126.com
  • 作者简介:2021-12-06
  • 基金资助:
    广东省自然科学基金;教育部留学基金;国家级大学生创新创业训练计划项目

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

ZHANG  Wen-qi YANG  Jie  WU  Jiang  LANG  Guan-cun  XIAO  Mei*   

  1. Department of Veterinary Medicine, College of Coastal Agricultural Sciences, Guangdong Ocean University, Guangdong Zhanjiang 524088, China
  • Received:2021-10-14 Revised:2021-12-08 Online:2022-06-06 Published:2019-06-06
  • Contact: XIAO Mei E-mail:xiao0812@126.com

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。

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

Abstract:

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.

Key words: Pancreatic ductal stem cell, Alltrans retinoic acid, Islet, Real-time PCR, Rat

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