中国医学科学院学报

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中国医学科学院学报

中国医学科学院学报 ›› 2013, Vol. 35 ›› Issue (3): 270-274.doi: 10.3881/j.issn.1000-503X.2013.03.006

• 论 著 • 上一篇    下一篇

悬滴和悬浮法相结合培养胰腺祖细胞

马凤霞,陈芳,池颖,杨少光,卢士红,韩忠朝   

  1. 中国医学科学院 北京协和医学院 血液病医院/血液学研究所实验血液学国家重点实验室,天津 300020
  • 收稿日期:2012-12-11 出版日期:2013-07-04 发布日期:2013-07-04
  • 通讯作者: 马凤霞 电话:022-23909186,电子邮件:mafengxia@gmail.com E-mail:mafengxia@gmail.com
  • 基金资助:

    天津市应用基础及前沿技术研究计划(09JCYBJC09700、12JCZDJC25000)Supported by the Tianjin Research Program of Application Foundation and Advanced Technology(09JCYBJC09700,12JCZDJC25000)

Culture of Pancreatic Progenitor Cells in Hanging Drop and on Floating Filter

MA Feng-xia,CHEN Fang,CHI Ying,YANG Shao-guang,LU Shi-hong,HAN Zhong-chao   

  1. State Key Laboratory of Experimental Hematology,Institute of Hematology and Hospital of Blood Diseases,
    CAMS and PUMC,Tianjin 300020,China
  • Received:2012-12-11 Online:2013-07-04 Published:2013-07-04
  • Contact: MA Feng-xia Tel:022-23909186,E-mail:mafengxia@gmail.com E-mail:mafengxia@gmail.com

摘要:

目的 建立悬滴和悬浮法相结合培养胰腺祖细胞的方法,研究该方法能否诱导胰腺祖细胞在体外分化为成熟的内分泌细胞。方法 体外分离小鼠12.5d胚胎胰腺,消化成单个细胞,悬滴法培养24h后细胞形成球体,将细胞球转移到下层为培养基漂浮的膜上培养6d。培养过程中,采用免疫组织化学方法检测胰腺十二指肠同源盒基因1(PDX-1)和神经源素3(Ngn3)的表达及胰岛素、胰高血糖素、羧肽酶(CPA)的表达,酶联免疫吸附法检测葡萄糖刺激后细胞球分泌胰岛素的变化,定量PCR检测培养过程中胰腺标志表达的变化。结果 细胞球培养1d后,有大量PDX-1表达,而且这些PDX-1阳性细胞增殖。细胞球培养3d后,Ngn3大量表达,Ngn3阳性细胞不增殖。培养7d后,分化的胰腺细胞表达成熟内、外分泌细胞的标志,而且胰腺标志的表达与体内表达模式一致。高糖能刺激细胞球分泌胰岛素。结论 悬滴和悬浮法相结合能诱导胰腺祖细胞在体外分化为成熟的内分泌细胞。

关键词: 胰岛素, 腺十二指肠同源盒基因1, 神经源素3, 胰腺祖细胞

Abstract:

Objective To construct a method to culture pancreatic progenitor cells in hanging drop and on floating filter,and to examine if pancreatic progenitor cells can differentiate into mature endocrine cells with this method.Methods Murine embryos at day 12.5 were isolated and digested into single cells,which were then cultured in hanging drop for 24h and formed spheres.Spheres were cultured on the filter for 6 days,which floated in the dish containing medium.During culture,the expressions of pancreas duodenum homeobox-1(PDX-1)and neurogenin3(Ngn3)were determined.The expressions of endocrine and exocrine markers,insulin,glucagon,and carboxypeptidase(CPA)were determined on day 7 by immunohistochemistry.Insulin secretion of spheres stimulated by glucose was detected by ELISA.The changes of pancreatic marker expressions during culture were monitored by real-time polymerase chain reaction(PCR).Results One day after the culture,there were still a large amount of PDX-1 positive cells in pancreatic spheres,and these cells proliferated.On day 3,high expression of Ngn3 was detected,and the Ngn3-postive cells did not proliferate.On day 7,The expressions of endocrine and exocrine markers in the differentiated pancreatic progenitor cells were detected,which were consistent with that in vivo.Insulin was secreted by spheres upon the stimulation of glucose.Conclusion In hanging drop and on floating filter,pancreatic progenitor cells can differentiate into mature endocrine cells.

Key words: insulin, pancreas duodenum homeobox-1, neurogenin3, pancreatic progenitor cell

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