胎儿胰岛样细胞团源上皮样细胞分离、纯化和鉴定

Isolation,Purification and Identification of Epithelial Cells Derived from Fet al Islet-like Cell Clusters

旨在优化胰腺干细胞分离、鉴定的方法和体系，为糖尿痛研究和治疗提供种子细胞。采用胶原酶消化法，分离培养出胰岛样细胞团（ICCs），对其进行贴壁培养，从中纯化出上皮样细胞。采用MTT法测定其生长情况并绘制生长曲线。采用免疫组织化学染色检测分离得到细胞的PDX-1、PCNA、CK-7、CK-19、Nesfin、Glut2、Vimenfin、Insulin表达情况。应用流式细胞仪检测其表面标志。由分离培养的ICCs成功纯化出上皮样细胞；传40代，每代冻存106～10s个细胞；生长曲线显示其传代第3天进入对数生长期，第5天进入平台期；免疫组织化学染色显示其表达PDX-1、PCNA、CK-7、CK-19、Nestin、Glut2、Vimentin；不表达Insulin；流式细胞仪分析表明其表达CD29、CD44、CD166，不表达CD11a、CD14、CD34、CD45、CD90、CD105、CD117。由胎儿胰腺能分离出具有自我更新能力的上皮样细胞，为导管来源，具干细胞特性。

The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase iV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTI＂. The expression of molecular markers in the cells was identified by immunohistocbemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 106 - 108 cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CDl17. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells.