通过G418处理离体纯化小鼠胰腺上皮细胞

Purification of Pancreatic Epithelial Cells Treated with G418 in Vitro

目的:探索一种能有效去除成纤维细胞,筛选有较强增殖能力上皮细胞的方法。方法:本研究利用成纤维细胞对G418敏感的特性,在成体小鼠胰腺细胞分离后,采用悬浮细胞直接接种到含30μg/mL G418的培养基中,或细胞贴壁生长汇合至50-70%后用30至100μg/mL之间不同浓度G418处理24h、48h或72h两种方案进行上皮细胞的纯化。结果:在直接接种法培养处理中,存活的大部分细胞为成纤维细胞,上皮细胞的生长受到抑制,无法得到纯化的胰腺上皮集落;而在细胞贴壁生长汇合至50-70%后经G418处理,成纤维细胞随着处理浓度的增加死亡率也在逐渐上升,其中50μg/mL G418处理72小时对去除成纤维细胞效果最佳。结论:G418处理能够有效去除成纤维细胞,分离纯化出一群在离体条件下具有强增殖能力、形成大上皮细胞集落的细胞。该分离纯化方法为今后进一步研究成体胰腺干/祖细胞增殖与分化调控机制等问题奠定基础。

Objective：To eliminate the fibroblasts and purify epithelial cells from adult mouse pancreas in vitro.Methods：Based on the sensitivity of fibroblasts to G418,this study designed two schemes：Scheme 1 was that the isolated single cells from pancreas were cultured directly in media with 30μ g/mL G418,and Scheme 2 was that the isolated single cells were cultured to 50-70% aggregation,and then they were treated with G418 at concentrations from 30 to 100 μ g/mL of G418 for 24h,48h or 72h.Results：The results showed that the growth of the epithelial cells was inhibited in scheme 1,but that of the fibroblasts was little affected.However,after the pancreatic cell grew 50-70% aggregation in scheme 2,G418 treatment could eliminate the fibroblasts,and the death ratio of fibroblasts increase with the elevation of G418 concentration.Among them,50μ g/mL G418 treatment for 72h was optimal to kill fibroblasts and preserve epithelial cells.Conclutions：G418 treatment could effectively purify a pancreatic epithelial cells with strong proliferative and colony-forming capability,thus provide a pathway for further studies on the proliferation and differentiation of adult pancreas stem/progenitor cells.