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目的:胰腺上皮细胞能诱导成表达胰岛素的细胞,成为细胞替代疗法治疗糖尿病的重要来源。胰腺细胞的分离多采用机械剪切后胶原酶消化,本文在以往研究基础上,探索一种能分离得到更纯净的胰腺上皮样细胞的新方法。方法:本研究采用胰腺整体消化的方法,将成体小鼠整个胰腺取下,摘除系膜及大的血管,置于胶原酶中消化20min,用PBS吹打胰腺组织,得到的细胞悬液,离心后去除上清与细胞碎片,用培养基重悬实质细胞,接种于6 cm培养皿中,培养7-10天后得到单细胞集落。结果:整体消化法不剪碎胰腺组织,从而避免多种胰腺细胞的参与,得到较为纯净的胰腺上皮细胞悬液,细胞总体数量小于部分消化法,但是单细胞比率远远高于部分消化法,得到的细胞集落更纯净,不需要去除成纤维细胞,方便筛选及进一步扩大培养。结论:整体消化法能够分离纯化出一群在离体条件下具有强增殖能力、形成大的上皮样集落的细胞。该分离方法方便、快捷,为今后进一步研究成体胰腺干细胞增殖与分化调控机制等问题奠定基础。
OBJECTIVE: Pancreatic epithelial cells can be induced into insulin-expressing cells and become an important source of cell replacement therapy in the treatment of diabetes. Pancreatic cells are mostly separated by mechanical shear after collagenase digestion. Based on the previous studies, this paper explores a new method to isolate more pure pancreatic epithelial-like cells. Methods: In this study, the whole pancreas of adult mice were removed and the mesangium and large blood vessels were removed and digested with collagenase for 20 minutes. The pancreatic tissue was blown with PBS, and the resulting cell suspension was centrifuged The supernatant and cell debris were removed, the parenchymal cells were resuspended in culture medium, inoculated into 6 cm petri dishes, and single cell colonies were obtained after 7-10 days of culture. Results: The whole digestion method did not cut the pancreas tissues, thus avoiding the involvement of multiple pancreatic cells, resulting in a more pure pancreatic epithelial cell suspension, the overall number of cells is less than the partial digestion, but the single cell ratio is much higher than the partial digestion, The resulting cell colonies are more pure, do not need to remove fibroblasts, to facilitate screening and further expansion of culture. Conclusion: The whole digestion method can isolate and purify a group of cells with strong proliferative ability in vitro and form large epithelial-like colonies. The separation method is convenient and fast, which lays the foundation for further research on the regulation of proliferation and differentiation of adult pancreatic stem cells in the future.