通过整体消化法分离具有集落形成能力的胰腺上皮样细胞  

Isolation of Adult Mouse Pancreatic Epithelial Cells in Vitro by Overall Digestion

作　　者：梁杰[1] 孔德麟 梁洋[1] 李丹[1] 冯锐成[1] 滕春波[1] 

机构地区：[1]东北林业大学生命科学学院发育生物学研究室,黑龙江哈尔滨150040

出　　处：《现代生物医学进展》2014年第12期2247-2250,2258,共5页Progress in Modern Biomedicine

基　　金：黑龙江省自然科学基金项目(C201215)

摘　　要：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 turn into the cells which expressing insulin by introduction and they are important source for cell replacement therapy treatment of diabetes.Base on the research of parting digestion,we try to explore a new separation method of pancreatic epithelial cells.Methods：Pancreas was carefully isolated from 4-8 week-old mice under a dissecting microscope.Fat,blood vessels,mesentery,and lymph nodes were carefully removed with pointed tweezers.Pancreatic tissue was incubated in 0.8 mg/ml collagenase IV at 37 ℃ for 20 min.At the conclusion of the digestion period,the pancreatic tissue were gently pipetted and washed to maximize the release of single cells.The single-cell suspension was transferred to a new tube and centrifuged for 5 min at 900 r/min.The supernatant,upper cell debris,DNA floc winding and the middle layer of blood cells were discarded.The milky white cells in the lower layer were resuspended in medium and then inoculated into 6 cm dish.Results：The cells isolated by overall digestion almost are pancreatic epidermal cells.In the 2% serum medium,adherent pancreatic epidermal cells growth into tight cells colony after 6 days.The number of cells isolated by overall digestion is less than parting digestion,but it is convenient to pick the colony and gain more cells.Conclusions：Overall digestion can isolate a group of pancreatic epithelial cells which have strong proliferation and large colony formation ability.This separation method is convenient and fast,for further research of pancreatic stem cells laid the foundation.

关 键 词：整体消化法 胰腺上皮样细胞 成纤维细胞 

分 类 号：Q95-3[生物学—动物学] R587.1[医药卫生—内分泌]