仔猪胰腺细胞体外培养体系的建立  

Establishment of a culture system for pancreatic cells in neonatal piglets in vitro

作　　者：张慧茹[1,2] 冯若鹏 窦忠英 

机构地区：[1]河南工业大学生物工程学院,河南郑州450001 [2]国家干细胞工程技术研究中心陕西省分中心,陕西杨凌712100

出　　处：《黑龙江畜牧兽医》2011年第5期1-5,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金："863"国家高新技术研究与发展计划项目(2005AA219050);国家自然科学基金项目(30671067);教育部重大项目(03160);河南工业大学人才引进项目(150214)

摘　　要：为了研究体外培养仔猪胰腺细胞的适宜条件,试验选取1日龄的仔猪胰腺组织置于冰上清洗、修剪,对消化酶、离心方式、培养皿铺被方式、培养液及添加不同促生长因子等多种因素进行筛选和优化。结果表明:0.25%胰酶消化所获得的细胞强于0.1%胶原酶消化获得的细胞,细胞清亮,易于生长;采用500 r/min离心5 min、多次离心所获得的细胞较Percoll不连续密度梯度离心和5%BSA等密度梯度离心所获得的细胞贴壁率高,生长能力强,易于培养传代;采用0.1%明胶或20μg/mL的层黏连蛋白铺被培养皿均比无铺被的培养皿获得更多的贴壁细胞;以RPMI 1640、M199、F12、L-DMEM多种培养液培养仔猪胰腺细胞,表现出多种细胞形态和生长趋势;在基础培养液中添加上皮生长因子、角质化生长因子、β-巯基乙醇和白血病细胞抑制因子、转铁蛋白与亚硒酸盐混合试剂等促干细胞增殖因子可促进不同形态细胞的增殖。说明按此方式培养可获得大量、多种细胞形态的仔猪胰腺祖/干细胞。To study suitable culture-conditions for pancreatic cells in neonatal piglets in vitro.The pancreatic tissue of neonatal piglets was collected,and washed bloodiness,and took off the fat,and cut into pieces all on the ice.Multiple factors were screened and optimized which were digestive-enzymes,centrifugal-methods,coated-mediums,basic-media,and many factors.The results indicated a culture system of pancreatic cells in neonatal piglets as follow： The digested cells with 0.25% trypsin were clearer than 0.1% collagenase,had higher growth capacity.The cells with low centrifugal speed（500 r/min,five minutes） and many times（above three times） were higher attachment efficiency than percoll centrifugal-liquid and 5% BSA centrifugal-liquid,which had better growth and short passage-period.The coated-media of 0.1% gelatin or 20 μg/mL laminin were benefited plating efficiency than no-coated.Many basic media such as RPMI 1640,M199,F12,L-DMEM could facilitate pancreatic cells to show various cell shapes and growth trends in neonatal piglets.The basic media with epidermal growth factor,keratinocyte growth factor,β-mercaptoethanol and leukemia inhibition factor,and Insulin-Transferrin-Selenium admixture could accelerate the multi-shapes cells proliferation.According above conditions,the culture system of pancreatic cells in neonatal piglets was established,it could proliferate large pancreatic progenitor/stem cells in vitro.

关 键 词：仔猪 胰腺细胞 体外培养体系 

分 类 号：Q813.11[生物学—生物工程]