【作者】 效梅； 安立龙； 张土保； 吴凌峰； 郭鸿波； 许英梅；

【Author】 Xiaomei,An lilong,Zhang TuBao,Wu LingFeng,GuoHongBo,Xu YingMei(college of Agriculture,GuangDong Ocean University,Zhanjiang Guangdong 524008,China)

【机构】 广东海洋大学农学院；

【摘要】 研究Ⅳ型胶原酶消化时间,血清浓度对小鼠胰腺干细胞分离培养的影响,确定小鼠上皮样胰腺干细胞分离培养适宜的Ⅳ型胶原酶消化时间和血清(NBS)浓度。胰腺组织用0.1%Ⅳ型胶原酶消化不同时间后,培养观察细胞生长状况及形态特征,并用含不同血清浓度的培养液培养胰腺干细胞,观察上皮样胰腺干细胞生长和增殖的情况。结果表明,用0.1%Ⅳ型胶原酶消化小鼠胰腺组织,消化17-20min,上皮样胰腺干细胞少,成纤维细胞生长旺盛,干细胞易被挤侵;消化21-24min,上皮样胰腺干细胞多,培养9-10d呈鹅卵石样铺于皿底。在细胞原代培养时,在培养液中添加5%或10%的小牛血清(NBS),上皮样胰腺干细胞8-10d生长成片;添加15%或20%的NBS,成纤维细胞生长旺盛,干细胞无增殖空间。在细胞继代培养时,在培养液中添加5%的NBS,胰腺干细胞生长增殖缓慢,添加10-20%的(NBS,胰腺干细胞生长增殖快,以15%的NBS浓度效果最好。小鼠胰腺干细胞分离培养适宜的Ⅳ型胶原酶消化时间为21-22min,适宜的NBS浓度为10%,按此法分离培养得到传至5代的上皮样胰腺干细胞,并可分化为胰岛团样细胞。更多还原

【Abstract】 The aim of this experiment was to study the impact of the mouse pancreas stem cells isolated and cultured by the action of the different Collagenase Type 4 digestion time and the different calf serum concentration.So that the rtght condition was determined to separate and sublime epithelioid pancreatic stem cell.First,pancreatic tissue were digested by 0.1% Collagenase Type 4 for different time,and then the groth and Morphology was observed.Second,Pancreatic stem cells was cultured by the Cell Culture media which have Different concentrations of serum,Epithelioid pancreatic stem cells Growth and proliferation of was observed.First,0.1% Ⅳ of collagenase was used to digest mouse pancreas for 17-20 mins.The number of Epithelioid pancreatic stem cells was not much.Fibroblast grew strongly.Stem cell was crowded invasion.If 0.1% Ⅳ of collagenase was used to digest mouse pancreas for 21-24mins,there are many Epithelioid pancreatic stem cells.pancreatic stem cells which was Cultured for 9-10 days pebble-like were grew in the bottom of the Containers.Second,primary culture,if 5% or 10% of the NBS to the medium was added,epithelioid pancreatic stem cells grow into a 8-10 d-chip.If 15 percent or 20 percent of the NBS was added,MEF strong growth,stem cell proliferation without space.Third,subculture,if 5 percent of the NBS was added to the medium,the growth and proliferation of pancreatic stem cells slowly,add 10% to 20% of the NBS,the growth and proliferation of pancreatic stem cells quickly,the effect of 15% of the serum concentration is the best.。 The most suitable time of mouse pancreatic stem cells isolated and cultured for the collagenase is 21-22 min,and the serum concentration is 10 percent.As this method,Epithelioid pancreatic stem cells which was cultured to spread to five generations can be produced,and differentiate to pancreatic Island Mission-like cells.更多还原