胎猪胰腺干细胞扩增及诱导

目的 寻找胰腺干细胞扩增方法,为研究糖尿病的细胞治疗提供种子细胞.方法无菌采集胎猪胰腺,经胰酶消化,以含10%血清的RPMI1640培养基培养.细胞长满皿底85%时,用胰酶消化传代,传代5～6次后细胞活力下降,细胞大量死亡漂浮,留下少数贴壁的小圆细胞.将培养基中的血清浓度提高到15%,贴壁的小圆细胞能快速增殖并形成细胞克隆,4～6d便可融汇成单层.采用免疫组织化学方法对所获得的的细胞进行胰腺干细胞特征检测.结果经上述方法处理的细胞具有旺盛的增殖能力,可以连续传代,目前已传至64代.采用免疫组织化学检测细胞表达胰腺干细胞特征性标志物,胰肠同源域因子1(PDX-1)、葡萄糖转运子2(GLUT-2)、巢蛋白、波形蛋白;经诱导后细胞表达胰腺内分泌细胞的标志为胰高血糖素、胰岛素、生长抑索、胰多肽.结论通过该方法可以获得大规模扩增的胰腺干细胞,该细胞在体外诱导可分化形成胰腺内分泌部4种主要细胞.

Method for expansion and induction of porcine fetus islet stem cell

Objective To find a method for proliferating porcine fetus islet stem cells. Methods Cells collected from porcine fetus pancreas were digested with trypsine and cultured in medium RPMI1640 with 10% FBS. Some cells survived after seven days, these cells could be passed for 5 to 6 times. Then, most of those cells were trend to die and float in the medium, only were a few little cells attached on culture plate. These little cells proliferated quickly when medium was changed to RPMI 1640 with 15% FBS and each of the little cells can formed cells’ clone in 4 days. The cell’s character of islet stem cell was detected by immunochemical method. Results Cells obtained by the method posses strong proliferation ability and it has been sub cultured over 64 passages till now. It expressed islet stem cell’s mark such as PDX-1，GLUT-2，nestin，vimentin et.al. After induction, it expressed insulin, glucagon, somatostatin and polypeptide. Conclusion Cells obtained by this method were determined as islet stem cells. They can differentiate into islet endocrine cell EM>in vitro/EM>. This study can provide reference for isolation and expansion of isl