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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、生长抑素、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。
Objective To search for methods of pancreatic stem cell expansion and to provide seed cells for the research of diabetes cell therapy. Methods Aseptically collected fetal pancreas was digested by trypsin and cultured in RPMI1640 medium containing 10% serum. When the cells covered 85% of the bottom of the dish, they were passaged with trypsin and passaged for 5 to 6 times. The viability of the cells decreased and the cells died in large numbers, leaving a few adherent small round cells. Serum concentration in the medium increased to 15%, adherent small round cells can rapidly proliferate and form cell clones, 4 ~ 6d can be integrated into a single layer. The obtained cells were examined for the characteristics of pancreatic stem cells by immunohistochemistry. Results The cells treated by the above method had strong proliferative capacity and could be continuously passaged, which has now passed on to the 64th generation. Immunohistochemistry was used to detect the expression of PDX-1, GLUT-2, Nestin and vimentin in pancreatic stem cells. The induced pancreatic endocrine The cells are labeled with glucagon, insulin, somatostatin, and pancreatic polypeptide. Conclusion This method can obtain large-scale expansion of pancreatic stem cells, the cells induced in vitro differentiation of the formation of pancreatic endocrine 4 kinds of major cells.