一种高效诱导胚胎胰腺细胞分化为内分泌细胞培养方法的建立

摘要

Objective To establish a new culture method to induce the differentiation of embryonic pancreatic cells into mature endocrine cells. Methods Mouse embryos at day 12. 5 were used and embryonic pancreata were isolated. The isolated embryonic pancreata were cultured on the filter for 7 days, which floated in the dish containing medium. During culture, the expression of pancreas duodenum homeobox-1A PDX-1) , a pancreatic stem cell marker, was examined at day 1. The expression of neurogenin 3 ( Ngn3 ) , a pancreatic progenitor cell marker, was examined at day 3. The expressions of endocrine and exocrine markers, insulin, glucagon, and carboxypeptidase ( CPA) were examined at day 7 by immunohistochemistry. The kinetics of pancreatic marker expression during culture was assayed by real-time PCR. Results Many pancreatic stem cells still existed in embryonic pancreata cultured for 1 day; meanwhile, these pancreatic stem cells proliferated in high rate. A large amount of pancreatic progenitor cells were found in embryonic pancreata cultured for 3 days. Pancrealic stem/progenitor cells differentiated into mature endocrine and exocrine cells in embryonic pancreata after having been cultured for 7 days. Furthermore, the expression pattern of pancreatic marker is consistent with that in vivo. Conclusion We successfully established a new culture method, with which embryonic pancreatic cells can efficiently differentiate into mature endocrine cell.%目的 建立一种使胚胎胰腺细胞体外高效分化为成熟内分泌细胞的培养方法.方法 体外分离小鼠12.5d的胚胎胰腺,培养于下层为培养基的漂浮膜上7d,采用免疫组织化学方法检测胰腺祖细胞标志胰腺十二指肠同源盒基因1 (PIDX-1)和神经源素3(Ngn3)的表达及内、外分泌细胞的标志胰岛素、胰高血糖素、羧肽酶表达,定量PCR检测培养过程中胰腺标志表达的变化.结果 胚胎胰腺培养1d后,有大量胰腺祖细胞标志表达,且这些胰腺祖细胞处于增殖状态.培养3d后,仍有胰腺祖细胞的标志大量表达.培养7d后,分化的胰腺表达成熟内、外分泌细胞的标志,且胰腺标志的表达与体内表达模式一致.结论 建立了一种使胚胎胰腺细胞体外高效分化为成熟内分泌细胞的培养方法.