胰腺干细胞体外培养扩增的方法

背景:胰腺干细胞在体外培养时极易分化,很难获得足够数量的胰腺干细胞。目的:探讨一种简便易行的胰腺干细胞体外扩增培养方法。方法:分离新生昆明小鼠胰腺,原代培养三四天,成纤维细胞中出现呈集落生长的胰腺干细胞后,以0.02%EDTA消减成纤维细胞的数量,降低其比例后继续培养,待细胞铺满培养瓶底的80%时,重复三四次,逐渐提高胰腺干细胞的数量。检测胰腺干细胞特异性标志Nestin。尼克酰胺诱导胰腺干细胞分化;双硫腙染色对胰岛样细胞团进行检测。结果与结论:消减胰腺成纤维细胞的数量及比例后,胰腺干细胞可持续表现出活跃的分裂增殖能力,胰腺干细胞的数量显著增加;细胞保持球形、单个大核、细胞质折光性强、表达(Nestin)。扩增的胰腺干细胞,以尼克酰胺诱导后分化为胰岛样细胞团,双硫腙染色阳性。提示,减少共培养中成纤维细胞的比例和数量,有利于胰腺干细胞的增殖,获得较多数量的胰腺干细胞。扩增后的胰腺干细胞仍保持未分化状态,在适当的体外诱导条件下能分化为胰岛样结构,具有一定的分化潜能。

A method of pancreatic stem cells culture and amplification in vitro

BACKGROUND:Pancreatic stem cells are differentiated easily when cultured in vitro,so it is difficult to obtain a sufficient number of pancreatic stem cells.OBJECTIVE:To explore a simple method which pancreatic stem cells were amplified and cultured in vitro.METHODS:The pancreases of newborn Kunming mice were isolated.After culturing in vitro for 3-4 days,the colony growing pancreatic stem cells were emerged in fibroblasts.The number of fibroblasts was reduced by 0.02% EDTA;the fibroblasts were cultured continuously after their proportions were reduced.When the cells of confluent culture reached 80% of bottom,repeat 3 or 4 times,the number of pancreatic stem cells was gradually increased.The specific marker(Nestin) of pancreatic stem cells was detected.Differentiation of pancreatic stem cells was induced by nicotinamide.Islet-like cell clusters were detected by dithizone(DTZ) staining.RESULTS AND CONCLUSION:After the number and proportion of fibroblasts were reduced,the pancreatic stem cells presented active proliferative ability.The number of the pancreatic stem cells was significantly increased.The amplificated pancreatic stem cells maintained their properties,including round appearance with single nucleus,strong diffraction,and expression(Nestin).After induced by nicotinamide,the amplificated pancreatic stem cells were differentiated to islet-like cell clusters with positive DTZ staining.The results demonstrated that the proportion and number of fibroblasts were reduced which were beneficial for proliferation of pancreatic stem cells.A large number of pancreatic stem cells were obtained.The amplificated pancreatic stem cells with undifferentition condition still had the potential of differentiation to pancreatic islet under suitable induction condition.