新生昆明小鼠胰腺干细胞体外分离、培养及自然分化的潜能

背景：关于胰腺干细胞在胰腺组织中的分布情况,以及如何有效的将其分离、体外优化培养,目前仍有一定的困难。目的：从新生昆明小鼠胰腺组织中分离出胰腺干细胞,在体外条件下培养观察其形态学和生物学特征,并进行初步鉴定。方法：取新生SPF级昆明小鼠的胰腺组织,使用Ⅴ型胶原酶消化,采用Percoll不连续密度梯度离心,使胰腺组织内、外分泌部细胞分离,分布在3个不同的密度界面;收集各界面的细胞,以无血清、添加碱性成纤维细胞生长因子和表皮细胞生长因子的DMEM培养基培养。结果与结论：通过细胞形态学和细胞生长特性的观察,结合双硫腙染色证实：采用Percoll不连续密度梯度离心,第一、二密度界面的细胞来源于胰腺内分泌部;第三密度界面细胞来源于胰腺外分泌部。分别从胰腺内、外分泌部获取胰腺组织细胞,在体外培养观察发现均存在一类大、圆、单个核的细胞,呈集落样附壁生长,具有较活跃的分裂增殖能力,碱性磷酸酶染色阳性,表达胰腺干细胞的特异性标志巢蛋白,即为胰腺干细胞;随着体外培养时间的延长,分别表达PDX-1和CK-19,呈现向胰腺内分泌部细胞和外分泌部细胞分化的趋势。

In vitro isolation, culture and natural differentiation potential of pancreatic stem cells in newborn Kunming mouse

BACKGROUND： Now, it is still difficult to obtain the distribution of pancreatic stem cells in pancreatic tissue, and to isolate them effectively and in vitro optimal culture. OBJECTIVE： To isolate pancreatic stem cells from pancreatic tissue in newborn Kunming mice and to culture them in vitro conditions for the morphological and biological characteristics observation and preliminary identification. METHODS： The newborn SPF Kunming mouse pancreatic tissue were digested by V collagenase. Percoll discontinuous density gradient centrifugation was used for the separation of pancreatic tissue cells and exocrine cells and distribution at three different density interfaces. Different interface cells were collected to culture in Dulbecco＇s modified eagle medium containing serum-free, basic fibroblast growth factor and epidermal growth factor. RESULTS AND CONCLUSION： Observation of the cell morphology and cell growth characteristics combined with dithizone staining confirmed that, pancreatic endocrine cells were located in the first and second density interface, whereas cells of pancreatic exocrine located in the third density interface after Percoll discontinuous density gradient centrifugation. A kind of large, round, mononuclear, colony-like grown cells were found in both exocrine and endocrine cells from pancreas, which showed active proliferation, positive for alkaline phosphatase staining and expressed specific markers of pancreatic stem cells-Nestin, these cells just were the pancreatic stem cells. With the extension of time in vitro, pancreatic stem cells from exocrine and endocrine part of pancreas expressed PDX-1 and CK-19 respectively, which showed the differentiation potential to pancreatic endocrine cells and exocrine cell respectively.