新生大鼠胰腺来源干细胞的分离培养与鉴定

Isolation, culture and identification of stem cells from the pancreas of newborn rats

目的从新生大鼠胰腺分离出一种胰腺干细胞,并对其进行初步鉴定。方法采集大鼠胰腺,5.5mg/mlⅣ型胶原酶消化,用含10%胎牛血清(FBS)的RPMI1640培养基培养,首次传代时利用差速贴壁分离上皮样细胞和成纤维样细胞。多次纯化上皮细胞,细胞密度为80%进行传代。对第4代的细胞进行免疫荧光染色,诱导向β胰岛细胞分化并利用高糖刺激胰岛素释放。结果新生大鼠胰腺中的胰腺干细胞具有较强的增殖能力,可连续传代。第4代细胞经免疫荧光染色,显示胰十二指肠同源框因子1(PDX-1)、巢蛋白(Nestin)、神经元素3(NGN3)、波形蛋白(Vimentin)、胰岛素(Insulin)和C肽(C-peptide)阳性;经诱导分化后,经双硫腙(DTZ)染色为阳性,同时免疫组织化学显示PDX-1和C-peptide阳性;在糖刺激实验中,当无糖刺激时,细胞组胰岛素释放量为(5.3±1.5)pIU/ml,而诱导后的细胞组胰岛素释放量为(30.0±11.5)pIU/ml,两者比较差异有统计学意义(P=0.035)。结论分离的胰腺细胞是胰腺干细胞。

Objective The aim of this study is to isolate and identify the pancreatic stem cells from the pancreas of neonatal rats. Methods The pancreatic tissue was collected from neonatal rats in aseptic environment, and then digested with 5.5 mg/ml collagenase IV. The organoid cultured in RPMI 1640 medium supplemented with 10%fetal bovine serum (FBS). Differential centrifugation method was carried out to isolate the epithelial cell and fibroblast. The epithelial cells were purified for several times. The 4th-generation cells were then induced to differentiate into β islet cells, and stained with immunofluorescent signal. High concentration of glucose was applied to induce insulin secretion. Results The pancreatic stem cells in the pancreas of the neonatal rats possessed high proliferative rate and can be applied for continuous passage. Immunofluorescence staining of the4th-generation cells showed positive Pancreatic duodenal homeobox-1 (PDX-1), Nestin, neurogenin3 (NGN3), Vimentin, insulin and C-peptide which were pancreatic stem/progenitor cell specific proteins; after inducing to differentiation, the cells displayed positive with Dithizone staining, while immunohistochemistry test showed cells were PDX-1 and C-peptide positive; for glucose stimulation assay, insulin secretion of induced ICCs [(30.0±11.5) pIU/ml] was significantly higher than that of non-induced ICCs [(5.3±1.5) pIU/ml, P=0.035). Conclusion The isolated pancreas cells were pancreas stem cells.