小鼠胰腺干细胞的分离、培养与鉴定

摘要

目的 从新生小鼠中分离培养并鉴定胰腺干细胞.方法 利用组织块法分离新生小鼠胰腺源性干细胞,经RPMI1640+ 5％胎牛血清+双抗+1 mmol/L丙酮酸钠+1 mmol/L非必需氨基酸+10 ng/mL表皮生长因子+ 10 ng/mL成纤维生长因子+2 mmol/L谷氨酰胺的溶液培养,利用免疫组化分析胰腺源性干细胞的兔抗巢蛋白抗体(Nestin)、胰肠同源域因子(PDX-1)和葡葡糖转运子(GLUT-2)抗原表达；经10 mmol/L尼克酰胺诱导产生胰岛样细胞团并利用双硫腙(DTZ)染色分析其胰岛素分泌情况.结果 分离培养的胰腺源性干细胞呈Nestin、PDX-1和GLUT-2阳性；诱导的胰岛样细胞团呈DTZ染色阳性.结论 分离获得的新生小鼠胰腺源性干细胞在体外可分化为类胰岛,并具有胰岛素分泌功能.%Objective To separate, culture and identify the pancreatic stem cells (PSCs) from newborn mouse. Methods The pancreas-derived stem cells were separated from newborn mouse by tissue block method and cultured in the liquid medium (RPMI1640 +5% FBS + double-antibiotics + 1 mmol/L sodium pyruvate + 1 mmol/L Non-essential amino acid + 10 ng/mL EGF + 10 ng/mL bFGF + 2 mmol/L Glutamine). The expressions of Nestin, PDX-1 and GLUT-2 antigens were identified and analyzed by immunohistochemistry. The pancreas-derived stem cells were induced by 10 mmol/L NIC and differentiated into pancreatic islet-like cell clusters, which was stained with DTZ for analyzing the secretion of insulin. Results In the isolated and cultured pancreas-derived stem cells, Nestin, PDX-1 and GLUT-2 expressed positively , in the induced pancreatic islet-like cell clusters, DTZ expressed positively. Conclusions The pancreas-derived stem cells isolated from newborn mouse are PSCs, which can differentiate into similar islets of pancreas, and have islets of pancreas function.