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目的探讨人胚胎干细胞(hESs)体外定向诱导分化胰岛细胞移植治疗非肥胖-联合免疫缺陷(NOD/SCID)糖尿病小鼠的可行性。方法体外分四阶段诱导hESs定向分化为胰岛细胞:第一阶段,予以活化素A(activin A)、渥曼青霉素(wortmannin)诱导分化形成定型内胚层;第二阶段,予以全反式维甲酸(RA)、NOGGIN、碱性成纤维细胞生长因子(bFGF)诱导胰腺细胞定向分化;第三阶段,予以表皮生长因子(EGF)扩增胰腺祖细胞;第四阶段,予以尼克酰胺(nicotinamide)、唾液素4(exendin-4)、bFGF及骨形成蛋白(BMP4)促进胰岛细胞成熟;观察诱导各阶段细胞形态变化、免疫荧光鉴定胰十二指肠同源异型盒基因(PDX-1)、胰高糖素、胰岛素、C肽、葡萄糖转运子2(Glut-2)的表达;四阶段分化成熟的胰岛细胞体外检测胰岛素释放反应并植入链脲菌素(STZ)诱导形成的NOD/SCID糖尿病小鼠一侧附睾脂肪垫内,观察血糖变化。结果诱导第四阶段14天时hESs出现胰高糖素荧光表达;20天时细胞出现PDX-1和C肽共表达;22天形成的成熟胰岛细胞出现Glut-2和胰岛素的阳性表达;流式鉴定胰岛素阳性细胞占17.1%,C肽阳性细胞占3.8%;体外检测有葡萄糖刺激的胰岛素释放反应。分化成熟胰岛细胞约(3～5)×106植入NOD/SCID糖尿病小鼠体内可以逆转其高血糖至少8周。结论体外定向诱导hESs分化形成的胰岛细胞植入NOD/SCID糖尿病小鼠附睾脂肪垫内可以逆转其高血糖。
Objective To investigate the feasibility of human embryonic stem cells (hESs) directed differentiation of islet cells transplantation in non-obese - combined immunodeficiency (NOD / SCID) diabetic mice. Methods The hESs were induced to differentiate into islet cells in four phases in vitro. In the first phase, activin A and wortmannin were induced to differentiate into definitive endoderm. In the second phase, all-trans retinoic acid RA), NOGGIN and basic fibroblast growth factor (bFGF) induce the differentiation of pancreatic cells. In the third phase, pancreatic progenitor cells were induced by epidermal growth factor (EGF). In the fourth phase, nicotinamide and saliva The expression of PDX-1, pancreatic isoform of pancreaticoduodenal homeobox gene (PDX-1) was identified by immunofluorescence in order to observe the morphological changes of cells induced by exendin-4, bFGF and BMP4. Glucagon, Insulin, C-peptide, Glucose transporter 2 (Glut-2) were detected in vitro. Insulin-releasing reaction was induced in vitro by four-phase differentiated islet cells and implanted with streptozotocin (STZ) Mouse epididymal fat pad, observed changes in blood glucose. Results Glucose-induced expression of hESs was observed in the 14th day of the fourth phase. PDX-1 and C-peptide were co-expressed in the cells at day 20, Glut-2 and insulin were expressed in 22-day mature islets, Positive cells accounted for 17.1%, C peptide positive cells accounted for 3.8%; in vitro detection of glucose-stimulated insulin release response. Differentiation of mature islet cells about (3 ~ 5) × 106 Implanted NOD / SCID diabetic mice can be hyperglycemia reversed for at least 8 weeks. Conclusion The islet cells formed by differentiation of hESs induced in vitro can reverse the hyperglycemia in the epididymal fat pad of NOD / SCID diabetic mice.