人胚胎干细胞体外定向诱导分化胰腺前体细胞及胰岛细胞移植治疗NOD/SCID糖尿病小鼠  

Transplantation of human embryonic stem cells derived pancreatic progenitors and islets corrects diabetes in NOD/SCID mice

作　　者：华秀峰[1] 孙强[4] 李华峰[1] 孟晓梅[1] 王延伟[2] 于胜强[3] 丛晋[1] 刘芙君[2] 靳少华[2] 王海燕[2] 李建远[2] 唐与晓[1] 

机构地区：[1]烟台毓璜顶医院内分泌科,264000 [2]烟台毓璜顶医院中心实验室,264000 [3]烟台毓璜顶医院泌尿外科,264000 [4]中国人民解放军海军烟台医院骨科,264000

出　　处：《中华内分泌外科杂志》2014年第1期57-61,共5页Chinese Journal of Endocrine Surgery

基　　金：烟台市科学技术发展计划资助项目（2008142-9,2011204）

摘　　要：目的 探讨人胚胎干细胞（human embryonic stem cells,hESCs）体外定向诱导分化胰腺前体细胞及胰岛细胞移植治疗非肥胖糖尿病/严重联合免疫缺陷（non-obese diabetic/severe combined immunodeficient,NOD/SCID）小鼠的可行性.方法 体外分4阶段诱导hESCs定向分化为胰岛细胞：①诱导分化形成定型内胚层;②诱导胰腺细胞定向分化;③扩增胰腺前体细胞;④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（glucagon）、胰岛素（insulin）、C肽（C-peptide）、葡萄糖转运子2（Glut-2）的表达.3阶段及4阶段分化形成的细胞分别植入链脲菌素（streptozotocin,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 hESCs诱导4阶段细胞表达胰高血糖素、胰岛素、Glut-2;共表达PDX-1和C肽;流式鉴定诱导4阶段胰岛素阳性细胞为17.1％;体外检测有葡萄糖刺激的胰岛素释放反应;3阶段及4阶段分化形成的细胞分别植入NOD/SCID 糖尿病小鼠体内可逆转其高血糖至少12周.结论 体外定向诱导hESCs分化形成的胰腺前体细胞及胰岛细胞分别植入NOD/SCID小鼠可逆转其高血糖.Objective To investigate whether pancreatic progenitors and islets differentiated from human embryonic stem cells（hESCs） could correct hyperglycemia in non-obese diabetic/severe combined immunodeficient （NOD/SCID） mice.Methods We obtained pancreatic progenitors and islets derived from hES cells line YT1 according to the optimized four-stage differentiation protocol.Stage 1：definitive endoderm formation; Stage 2：pan creatic specialization; Stage 3：amplification of pancreatic progenitors,Stage 4：maturation of pancreatic islets.To observe the morphological changes of each stage and immunofluorescent expression of pancreatic and duodenal homeobox gene（PDX-1）,glucagon,insulin,C-peptide,glucose transporter-2（Glut-2）.The differentiated cells from stage 3 and stage 4 were then transplanted into one of the epididymal fat pads（EFP） of NOD/SCID mice.The survival and function of the graft were measured by immunohistochemistry and blood glucose monitor.Results The stage 4-differentiated pancreatic islets expressed mature β cell-specific markers such as glucagon,insulin and Glut 2,and even PDX-1 and C-peptide-double-positive.The stage 4-pancreatic islets had nearly 17.1％ insulin-positive cells as assayed by flow cytometry analysis.Differentiated pancreatic islets released insulin/C-peptide in response to glucose stimulation.After implantation into EFP of NOD/SCID mice,hES cell-derived human pancreatic progenitors and islets corrected hyperglycemia for at least 12 weeks.Conclusions Pancreatic progenitors and islets differentiated from hESCs can correct hyperglycemia in NOD/SCID mice.

关 键 词：胚胎干细胞 分化 胰腺前体细胞 胰岛细胞 胰岛素 C肽 NOD SCID 

分 类 号：R587.1[医药卫生—内分泌]