胰岛新生相关蛋白肽方案诱导人脂肪源性干细胞体外分化成为胰岛样细胞团的效果评价  

Evaluation of in vitro differentiation of human adipose-derived stem cells into islet-like cell clusters induced by INGAP-pp protocol

作　　者：任莉莉[1,2] 陈丽娟 齐晖 李富荣 龚非力[1] 

机构地区：[1]华中科技大学同济医学院免疫学系,湖北武汉430030 [2]广东省深圳市人民医院临床医学研究中心,广东深圳518020

出　　处：《吉林大学学报（医学版）》2010年第6期1189-1195,1208,共8页Journal of Jilin University：Medicine Edition

基　　金：深圳市科技计划重点项目资助课题(200901001)

摘　　要：目的:探讨胰岛新生相关蛋白(INGAP)联合细胞因子诱导人脂肪源性干细胞(hASCs)分化为胰岛样细胞团的效果。方法:分离、纯化、鉴定人脂肪源性干细胞。应用胰岛新生相关蛋白肽(INGAP-pp)联合细胞因子进行四步法诱导,RT-PCR、实时定量PCR及细胞免疫荧光检测诱导后细胞胰腺发育及功能基因的表达变化,在不同浓度(5.6和25.0mmol·L-1)葡萄糖刺激下,检测分化细胞胰岛素及C肽分泌量。结果:在诱导过程中,干细胞相关基因Oct3/4表达降低至原始水平的1/20,而与胰腺系分化发育相关的基因(Ck19、nestin、pdx-1及nkx2.2均显著升高。在诱导分化后期,INGAP-pp/Scramble-pp诱导组均出现了胰岛样细胞团,INGAP-pp诱导组细胞团的直径为150～200μm,而Scramble-pp诱导组细胞团的直径为50～100μm,前者更接近天然胰岛直径。免疫荧光检测显示,胰岛样细胞团中存在胰岛素和C肽双阳性的细胞群,对不同浓度的葡萄糖刺激有反应,分泌量约占天然胰岛细胞的1/15～1/10。结论:INGAP-pp诱导方案可诱导hASCs向胰腺系细胞分化,在体外形成具有胰岛素分泌功能的胰岛样细胞团。提示hASCs有望替代胚胎干细胞及骨髓来源间充质干细胞,成为细胞移植治疗糖尿病的种子细胞。Objective To evaluate the effects of the INGAP-related pentadecapeptide（INGAP-PP）combined with chemical molecules and cytokines on the differentiation of human adipose-derived stem cells（hASCs）into islet-like clusters.Methods The hASCs were isolated,purified and expanded in vitro and differentiated using a four-step protocol that included TSA,INGAP-PP/Scrambled peptide（Scrambled-P）,nicotinamide and exendin-4.By using real-time PCR and immunofluorescence technology,the expression levels of genes related with pancreas development were detected.The insulin and C-peptide released by differentiated cells in response to high/low glucose level were detected by ELISA.Results Undifferentiated hASCs expressed oct3/4,key markers of embryo stem cells and at the end of induction,the Oct3/4expression level decreased to 1/20of original.The expression levels of genes involved in early pancreas development（Ck19,nestin,pdx-1,and nkx2.2）were increased in both INGAP-PP/Scrambled peptide cultures significantly.The islet-like clusters were obtained in both INGAP-PP/Scrambled peptide groups.The diameters of clusters in the INGAP-pp group were much closer to native islets, which were about 150-200μm,whereas those in Scramble-pp group were about 50-100μm.Immunocy-tochemical analysis showed C-peptide/insulin positive cells in INGAP-pp induced group but not in undifferentiated hMSCs.Conclusion By INGAP-pp protocol,the hASCs could be differentiated into islet-like clusters secreting the insulin.It suggests that the hASCs could be substitution of embryo or bone-marrow derived stem cells for future cell therapy applications.

关 键 词：胰岛新生相关蛋白 脂肪源性干细胞 分化 胰岛 

分 类 号：Q813.1[生物学—生物工程] R587.1[医药卫生—内分泌]