体外诱导人羊膜间充质干细胞分化为胰岛素分泌细胞  

Inducing differentiation of human amnion-derived mesenchymal stem cells into insulin-secreting cells in vitro

作　　者：赵玉洁[1] 方宁[1] 陈代雄[1] 余丽梅[1] 喻皇飞[1] 万卫红[1] 赵春华[2] 

机构地区：[1]遵义医学院附属医院贵州省细胞工程重点实验室,563003 [2]中国医学科学院基础医学研究所组织工程中心

出　　处：《中华内分泌代谢杂志》2011年第12期1013-1016,共4页Chinese Journal of Endocrinology and Metabolism

基　　金：“重大新药创制”国家科技重大专项（2009ZX09503-025）贵州省科技计划发展项目（黔科合字2051号;黔科合SZ字3017号）

摘　　要：目的 探讨体外诱导人羊膜间充质干细胞（hAD-MSCs）向胰岛素分泌细胞分化潜能.方法 采用胰蛋白酶-胶原酶消化法分离提取hAD-MSCs,流式细胞术分析和免疫细胞化学染色行表型鉴定.取第3代按2.5× 106个/ml或5×105个/ml细胞密度接种6孔培养板或预置盖玻片的24孔培养板,在含10mmol/L尼克酰胺和N2补充物的无血清HG-DMEM培养基培养,未诱导组为含10％胎牛血清的LG-DMEM基础培养基.分别于体外诱导第7、14、21天采用免疫细胞化学法检测胰岛素和β2微球蛋白的表达,采用放射免疫法检测上清液中胰岛素含量,采用RT-PCR检测胰岛素mRNA和胰十二指肠同源异型盒因子1（PDX-1）mRNA的表达.结果 （1）hAD-MSCs高表达间充质干细胞表面标志CD29、CD44、CD73、CD166及波形蛋白；（2） hAD-MSCs诱导第7、14、21天胰岛素阳性细胞百分率分别为74.67％±1.53％、75.00％±1.00％、74.33％±1.53％,培养物上清液中胰岛素含量分别为（331.62±1.76）、（330.50±1.22）和（331.65±0.48） μIU/ml,各时点间比较无显著性差异（均P＞0.05）,而未诱导组仍未见胰岛素阳性细胞；培养上清也未检测到胰岛素；（3） hAD-MSCs诱导前后均有PDX-1 mRNA和蛋白表达,胰岛素基因mRNA表达仅见于诱导组；（4） hAD-MSCs诱导组和未诱导组各时点均有β2微球蛋白表达,其阳性细胞百分率组间差异无显著性（均P＞0.05）.结论 hAD-MSCs具有向胰岛素分泌细胞分化的能力,可能成为1型糖尿病细胞移植治疗的新的细胞供源.Objective To investigate the potential of human amnion-derived mesenchymal stem cells to differentiate into insulin secreting cells in vitro.Methods The hAD-MSCs were isolated from human amnion by trypsin-collagenase digestion.The phenotype of the isolated cells was identified by flow cytometry and immunocytochemical staining.The 3rd generation cells were inoculated at density of 2.5 × 106 unit/ml or 5 × 105 unit/ml in 6 well plates or preset coverslip 24 well plates.Induced groups were treated in serum-free HG-DMEM with 10 mmol/L nicotinamide and N2 supplement.The cells in the non-induced groups were incubated in LG-DMEM supplemented with 10% fetal bovine serum.At days 7,14 and 21 after induction,insulin and β2 microglobulin was determined by immunocytochemical stain,the content of insulin in the culture supernatant was assayed by radioimmunoassay,and insulin mRNA and PDX-1 mRNA were detected by reverse transcriptase-polymerase chain reaction.Results （ 1 ） The hAD-MSCs highly expressed CD29,CD44,CD73,CD166,and vimentin.（ 2 ） At 7,14,and 21 days,the percentages of insulin-positive cells in hAD-MSCs induced groups were 74.67% ± 1.53%,75.00%：1.00%,and 74.33% ±1.53%,respectively.Contents of insulin in the supematant of hAD-MSCs induced groups were （ 331.62 ± 1.76 ）,（ 330.50 ± 1.22 ）,and （ 331.65 ± 0.48 ） μIU/ml,respectively,but non-induced groups were negative.（3） PDX-1 mRNA and PDX-1 protein were expressed before and after the induction of hADMSCs,but insulin mRNA was expressed only in the induced groups.（ 4 ） Both hAD-MSCs induced groups and non-induced groups expressed β2 microglobulin （ all P 〉 0.05 ）.Conclusion The hAD-MSCs have a potential of differentiating into ISCs and thus may become a new cell source of therapy for type 1 diabetes.

关 键 词：人羊膜间充质干细胞 胰岛素分泌细胞 诱导分化 

分 类 号：R587.1[医药卫生—内分泌]