体外诱导人羊膜上皮细胞分化为胰岛素分泌细胞的研究

Study on inducing differentiation of human amniotic epithelial cells into insulin secreting cells in vitro

目的:通过体外诱导分化实验,探讨人羊膜上皮细胞(hAECs)向胰岛素分泌细胞(ISCs)分化的能力。方法:采用胰蛋白酶消化法从人羊膜组织分离提取hAECs,用流式细胞仪和免疫细胞化学法进行鉴定。取第3代hAECs在含尼克酰胺和N2补充物的无血清培养基中诱导培养,分别于诱导不同时间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:①hAECs高表达CD29、CD73、CD166和CK19;②hAECs诱导组第7、142、1天胰岛素阳性细胞百分率分别为74.00%±1.73%、75.33%±1.15%和75.67%±0.58%,而对照组未见胰岛素阳性细胞;③hAECs诱导组第7、14、21天培养物上清液中胰岛素含量分别达(328.47±3.22)μIU/ml、(332.26±1.22)μIU/ml和(329.68±2.57)μIU/ml,均显著高于对照组(P均<0.01);④hAECs诱导前后均有PDX-1 mRNA和β2微球蛋白表达,胰岛素mRNA表达仅见于诱导组。结论:hAECs能分化为ISCs,在Ⅰ型糖尿病细胞移植治疗方面具有潜在应用前景。

Objective： To investigate the differentiation of human amniotic epithelial cells（hAECs） into insulin secreting cells（ISCs） in vitro.Methods： The hAECs were isolated from human amnion by trypsin digestion,and the phenotype of the isolated cells were identified by flow cytometry and immunocytochemical staining.The hAECs at passage 3 were treated with nicotinamide and N2 supplement to investigate their differentiation into ISCs.At different times after differentiation,the expression of insulin and β2 microglobulin（β2-MG） was determined by immunocytochemical staining,while the content of insulin in supernatant from cultured hAECs was detected by radioimmunoassay and the expressions of insulin, pancreatic and duodenal homeobox factor-1（PDX-1） mRNA were detected by reverse transcriptase-polymerase chain reaction（RT-PCR）.Results： ①hAECs expressed high percent of CD29,CD73,CD166 and CK19.②At 7,14 and 21 days,the percentages of insulin-positive cells in induced groups were 74.00%±1.73%、75.33%±1.15%和75.67%±0.58% respectively,which were negative in control groups.③At 7,14 and 21 days,contents of insulin in supernatant from induced groups were（328.47±3.22）μIU/ml,（332.26±1.22）μIU/ml and（329.68±2.57）μIU/ml respectively,they were significantly higher than those in control groups（All P0.01）.④PDX-1 mRNA and β2-MG were expressed before and after the induction of hAECs,but insulin mRNA was expressed only in the induced groups.Conclusion： hAECs can differentiate into ISCs,having the potential application for therapy of type Ⅰ diabetes.