摘要
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.
出处
《中国应用生理学杂志》
CAS
CSCD
2012年第2期139-143,I0002,共6页
Chinese Journal of Applied Physiology
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)
关键词
人羊膜上皮细胞
胰岛素分泌细胞
诱导分化
human amniotic epithelial cell
insulin secreting cells
differentiation