LY294002与Exendin-4体外诱导人胚胎干细胞分化为胰岛素分泌细胞

Differentiation of insulin-secreting cells derived from human embryonic stem cells induced by LY294002 or Exendin-4 in vitro

摘要:

目的：探讨磷脂酰肌醇3激酶（phosphatidylinositol 3 -kinase，PI3K）抑制剂LY294002与胰升血糖素样肽-1（glucagon-like peptide-1，GLP-1）拟似物Exendin-4体外诱导人胚胎干细胞（human embryonic stem cells，HESc）分化为成熟胰岛素分泌细胞的影响。方法：利用“五步法”包括：HESc体外培养；拟胚体（embryoid bodies，EBs）形成；筛选巢蛋白阳性细胞；扩增巢蛋白阳性细胞；第5阶段分别加入PI3K抑制剂LY294002为实验组（LY组）和GLP-1拟似物Exendin-4为对照组（Ex组）诱导HESc定向分化。光镜下观察各阶段细胞分化特点，细胞免疫荧光染色鉴定胰岛样细胞特异蛋白表达。结果：C肽、胰高血糖素、生长抑素单染细胞阳性率及胰岛素/生长抑素、胰岛素/C肽双染细胞阳性率Ex组均高于LY组：C肽（85.7±2.1）% vs. （74.5±2.7）%，P=0.005；胰高血糖素（38.1±3.4）% vs.（27.2±2.4）%，P=0.018；生长抑素（75.4±3.7）% vs.（31.3±4.7）%，P=0.000；胰岛素/生长抑素（21.5±2.2）% vs. （3.5±0.9）%，P=0.000；胰岛素/C肽（87.5±2.4）% vs. （76.7±3.1）%，P=0.013。结论：在无血清培养体系下，LY294002较Exendin-4诱导HESc分化的胰岛素分泌细胞更加成熟。

Abstract:

Objective：To investigate whether adding different inducible factors：phosphatidylinositol 3-kinase（PI3K） inhibitor LY294002 and long-acting glucagon-like peptide-1（GLP-1） mimetic Exendin-4 could efficiently induce human embryonic stem cells（HESc） to differentiate into mature insulin-producing cells. Methods：A five-stage culturing strategy was used in this study. Human embryonic stem cells were amplified in vitro for 6－7 d at stage 1. Then，the HESc were cultured in the condition of suspension culture for forming embryoid bodies（EBs）（stage 2）. The nesting positive cells were selected in condition of ITSF medium（stage 3） and amplified in N2 and B27 medium（stage 4）. Finally，LY294002 was taken as experimental（group LY） and Exendin-4 was taken as control group（group Ex），which was utilized to induce the nesting positive cells into mature insulin-producing cells. The morphology and char－acteristics of each stage were observed under the microscope，and the specific markers were identified by immunofluorescence staining. Results：The positive rates of C-peptide staining（85.7±2.1） % vs. （74.5±2.7）%，P=0.005；somatostatin staining（75.4±3.7）% vs. （31.3±4.7）%，P=0.000；glucagon staining（38.1±3.4） % vs. （27.2±2.4）%，P=0.018；insulin/c-peptide（87.5±2.4）% vs. （76.7±3.1）%，P=0.013 and insulin/somatostatin co-staining（21.5±2.2）% vs. （3.5±0.9）%，P=0.000 in group Ex were significantly higher than those of group LY. Conclusion：LY294002 could induce HESc to differentiate into more mature insulin-produc－ing cells in serum-free culture medium.