糖原合成酶激酶3β抑制剂CHIR99021诱导人胚胎干细胞分化为限定性内胚层细胞

Glycogen synthase kinase 3β inhibitor CHIR99021 induces human embryonic stem cells to differentiate into definitive endoderm cells

目的探讨糖原合成酶激酶(GSK)3β抑制剂CHIR99021诱导人胚胎干细胞分化为限定性内胚层细胞的可行性。方法将人胚胎干细胞H1细胞置于无滋养层培养基(Essential 8)培养。采用免疫荧光法检测人胚胎干细胞多能性标志物Oct4、Nanog的表达。于细胞中加入0.33、1.00、3.00、9.00、27.00μmol/L浓度的CHIR99021培养3 d,以不加CHIR99021的细胞作对照。采用荧光定量聚合酶链反应(PCR)检测细胞多能性相关基因OCT4、SOX2和限定性内胚层相关基因GATA4、SOX17的表达水平。实验数据比较采用单因素方差分析和LSD-t检验。结果免疫荧光法能够检测到人胚胎干细胞多能性标志物Oct4、Nanog的表达。经0.33、1.00、3.00μmol/L CHIR99021处理后的人胚胎干细胞生长状态良好,CHIR99021浓度较高时细胞生长状态较差或死亡。经1.00、3.00μmol/L CHIR99021处理后的细胞多能性相关基因OCT4表达均下调(LSD-t=-40.54,-59.12;P<0.05),SOX2表达亦明显下调(LSD-t=-20.46,-3.87;P<0.05)。经1.00、3.00μmol/L CHIR99021处理后的细胞限定性内胚层相关基因GATA4表达上调(LSD-t=137.21,65.29;P<0.05),SOX17表达亦明显上调(LSD-t=50.93,6.56;P<0.05)。结论人胚胎干细胞应用无滋养层培养仍然保持良好的干细胞生物学特性。CHIR99021可以高效诱导人胚胎干细胞向限定性内胚层细胞分化。

Objective To investigate the feasibility of glycogen synthase kinase （GSK） 3βinhibitor CHIR99021 induces human embryonic stem cells to differentiate into definitive endoderm cells. Methods Human embryonic stem cells H1 cells were cultured in feeder-free medium （Essential 8）. The expression of human embryonic stem cell pluripotency markers Oct4, Nanog were detected by immunolfuorescence. Human embryonic stem cells were treated with CHIR99021 of different concentrations （0.33, 1.00, 3.00, 9.00, 27.00μmol/L） and cultured for 3 d respectively. The untreated cells were taken as control. The expression levels of pluripotency related genes OCT4, SOX2 and deifnitive endoderm related genes GATA4, SOX17 were detected by lfuorescent quantitative polymerase chain reaction （PCR）. The experimental data were compared using one-way analysis of variance and LSD-t test. Results The expression of human embryonic stem cell pluripotency markers Oct4, Nanog could be detected by immunofluorescence. The human embryonic stem cells were observed in good growth when treated with CHIR99021 of different concentrations （0.33, 1.00, 3.00μmol/L） . The human embryonic stem cells were observed in poor growth or dead when treated with the higher concentrations of CHIR99021. The expression of pluripotency related gene OCT4 decreased after treated with CHIR99021 of 1.00, 3.00μmol/L concentration （LSD-t=-40.54,-59.12;P〈0.05）. The expression of SOX2 also decreased significantly （LSD-t=-20.46,-3.87;P〈0.05）. The expression of definitive endoderm related genes GATA4 increased after treated with CHIR99021 of 1.00, 3.00μmol/L concentration （LSD-t=137.21, 65.29;P〈0.05）. The expression of SOX17 also increased significantly （LSD-t=50.93, 6.56;P〈0.05）. Conclusions Human embryonic stem cells can maintain a good pluripotent state in the feeder-free culturing system. CHIR99021 can effectively induce the human embryonic stem cells to differentiate into deifnitive endoderm cells.