摘要
目的探讨不同浓度糖原合酶激酶-3(GSK3)抑制剂CHIR99021对人诱导多能干细胞(i PSC)诱导分化为运动神经元的影响,分析其促进分化的最适浓度。方法在分化早期加入不同浓度CHIR99021培养i PSC 6 d,并依此将其分为对照组(0μmol/L)及1、2、3、4μmol/L组。分别在诱导分化早期、中期、晚期对各组细胞进行光镜观察和免疫荧光鉴定。结果在分化早、中、晚各期,细胞计数显示CHIR99021浓度越高细胞数量越多。早期,0~3μmol/L组神经干细胞标志物SOX1表达量无差异,4μmol/L组低于其余各组(P均<0.05);中期,0~3μmol/L组运动神经元前体标志物Olig2的表达量随CHIR99021浓度增加而增加(P<0.05),而4μmol/L组则稍低于3μmol/L组(P<0.05);晚期,2~4μmol/L组的细胞成功表达成熟运动神经元标志物胆碱乙酰转移酶(Ch AT),而0~1μmol/L组未表达。结论在神经分化的早期应用一定浓度的CHIR99021可以促进i PSC的细胞增殖,提高分化效率,并最终诱导其成为成熟运动神经元,其中3μmol/L为诱导最佳浓度。
Objective To evaluate the effect of different concentrations of glycogen synthase kinase-3（GSK3） inhibitor CHIR99021 upon regulating the differentiation of human induced pluripotent stem cells （iPSCs） into motor neurons, aiming to investigate the optimal concentration. Methods During the early stage of differentiation, iPSCs were co-cultured with different concentrations of CHIR99021 for 6 days and were divided into the control （0 μmol/L）, 1, 2, 3 and 4 μmol/L groups. Immunofluorescence identification was observed under light microscope during the early, middle and advanced stages of induction and differentiation. Results Throughout the early, middle and advanced stages of differentiation, cell counting revealed that the cell quantity was increased along with the elevated concentration of CHIR99021. During the early stage, the expression level of nerve stem cell marker SOX1 did not significantly differ among 0, 1, 2 and 3 μmol/L groups. The expression of SOX1 in the 4 μmol/L group was significantly lower than that in the other groups （all P 〈 0.05）. In the middle stage, the expression level of motor neuron precursor marker Olig2 was significantly increased along with the elevated concentration of CHIR99021 in 0, 1, 2 and 3 μmol/L groups （all P 〈 0.05）. The expression of Olig2 in 4 μmol/L group was slightly lower than that in 3 μmol/L group （P 〈 0.05）. During the advanced stage, the cells in 2, 3 and 4 μmol/L groups expressed mature motor neuron marker choline acetyl transferase （ChAT） rather than in control and 1 μmol/L groups. Conclusion During the early stage of nerve differentiation, a proper concentration of CHIR99021 can promote the proliferation of iPSCs, enhance the efficiency of cell differentiation and eventually induce iPSCs into motor neurons. The optimal concentration of CHIR99021 is 3 μmol/L.
出处
《新医学》
2017年第4期217-223,共7页
Journal of New Medicine
基金
国家自然科学基金(81271327
81570874)
广东省自然科学基金(2015A03013167)
广东省科技计划项目(2014A020211008
2015A020212011)
广州市科技计划项目(1563000227)
