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目的探讨单层细胞分化法结合Smad信号双抑制剂,诱导人诱导多能干细胞(hi PSCs)分化为神经干细胞,并进一步分化为运动神经元的可行性。方法无血清E8培养基培养hi PSCs,当其接近70%融合度时改用神经诱导培养基,并加入SB431542和DMH1。9 d后将贴壁细胞消化,在含b FGF和EGF的培养基中悬浮培养,免疫荧光检测神经干细胞标记物的表达。在神经干细胞培养液中添加视黄酸(RA)和音猬因子(SHH),免疫荧光检测各分化阶段运动神经元相关标记物的表达。结果经9 d神经诱导,hi PSCs分化为表达Nestin、Sox1和Sox2的神经干细胞。神经干细胞在RA和SHH等的作用下进一步分化为运动神经元,免疫荧光检出各不同阶段标志物的表达。结论单层分化法结合Smad信号双抑制剂,可将hi PSCs诱导为神经干细胞,并在RA和SHH等作用下进一步分化为运动神经元。
Objective To investigate the feasibility of inducing the differentiation of human induced pluripotent stem cells (hpSCs) into neural stem cells and further differentiate into motor neurons by monolayer differentiation combined with Smad double inhibitors. Methods hi PSCs were cultured in serum-free E8 medium, and when it was near 70% confluency, the neural induction medium was used instead. When SB431542 and DMH were added for 1.9 days, adherent cells were digested and cultured in medium containing bFGF and EGF Suspension culture, immunofluorescence detection of neural stem cell marker expression. Retinoic acid (RA) and sonicated sonic hedgehog (SHH) were added to the culture medium of neural stem cells, and the expression of related markers of motor neuron in different stages of differentiation was detected by immunofluorescence. Results After induced for 9 d, hi PSCs differentiated into neural stem cells expressing Nestin, Sox1 and Sox2. Neural stem cells were further differentiated into motor neurons under the action of RA and SHH, and the expression of different stages of markers was detected by immunofluorescence. Conclusion Monolayer differentiation combined with Smad double inhibitor can induce hi PSCs to become neural stem cells and differentiate into motor neurons under the action of RA and SHH.