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本研究目的为探究γ-分泌酶抑制剂DAPT(3,5-二氟苯乙酰-L-丙氨酰-S苯基甘氨酸t-丁酯)对神经元前体细胞系分化的影响,检验经DAPT诱导产生更多可用于移植的神经元的可能性。培养神经元前体细胞系GE6时,以培养基中加入4μmol/L DAPT的为实验组,不加DAPT的为对照组,分化4d,采用免疫荧光染色的方法分别检测两组细胞Tuj1、GFAP、O4的阳性率,采用qRT-PCR分别检测两组细胞Tuj1mRNA、GFAP mRNA相对表达量。免疫荧光染色表明实验组Tuj1阳性率较对照组增加,而GFAP和O4的阳性率较对照组减少,这些差异均有统计学意义(P<0.05)。qRT-PCR中两组Tuj1和GFAP mRNA的变化趋势与免疫荧光染色结果一致。结果提示DAPT能促进神经元前体细胞系向神经元分化,抑制其向胶质细胞分化,可以用DAPT诱导产生更多的可用于移植的神经元。
The purpose of this study was to investigate the effect of the γ-secretase inhibitor DAPT (3,5-difluorophenylacetyl-L-alanyl-S-phenylglycine t-butyl ester) on the differentiation of neuronal precursor cell lines DAPT induces the possibility of producing more neurons that can be used for transplantation. Cultured neuronal precursor cell line GE6, 4μmol / L DAPT was added into the culture medium as experimental group, without DAPT as control group, and differentiated for 4 days. Immunofluorescence staining was used to detect the expression of Tuj1, GFAP, O4 positive rate, qRT-PCR were used to detect the two groups of cells Tuj1mRNA, GFAP mRNA relative expression. Immunofluorescence staining showed that the positive rate of Tuj1 in experimental group was higher than that in control group, while the positive rate of GFAP and O4 was lower than that in control group, all of these differences were statistically significant (P <0.05). The trend of Tuj1 and GFAP mRNA expression in qRT-PCR was consistent with that of immunofluorescence staining. The results suggest that DAPT can promote the differentiation of neuronal progenitor cells into neurons and inhibit its differentiation into glial cells. DAPT can induce more neurons for transplantation.