CAJ | 학술논문

目的探讨γ-分泌酶抑制剂3,5-二氟苯乙酰-L-丙氨酰-S苯基甘氨酸t-丁酯(DAPT)与神经干细胞增殖、分化的关系.方法分离和体外培养大鼠脑神经干细胞(NSC),采用γ-分泌酶抑制剂DAPT处理后,细胞计数和CCK8检测法观察NSC增殖能力的变化;特异性荧光免疫染色检测NSC诱导定向分化能力的变化;逆转录聚合酶链反应(RT-PCR)检测Notch信号途径节点分子基因RBP-Jk和Hes1的表达.结果 DAPT能够明显抑制NSC的增殖能力;与对照组相比,NSC诱导分化为神经元和少突胶质细胞的比例分别由(3.7±1.0)%和(4.8±1.2)%上升为(13.8±1.2)%和(14.8±1.6)%,而分化成星形胶质细胞的比例由(82.8±3.7)%下降为(63.4 ±1.2)%;DAPT能下调NSC的RBP-Jk和Hes1 mRNA的表达.结论 γ-分泌酶抑制剂DAPT能抑制大鼠脑NSC的增殖,改变NSC定向分化的能力,这些作用可能是通过抑制γ-分泌酶而下调Notch信号所致.
목적 탐토γ-분비매억제제3,5-이불분을선-L-병안선-S분기감안산t-정지(DAPT)여신경간세포증식、분화적관계.방법 분리화체외배양대서뇌신경간세포(NSC),채용γ-분비매억제제DAPT처리후,세포계수화CCK8검측법관찰NSC증식능력적변화;특이성형광면역염색검측NSC유도정향분화능력적변화;역전록취합매련반응(RT-PCR)검측Notch신호도경절점분자기인RBP-Jk화Hes1적표체.결과 DAPT능구명현억제NSC적증식능력;여대조조상비,NSC유도분화위신경원화소돌효질세포적비례분별유(3.7±1.0)%화(4.8±1.2)%상승위(13.8±1.2)%화(14.8±1.6)%,이분화성성형효질세포적비례유(82.8±3.7)%하강위(63.4 ±1.2)%;DAPT능하조NSC적RBP-Jk화Hes1 mRNA적표체.결론 γ-분비매억제제DAPT능억제대서뇌NSC적증식,개변NSC정향분화적능력,저사작용가능시통과억제γ-분비매이하조Notch신호소치.
Objective To investigate the role of N-[N-(3,5-Difluorophenacetyl-L-alanyt)-S-phenylglycine t-butyl ester(DAPT),a gamma-secretases inhibitor,on the proliferation and differentiation of neural stem cells(NSCs).Methods NSCs were isolated from Sprague-Dawley rat brain,cuhured,and treated with DAPT for 6 weeks.Cell counting was conducted every 24 h.CCK8 assay was used to draw the growth curve.Immunonuoresence staining was performed to observe the proportions of β-tubulin Ⅲ positive cells(neurons),glial fibrillary acidic protein (GFAP) positive cells (astrocytes),and 2',3'-cyclic nucleotide3' phosphohydrolase (CNPase) positive cells (oligodentrocytes).RT-PCR was employed to assay the mRNA expression of RBP-Jk and Hesl genes,downstream genes of the Notch pathway.Results Cell counting and CCK-8 assay showed that DAPT reduced the rate of NSC proliferation.Addition of DAPT altered NSC differentiation in vitro,percentage of The proportions neurons of the DAPT group was(13.84±1.22)%,significantly higher than that ofthe control group[(3.7±1.04)%,P＜0.01],the proportion of the oligodendrocytes of the DAPT group was(14.75±1.58)%,significantly higher than that pf the control group[(4.8±1.22)%,P＜0.01].However,the proportion of astrocytes of the DAPT group was (63.41±1.20)%,significantly lower than that of the control group[(82.84±3.68)%,P＜0.01].The expression levels of RBP-Jk and Hes1 mRNA(RBP-Jk/GAPDH and Hes1/GAPDH)in the NSC treated with DAPT were 0.52±0.13 and 0.66±0.18 respectively.both significantly lower than those of the control group(0.28±0.06 and 0.16±0.08 respectively,both P＜0.05).Conclusion DAPT inhibits the NSC proliferation and alters the NSC committed differentiation.These effects are mediated via Notch signaling down regulation as a result of the inhibition of gamma-secretase.