γ-分泌酶在神经干细胞分化过程中的调节作用

Regulatory effects of gamma-secretase in neural stem cell differentiation

背景:Notch信号通路在神经干细胞分化过程中起着关键作用,而γ-分泌酶是Notch信号通路调节的核心环节。目的:对神经干细胞分化过程中γ-分泌酶的活性、酶解产物Notch胞内段的生成量以及活性中心早老素1的表达进行检测。设计、时间及地点:细胞学基因水平检测,于2008-10/2009-01在山西医科大学生物化学与分子生物学实验室完成。材料:清洁级胚龄15d的BALB/c胎鼠由中国药品生物制品检定所提供,质粒Notch1△E-GVP和MH100由瑞典斯德哥尔摩诺贝尔医学学会的Urban Lendahl教授惠赠,荧光素酶质粒pRL-CMV为Promega公司产品。方法:体外分离培养胎鼠脑皮质神经干细胞,传至第5代后,按5×104/cm2密度接种到24孔板,每孔转染200ng MH100,100ng Notch1△E-GVP和2ng pRL-CMV质粒。主要观察指标:利用Gal4-VP16/UAS系统和双荧光素酶报告基因系统测定神经干细胞分化过程中γ-分泌酶的活性;通过Western blot技术检测酶解产物Notch胞内段的生成量;采用实时荧光定量PCR法测定γ-分泌酶活性中心早老素1mRNA的表达。结果:在γ-分泌酶抑制剂DAPT作用下,γ-分泌酶活性呈剂量依赖性降低,与对照组相比,0.1μmol/L DAPT即可对γ-分泌酶活性产生明显的抑制作用(P<0.05),至50μmol/L DAPT时γ-分泌酶活性降低近100倍(P<0.001)。Notch胞内段的生成量也同步减少,与DAPT抑制γ-分泌酶活性呈剂量依赖性的结果一致。与对照组相比,早老素1mRNA表达水平经0.1μmol/L DAPT干预后开始升高,并随着DAPT浓度增加而逐渐升高。结论:神经干细胞分化过程中,γ-分泌酶抑制剂干预后γ-分泌酶活性与酶解产物生成量呈剂量依赖性降低,活性中心基因表达则呈反馈性的同步增高。

BACKGROUND： Notch signaling pathway plays an important role in the differentiation of neural stem cells （NSCs）, while y-secretase is the key element in the accommodation of Notch signaling pathway. OBJECTIVE： To detect the activity, enzymatic product Notch expression and presenilin 1 expression of γ-secretase during NSC differentiation. DESING, TIME AND SETTING： The cytology, gene study was performed at the Laboratory of Biochemistry and Molecular Biology, Shanxi Medical University from October 2008 to January 2009. MATERIALS： Clean BALB/c fetal mice at embryonic day 15 were obtained from National Institute for the Control of Pharmaceutical and Biological Products. Plasmids Notch1 △E-GVP and MH100 were gifted by Professor Urban Lendahl from the Swedish Medical Association, Stockholm, Sweden. Luciferase plasmid pRL-CMV was purchased from Promega Company. METHODS： NSCs from fetal rat cerebral cortex were in vitro isolated. At the fifth passage, NSCs were incubated in a 24-well plate at a density of 5 × 10^4/cm2. NSCs in each well were transfected with 200 ng MH100, 100 ng Notch1 △E-GYP and 2 ng pRL-CMV plasmid. MAIN OUTCOME MEASURES： The activity of γ-secretase was detected by Ga14-VP16/UAS system and Dual Luciferase Reporter Assay System. The productions of the intracellular domain of Notch1 （NICD） were analyzed by Western blot assay. Presenilin 1 mRNA expression was determined by Real-Time fluorescent quantitative PCR. RESULTS： y-secretase inhibitor DAPT decreased luciferase activities in a dose-dependent manner. Compared with the control group, 0.1 μmol/L DAPT could significantly inhibit y-secretase activity （P 〈 0.05）. At 50 μ mol/L DAPT, y-secretase activity could decrease about 100 times （P 〈 0.001 ）. DAPT inhibited the productions of NICD, which showed a dose-dependent relation with inhibition of γ-secretase activity. Compared with the control group, Presenilin 1 mRNA expression began to increase following 0.1 μmol/L DAPT intervention, and gradually increased with the increased DAPT concentration. CONCLUSION： Following γ-secretase inhibitor DAPT intervention, γ-secretase activity and enzymatic product decreased with dose-dependent relationship, but gene expression increased.