脑源性神经营养因子在体外诱导SH-SY5Y细胞分化过程中的作用

目的 探讨不同浓度的脑源性神经营养因子(BDNF)在体外诱导SH-SY5Y细胞分化时,对G2期及其前后细胞周期相关蛋白mRNA表达的影响.方法 采用全反式维甲酸(RA)和低(1μg/L)、中(10μg/L)、高(100μg/L)3种不同浓度BDNF在无血清培养液中相继诱导SH-SY5Y细胞分化,形成均一的具有神经元形态的细胞.以一步法实时定量RT-PCR检测其G2期细胞周期相关蛋白mRNA的表达,荧光激活细胞分类术(FACS)检测各组细胞时相.结果 在各浓度BDNF组中周期蛋白B1(cyclin B1)的mRNA含量与对照组及RA组比较均明显降低(P<0.05);仅高浓度组cyclin B2和周期蛋白依赖性激酶1(Cdk1)的mRNA含量显著降低(P<0.05),;低浓度和中浓度BDNF组Cdk5 mRNA含量均高于RA组(P<0.05).BDNF各浓度组处于G1期的细胞百分率均显著高于RA和对照组(P<0.01),而处于S期的细胞百分率均显著低于对照组(P<0.01),且低浓度及高浓度组同时低于对照组(P<0.01).结论 提示BDNF在有效促进SH-SY5Y细胞进一步分化的同时,没有引起G2期及其前后细胞周期相关蛋白mRNA表达的异常升高,BDNF无诱导细胞凋亡的危险,可能有减缓AD进程的作用,并且呈剂量依赖性.

THE EFFECTS OF BRAIN-DERIVED NEUROTROPHIC FACTOR ON THE INDUCE-DIFFERENTIATED SH-SY5Y CELLS IN VITRO

Objective To study the effects of different contents of brain-derived neurotrophic factor(BDNF) on the expression of cell cycle\|related proteins in G2 phase in differentiated SH\|SY5Y cells in vitro. Methods Sequential exposure of SH\|SY5Y cells to retinoic acid(RA) for 5 days and various contents of brain\|derived neurotrophic factor(BDNF 1μg/L, 10μg/L, 100μg/L) for 2 days yielded homogeneous populations of cells with neuronal morphology. At the same time, control group was incubated in insulin-transferrin selenium-X(ITS) alone for 7 days; RA group was incubated in RA only for 7 days. All media were prepared with DMEM\|F12 containing 1% ITS without serum. Real time one\|step RT\|PCR was carried out to determine the expresson of cell cycle\|related protein in G2 phase. Fluorescence activated cell sorting (FACS) was used to analyze the phase of cell cycle. Results The results were shown that the expression of cyclin B1 was downregulated significantly comparing to RA group and ITS group (P<0.05) in all BDNF groups; The expressions of cyclin B2 and Cdk1 were decreased only in 100μg/L BDNF group comparing to ITS group and RA group(P<0.05)； No change of the expression of Cdk5 was found in all BDNF groups comparing to ITS group. The cell percent of G1 phase in all BDNF groups was increased significantly comparing to RA group and ITS group(P<0.01); the cell percent of S phase was decreased significantly comparing to RA group (P<0.01) in all BDNF groups, and decreased significantly comparing to ITS group in 1μg/L BDNF group and 100μg/L BDNF group(P<0.01).Conclusion The results suggest that BDNF doesn’t enhance the mRNA level of cell cycle\|related proteins of G2 phase, combining the change of cell percent in G1 and S phase certifies that