嗅鞘细胞分泌蛋白IGFBP-2对神经干细胞分化的作用

目的:探讨嗅鞘细胞分泌蛋白IGFBP-2在诱导神经干细胞(NSCs)分化过程中的作用.方法:采用新生1 d昆明小鼠嗅球培养嗅鞘细胞(OECs),并制备无血清上清液.C17.2 NSCs置于含10%FBS的DMEM/F12培养基培养,传至第3代,待细胞生长至80%融合时,分别加入OECs无血清上清液(实验组)、H-DMEM/F12培养基(对照组)培养;实验组和对照组均分别加入终浓度为0 ng/mL、125 ng/mL、250 ng/mL、500 ng/mL的IGFBP-2.倒置显微镜下观察诱导后细胞生长情况,于诱导5d后收集各组细胞,行Western blot检测细胞巢蛋白(Nestin)、神经胶质酸性蛋白(GFAP)及 β-微管蛋白Ⅲ(TUJ-1),蛋白激酶1/2(ERK 1/2)表达情况,免疫荧光染色鉴定GFAP,TUJ-1阳性细胞并计数,流式分析GFAP,TUJ-1阳性细胞.结果:Western blot、流式分析示对照组中各浓度组GFAP、TUJ-1表达差异无统计学意义;Western blot示实验组随IGFBP-2浓度升高,GFAP表达增多,TUJ-1表达减少,且加入IGFBP-2后ERK1/2表达迅速增多,免疫荧光鉴定表明,随着IGFBP-2浓度升高,GFAP阳性细胞增多,TUJ-1阳性细胞减少,结果有统计学意义.结论:IGFBP-2可以促进OCM诱导NSCs向星形胶质细胞分化,且与浓度呈正相关,其机制与ERK1/2通路有关.

Effect of OECs secreted protein IGFBP-2 on differentiation of neural stem cells

Objective To study the effect of OECs secreted protein IGFBP-2 on differentiation of neural stem cells. Methods OECs were isloated and cultured from the olfactory bulbs of 1-day-old postnatal mouse to prepare serum-free con-dition medium of OECs. After C17.2 NSCs were cultured with H-DMEM/F12 medium containing 10% FBS and the cell fu-sion reached 80%, the 3rd passage cells were induced by serum-free condition medium of OECs in the experimental group, by H-DMEM/F12 in the control group. Both the experimental group and control group were treated with 0, 100, 300, 500 ng/mL IGFBP-2. The growth condition of cells was observed with inverted microscope. After 5 days, analyzing expression patterns of neuroectodermal stem cell marker (Nestin), glial fibrillary acidic protein (GFAP), neuron specific class Ⅲ beta tubulin (TUJ-1), ERK1/2 by western blot. GFAP and TUJ-1 positive cells were identified and counted by immunofluorescence staining. Mean-while, flow cytometry were uesd to analyse the GFAP and TUJ-1 positive cells. Results In the control group, Western blot and flow cytometry analysis showed that there was no significant difference in the expression of GFAP and TUJ-1 between each concentration group. In the experimental group, Western blot analysis showed that With the increase of IGFBP-2concentration, the expression of GFAP was increased, the expression of TUJ-1 was decreased, and the expression of ERK1/2 increased rapidly after the addition of IGFBP-2. The immunofluorescence staining showed that GFAP positive cells increased and TUJ-1 positive cells decreased with the increase of IGFBP-2 concentration. Conclusion IGFBP-2 can promote the differentiation of NSCs into astrocyte induced by OCM in a dose-related way, and its mechanism is related to ERK1/2 pathway.