成年和幼年大鼠海马微环境星形胶质细胞对神经干细胞增殖的作用

Effects of astrocytes on the proliferation of neural stem cells in the hippocampal microenvironment of old and young adults

目的 探讨成年和幼年大鼠海马微环境中星形胶质细胞对神经干细胞(NSCs)增殖的作用及其机制。方法 分别取出生后1 d及30周的雌性SD大鼠各5只,分离培养海马星形胶质细胞;孕15 d SD大鼠1只分离培养胚胎海马NSCs;收取星形胶质细胞条件培养基(CM)用于培养NSCs。应用流式细胞术和CCK-8检测星形胶质细胞CM培养下NSCs增殖情况;收取星形胶质细胞CM进行质谱分析筛选神经发生相关的差异表达蛋白集落刺激因子1 (CSF-1);应用Western blotting和ELISA验证质谱结果;应用免疫荧光、流式细胞术和CCK-8等技术检测不同浓度CSF-1重组蛋白(20μg/L、100μg/L、1 mg/L及5 mg/L)处理后NSCs增殖情况。结果 与成年组海马星形胶质细胞CM相比,幼年组星形胶质细胞CM可促进NSCs增殖(P<0.01);与幼年组海马星形胶质细胞CM相比,成年组海马中CSF-1表达明显上调(P<0.01);20μg/L CSF-1可促进NSCs增殖(P<0.01),5 mg/L CSF-1明显抑制NSCs增殖(P<0.01)。结论 随着年龄增长,海马微环境中星形胶质细胞分泌CSF-1增加,可调控NSCs增殖。

Objective To investigate the effect and mechanism of astrocytes on the proliferation of neural stem cells(NSCs)in adult and juvenile hippocampus microenvironment.Methods Hippocampal astrocytes were isolated and cultured from 5 female SD rats at day 1 and week 30 postnatal,respectively;Embryonic hippocampus NSCs was isolated and cultured from 1 SD rat at day 15 of gestation;Conditioned astrocyte culture medium(CM)was collected for NSCs culture;Flow cytometry and CCK⁃8 were used to detect the proliferation of NSCs cultured in CM.Colony stimulating factor 1(CSF⁃1)with differential expression was screened by mass spectrometry after cultured astrocyte CM.Western blotting and ELISA were used to verify the result of mass spectrometry.Immunofluorescence,flow cytometry and CCK⁃8 were used to detect the proliferation of NSCs treated with different concentrations of CSF⁃1 recombinant protein(20μg/L,100μg/L,1 mg/L and 5 mg/L).Results Compared with the adult group,the CM of hippocampal astrocytes in the young group could promote the proliferation of NSCs(P<0.01);Compared with the conditioned medium of hippocampal astrocytes in the juvenile group,the expression of CSF⁃1 in the hippocampus of the elder group was significantly up⁃regulated(P<0.01);At 20μg/L,CSF⁃1 promoted the proliferation of NSCs(P<0.01),and 5 mg/L CSF⁃1 inhibited significantly the proliferation of NSCs(P<0.01).Conclusion The secretion of CSF⁃1 by astrocytes in hippocampal microenvironment can regulate the proliferation of NSCs with the development of the times.