摘要
目的:探索新生大鼠海马神经干细胞培养及鉴定方法,观察高浓度的皮质酮和抗抑郁剂氟西汀对其增殖的影响。方法:2004-02/06在解放军军事医学科学院毒物药物研究所完成。分离新生第1天大鼠的海马组织,将其制成单细胞悬液后置于无血清条件培养基中进行培养,用免疫细胞化学技术对所培养的细胞特性进行鉴定,用四甲基氮唑兰法观察高浓度的皮质酮和氟西汀对其增殖的影响。结果:从新生大鼠海马分离得到的细胞接种于含有表皮生长因子、碱性成纤维细胞生长因子两种丝裂原刺激因子的条件培养基中,可以持续分裂增殖形成细胞克隆(神经球),该神经球可以表达神经上皮干细胞蛋白(巢蛋白)。神经球中可以探测到外源性给与的细胞增殖特异性的标记物BrdU。撤除培养基中的丝裂原后,所培养的细胞可以分化,并且分化后可以表达成熟神经元的标志物神经元特异性烯醇化酶或星形胶质细胞标志物胶质纤维酸性蛋白。四甲基氮唑兰法显示,高浓度的皮质酮(100～500μmol/L)可以使吸光度(A570nm)明显降低。而氟西汀(0.5～1.0μmol/L)则使之升高。结论:用此方法分离得到的细胞具有神经干细胞的特性。高浓度的皮质酮可以抑制新生大鼠神经干细胞的增殖,氟西汀可以促进新生大鼠神经干细胞的增殖。
AIM:To investigate a practical method to culture and identify neural stem cells from hippocampus of neonatal rat,and observe the effects of high concentration corticosterone and fluoxetine on its proliferation. METHODS:The experiment was done in the Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences of Chinese PLA from February to June 2004.Hippocampus was isolated from newborn rats at the 1st day,and then it was made into single cell suspension and cultured on the serum free medium,the property of the cells was identified with immunocytochemistry method,and then the effects of high concentration corticosterone or fluoxetine on cell proliferation were studied by using the mononuclear cell direct cytotoxicity(MTT) reduction assay. RESULTS:With the presence of epidermal growth factor(EGF) and basic fibroblast growth factor(bFGF) in the medium, the cells isolated from the hippocampus of newborn rats could continuously proliferate and form cell clone(neurospheres) after inoculation,and the neurospheres could express as the neuroepithelial stem cell protein(nidogen).Bromodeoxyuridine(BrdU),the marker for cell proliferation, which was given exogenously,could be tracked in the neurospheres.After withdrawing the mitosis,cells were induced to differentiate and the differentiated cells could express the marker of mature neuron of neuron specific enolase(NSE) or the marker of mature astrocyte,glial fibrillary acidic protein(GFAP).MTT showed that high concentration corticosterone(100 to 500 μmol/L) could significantly decrease the absorbance(A570 nm) of hippocampal neural stem cells,which could be decreased by high concentration fluoxetine (0.5-1.0 μmol/L). CONCLUSION:Cells cultured with this method possess the property of stem cell.High concentration corticosterone can significantly inhibit the proliferation of hippocampal neural stem cells of newborn rats, while it can be promoted by fluoxetine.
出处
《中国临床康复》
CSCD
北大核心
2005年第13期53-55,i002,共4页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助项目(30300419)
北京市自然科学基金资助项目(7042052)~~
