叶酸对胎鼠神经干细胞体外增殖的影响  

EFFECTS OF FOLATE ON PROLIFERATION OF NEURAL STEM CELLS FROM FETAL RATS IN VITRO

作　　者：张绪梅[1] 黄国伟[1] 姬长珍[1] 张文治[2] 苏心[2] 

机构地区：[1]天津医科大学公共卫生学院,天津300070 [2]天津市神经外科研究所,天津300060

出　　处：《营养学报》2008年第2期144-147,152,共5页Acta Nutrimenta Sinica

基　　金：国家自然科学基金(No.30571563)

摘　　要：目的探讨叶酸对体外培养的胚胎大鼠神经干细胞(neural stem cells,NSCs)增殖的影响。方法本研究采用显微解剖、机械吹打、无血清悬浮培养方法分离培养鼠胚大脑NSCs,用巢蛋白(nestin)免疫荧光染色对其进行鉴定,5'-溴脱氧尿嘧啶(BrdU)掺入法检测细胞增殖状态;设立正常对照组,叶酸低、高剂量(培养液中添加叶酸4.0 40 mg/L)和叶酸缺乏组(培养液中添加叶酸拮抗剂甲氨蝶呤0.4mg/L);通过nestin+BrdU免疫荧光双标记和绘制细胞生长曲线(MTT法)检测叶酸对细胞增殖状态的影响。结果无血清培养液中可形成大量呈nestin抗原阳性细胞组成的神经球,BrdU免疫标记证实培养的NSCs具有增殖能力。免疫荧光双标记和MTT结果显示,两个叶酸干预组NSCs增殖能力较对照组显著增强。结论体外培养的胚胎大鼠NSCs具有增殖和自我更新的能力;叶酸可以在一定程度上增强NSCs的增殖能力,促进神经干细胞的生长。Objective To explore the effect of folic acid on neural stem cells （NSCs） proliferation from fetal rats in vitro. Method NSCs were isolated and cultured by microdissection, mechanical blowing and serum-free suspension culture, and identified by immunofluorescent staining using antibody against nestin. BrdU（5＇bromo-2＇deoxyuridine） was used to mark dividing neural stem cells. Cultured NSCs were divided into four groups： control group, low, high dose group （liquid media with added 4, 40 mg/L folic acid）, and deficiency group（liquid media with added 0.4 mg/L methotrexate, MTX）. Monotetrazolium （MTT） and double-label immunofluorescence technique detected NSCs proliferation under the condition of folic acid. Results In the serum-free suspension medium, neurospheres that consisted of a great number of nestin-positive cells could be obtained. The proliferative ability of NSCs were observed by BrdU labeling methods. MTT assay and double-label immunofluorescence for nestin＋BrdU showed that the growth tendency was increased with folate concentration in the medium. Compared with control group, NSCs growth rate of folate group was significantly increased in vitro. Conclusion The culture of NSCs isolated from fetal rats possesses the abilities of proliferation and self-regeneration. Folic acid may stimulate proliferation of NSCs efficiently.

关 键 词：叶酸 神经干细胞 NESTIN BRDU 免疫荧光双标记 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]