缺氧对成年大鼠SVZ组织分离培养神经干细胞的影响  

Effect of hypoxia on neural stem cells isolated from adult rat SVZ and cultured in vitro

作　　者：祁存芳[1,2] 张军峰[1,3] 陈新林[1] 焦倩[1] 张建水[1] 刘勇[1] 

机构地区：[1]西安交通大学医学院神经生物研究所,教育部环境和基因相关疾病重点实验室,陕西西安710061 [2]青海大学医学院基础医学部解剖学教研室,青海西宁810001 [3]西安医学院人体解剖学教研室,陕西西安710021

出　　处：《西安交通大学学报（医学版）》2013年第6期758-763,772,共7页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.30960107;81070998)~~

摘　　要：目的探讨缺氧对体外培养的大鼠神经干细胞(neural stem cells,NSCs)增殖、分化和细胞凋亡的影响。方法分离成体SD大鼠室管膜室下区(subventricular zone,SVZ)组织,NSCs在常氧和低氧培养箱培养,光镜观察细胞形态;采用NSCs标记物(nestin)免疫荧光染色鉴定细胞,未成熟神经元标记物(β-tubulinⅢ/Tju-1)、星形胶质细胞标记物(GFAP)和少突胶质细胞标记物(O4)免疫荧光染色和细胞计数观察细胞的分化能力;WST-1检测细胞增殖能力;流式细胞仪和TUNEL法检测细胞凋亡。结果从成体SVZ组织中成功分离培养出NSCs;无血清诱导下细胞培养至第4代有部分细胞分化,多为胶质细胞;血清诱导后NSCs可以向神经元、星形胶质细胞和少突胶质细胞分化,大部分干细胞分化为Tju-1阳性细胞(53.8%±6.8%),部分为GFAP阳性细胞(21.6%±5.4%),少部分为O4阳性细胞(11.4%±4.5%)。一定时间(<24h)的低氧(3mL/L氧浓度)可以促进NSCs增殖(P<0.05),但长时间缺氧(>24h),细胞增殖能力减弱而细胞凋亡比例增加(P<0.01)。结论正常成体组织中存在NSCs;体外培养条件下分离培养的NSCs可以分化为多种类型细胞;短时间低氧可促进NSCs的增殖,而长时间缺氧导致细胞凋亡比例增加。Objective To explore the effect of hypoxia on the proliferation, differentiation and apoptosis of neural stem cells （NSCs） isolated from adult rat subventricular zone （SVZ） and cultured in vitro. Methods The NSCs isolated from adult rat SVZ were cultured under normoxia condition or hypoxia condition （hypoxia for 6, 12, 24, 48 or 72 h） in vitro. The nestin＋ cells, Tju-1＋ cells, GFAP＋ cells and 04＋ cells were detected by immunofluorescence staining, and the percentage of the positive cells was measured respectively. The proliferation of the NSCs was assayed with cell proliferation reagent （WST-1） and the cell cycle was detected by flow cytometry （FCM）. The apoptosis of the NSCs was detected by TUNEL and flow cytometry. Results The NSCs from adult rat SVZ were successfully isolated and cultured in vitro. Induced by serum, the NSCs differentiated at the 4th generation and most of them were Tju-l-positive （53.8± 6.8%） while some of them were GFAP-positive （21.6 ± 5.4 %）. Besides, a small number of them were O4-positive （11.4± 4.5 % ）. Hypoxia （0.3 % 02 ） for a short period （~24 h） promoted the proliferation of the NSCs （P~ 0. 05）. But prolonged hypoxia （~ 24 h） reduced the proliferation capacity of the NSCs and increased the percentage of apoptotic cells （P〈0.01）. Gonclusion NSCs are present in normal adult tissues; NSCs isolated and cultured in vitro can differentiate into various types of cells. Short-term hypoxia can promote the proliferation of NSCs, but prolonged hypoxia induces apoptosis of NSCs.

关 键 词：缺氧 室管膜室下区 神经干细胞 增殖 凋亡 大鼠 

分 类 号：R322.8[医药卫生—人体解剖和组织胚胎学]