脑微血管内皮细胞对体外缺氧神经干细胞增殖及凋亡的影响  

Effects of brain microvascular endothelial cells on the proliferation and apoptosis of neural stem cells under hypoxia in vitro

作　　者：朱晓峰[1] 王银妹[1] 金玉玲[1] 

机构地区：[1]佳木斯大学神经科学研究所,黑龙江省佳木斯市154007

出　　处：《中国组织工程研究与临床康复》2007年第20期3882-3885,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然基金项目(批准号30450062);黑龙江省自然基金重点项目(批准号ZJY0508)~~

摘　　要：目的:观察脑微血管内皮细胞对体外培养缺氧神经干细胞增殖、凋亡的影响。方法:实验于1996-01/1996-12在佳木斯大学神经科学所完成。实验材料:同一基因背景新生的Wistar大鼠由哈尔滨医科大学实验动物学部提供(黑动字第99102001)。实验方法:①取新生24hWistar大鼠大脑培养神经干细胞,采用免疫组织化学方法鉴定神经干细胞。②取出生1～7dWistar大鼠大脑培养脑微血管内皮细胞,采用免疫细胞化学方法鉴定。③将培养传代纯化的脑微血管内皮细胞,用0.25%胰酶消化成单细胞液后接种于涂有多聚赖氨酸包被的盖玻片上,接种传3代后用胰酶消化的神经干细胞,以1∶10接种比例加入神经细胞完全培养液共培养。④用无菌石蜡油覆盖于低糖细胞培养液表面,制备低氧低糖溶液。对照组:正常神经干细胞置于37℃、体积分数为0.05的CO2饱和湿度培养。缺氧共培养组:取共培养3d细胞,吸出培养液,用无菌Hank’s缓冲液清洗2次,加入低氧低糖溶液共同作用2,4,8,16h后换液,用PBS缓冲液冲洗3次后,换神经细胞完全培养液,置于细胞培养箱中复氧培养。缺氧单纯神经干细胞组:取传3代神经干细胞,用无菌Hank’s缓冲液清洗2次后,方法同上。实验评估:免疫组织荧光鉴定缺氧条件下神经干细胞的增殖与凋亡。结果:①免疫组织化学方法检测Nestin及Ⅷ因子相关抗原分别鉴定为神经干细胞及脑微血管内皮细胞。②对照组细胞胞体饱满,折光性强,周围有光晕。缺氧单纯神经干细胞组细胞胞体肿胀,折光性差,有大量细胞碎屑,仅有少量活细胞,并出现悬浮死细胞。缺氧共培养组细胞形态有所改善,细胞折光性仍较好,大部分细胞突起仍未见回缩,仅少数细胞肿胀,坏死。③随着缺氧时间的延长,细胞死亡明显增多,存活数明显下降。缺氧共培养组细胞存活数明显高于缺氧单纯神经干细胞组与对照组(321.76±22.2AIM： To observe the effects of brain microvascular endothelial cells （MVECs） on the proliferation and apoptosis of neural stem cells （NSCs） under the hypoxic condition in vitro. METHODS： The experiment was conducted in the Institute of Neuroscience at Jiamusi University from January to December 1996. Experimental materials： Newborn Wistar rats with identical gene background were provided by Experimental Animal Education Ministry of Harbin Medical College （number 99102001）. Experimental technique： （1）NSCs were harvested from the cerebrum of newborn 24-hour Wistar rats for culture. NSCs were identified by immunohistochemical method. （2）Cerebrum of 1-7 day Wistar rat was harvested to culture MVECs that was determined by immunohistochemical method. （3）After culture, passage and purification, MVECs were digested with 0.25% trypsin into monoplast liquor and then inoculated on cover slips coated with polylysine. After inoculating for 3 generations, NSCs were co-cultured with complete culture solution at vaccination proportion of 1：10. （4）Sterile paraffin oil was coated on the surface of low-glucose culture fluid to prepare hypoxia low-glucose solution. Control group： The normal NSCs were cultured in saturated humidity medium containing CO2 of 0.05 volume fraction at 37 %. Hypoxia co-culture group： NSCs co-cultured for 3 days were obtained and then culture medium was sucked out. NSCs were washed sterilely with Hank＇s buffer solution twice where hypoxia low-glucose solution was added for 2, 4, 8 and 16 hours, and then medium was changed. After NSCs were washed with PBS three times and complete culture solution was changed, the medium was laid in an incubator with oxygen. Hypoxia simple NSCs group： 3 generation of NSCs were obtained and washed sterilely with Hank＇s buffer solution twice, and the methods were the same as mentioned above. Experimental evaluation： Proliferation and apoptosis of NSCs were identified by immunohistofluorescence under hypoxic condition. RESULTS：

关 键 词：缺氧 神经干细胞 鼠脑微血管内皮细胞 增殖 凋亡 

分 类 号：R394.2[医药卫生—医学遗传学]