摘要
目的探讨星形胶质细胞缺氧复氧损伤后细胞周期蛋白Dl（CyclinD1）的表达以及亚低温干预对其的影响。方法原代培养取材于新生sD大鼠皮层脑组织的星形胶质细胞（astrocytes，AC），传至第3代自然纯化后采用神经胶质纤维酸性蛋白（GFAP）化学染色法对其进行鉴定，并将其分为正常（37oC）对照组（C）、常温（37℃）缺氧复氧组（H／R）、亚低温（32℃）干预缺氧复氧组（M＋H／R）。以95％N2和5％CO2的培养条件致细胞缺氧8h，其后恢复正常供氧，分别复氧4，12，20h等，建立缺氧复氧模型。观察各时点AC的形态学变化、台盼蓝染色法测定AC的存活率、细胞免疫荧光及Western—blot检测CyclinD1的表达变化。结果1．自然纯化后的第3代AC95％以上呈GFAP阳性；2．缺氧8h后，常温组及亚低温组AC的形态和存活力变化均不明显，但随着复氧时间延长，两组细胞死亡数均显著增高（按复氧时间常温组分别为：12．87±2．76、31．55±3．00、46．40±8．50；低温组分别为：6．77±1．53、15．97±4．00、28．33±5．69），但亚低温组AC较常温组AC损伤明显减轻（P〈0．05），细胞存活力增强。3．免疫荧光和Western—blot检测示AC正常氧供时CyclinD1表达无明显变化，缺氧8h后两组AC的CyclinD1表达均增加，且随复氧时间延长呈明显增高趋势，亚低温组CyclinD1的免疫表达较常温组显著减少。结论CyclinD1是星形胶质细胞缺氧复氧损伤的敏感指标。
Objective To explore the changes of astrocytes in vitro on the expression of Cyclin D1 under normal and hypoxia/reoxygenation conditions. Methods The primary astrocytes were isolated from the cortex of SD fetal rats（ less than 24 hours） and identified by immunosytochemical method with anti-GFAP antibody after 3 passages. Astrocytes of passage 3 were divided into normal group （ control group）, hypoxia/reoxygenation group （H/R 37℃ ）, and mild hypothermiaintervention group（ H/R 32℃ ） separately. Astrocytes from the later tow groups were reoxygenated with 4,12, and 20 hours separately after exposed to hypoxia conditions for 8 hours. Trypan blue staining was employed to detect the survival rates and immunofluorescence, western-blot were used to analyse the expressin of Cyclin D1 of of astrocytes of different groups and time points. Results 1. The GFAP positive astrocytes from passage 3 exceeded 95%. 2. With regard to morphology and survival rates,there is no difference between astrocytes of normal and hypothermia groups after 8 hours exposure to hypoxia conditions. Reoxygenation could ob- viously rise astrocytes mortality with time went by （ H/R 37℃group ： 12.87 ± 2.76 （ R4）, 31.55 ± 3.00 （ R12 ）, 46.40± 8.50（R20） ;H/R 32℃ group ：6.77 ± 1.53 （R4）, 15.97 ± 4.00 （R12）, 28.33 ± 5.69 （R20） ;all P 〈 0.05）. 3. Immunofluorescence and western-blot revealed that reoxygenation increased Cyclin D1 expression markedly, which was proportional to the duration of reoxygenation. Mild hypothermia could reduce Cyelin D1 expression of astrocytes severely under reoxygenation condition. Conclusion Cyclin D1 expression can be regarded as a sensitive index of damaze to astrocvtes caused by hvooxia/reoxwenation conditions.
出处
《中华行为医学与脑科学杂志》
CAS
CSCD
北大核心
2012年第9期793-796,共4页
Chinese Journal of Behavioral Medicine and Brain Science
基金
：江苏省麻醉学重点实验室开放课题（K0798）
