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低氧预适应大鼠脑脊液减轻氧糖剥夺对培养新生鼠海马神经元的损伤及可能的机制

牛敬忠^*, 张颜波, 李美艺, 刘莉莉

泰山医学院附属医院神经内科，泰安 271000；山东泰山慢性病医院神经内科，泰安 271000

摘要

本文旨在观察低氧预适应(hypoxic preconditioning, HPC) Wistar大鼠脑脊液对新生鼠海马神经元氧糖剥夺(oxygen glucose deprivation, OGD)损伤的影响及机制。原代培养大鼠新生鼠(出生12 h)海马神经元，随机分为正常对照组、OGD组(OGD培养1.5 h)、正常脑脊液组(正常大鼠脑脊液培养1 d + OGD培养1.5 h)和HPC脑脊液组(HPC大鼠脑脊液培养1 d + OGD培养1.5 h)，各组n = 6。用含1 mmol/L Na2S2O4的无糖Earle’s液培养大鼠新生鼠海马神经元1.5 h建立OGD模型。应用激光共聚焦显微镜和流式细胞术检测各组神经元凋亡情况，用免疫荧光染色法检测各组神经元Bcl-2/Bax表达水平。结果显示，正常对照组偶见凋亡早期细胞，OGD组见大量中晚期凋亡细胞；与OGD组和正常脑脊液组相比，HPC脑脊液组细胞凋亡显著减少(P < 0.01)，细胞生存率提高(P < 0.01)。OGD组和正常脑脊液组细胞Bcl-2蛋白荧光强度分别为0.040 ± 0.003，0.457 ± 0.044，而HPC脑脊液组细胞Bcl-2表达荧光强度增至1.923 ± 0.078 (P < 0.01)，且阳性细胞数量亦明显增加(P < 0.01)；Bax表达则呈相反趋势，相对OGD组(1.548 ± 0.068)和正常脑脊液组(1.072 ± 0.219)，HPC脑脊液组荧光强度(0.411 ± 0.039)明显降低(P < 0.01)，且阳性细胞数量亦明显减少(P < 0.01)。以上结果提示，HPC脑脊液能减轻OGD损伤诱导的神经元凋亡，该神经保护效应与上调Bcl-2表达、下调Bax表达，提高Bcl-2/Bax有关。

关键词： 低氧预适应; 脑脊液; 海马神经元; Bcl-2/Bax

分类号：R363.2

[Lessening effect of hypoxia-preconditioned rat cerebrospinal fluid on oxygen-glucose deprivation-induced injury of cultured hippocampal neurons in neonate rats and possible mechanism.] [Article in Chinese]

NIU Jing-Zhong^*, ZHANG Yan-Bo, LI Mei-Yi, LIU Li-Li

Department of Neurology, Affiliated Hospital of Taishan Medical College, Taian 271000, China； Department of Neurology, Taishan Chronic Disease Hospital, Taian 271000, China

Abstract

The present study was to investigate the effect of cerebrospinal fluid (CSF) from the rats with hypoxic preconditioning (HPC) on apoptosis of cultured hippocampal neurons in neonate rats under oxygen glucose deprivation (OGD). Adult Wistar rats were exposed to 3 h of hypoxia for HPC, and then their CSF was taken out. Cultured hippocampal neurons from the neonate rats were randomly divided into four groups (n = 6): normal control group, OGD group, normal CSF group and HPC CSF group. OGD group received 1.5 h of incubation in glucose-free Earle’s solution containing 1 mmol/L Na2S2O4, and normal and HPC CSF groups were subjected to 1 d of corresponding CSF treatments followed by 1.5 h OGD. The apoptosis of neurons was analyzed by confocal laser scanning microscope and flow cytometry using Annexin V/PI double staining. Moreover, protein expressions of Bcl-2 and Bax were detected by immunofluorescence. The results showed that few apoptotic cells were observed in normal control group, whereas the number of apoptotic cells was greatly increased in OGD group. Both normal and HPC CSF could decrease the apoptosis of cultured hippocampal neurons injured by OGD (P < 0.01). Notably, the protective effect of HPC CSF was stronger than that of normal one (P < 0.01). Compared to OGD group, normal and HPC CSF groups both showed significantly higher levels of Bcl-2 (P < 0.01), and Bcl-2 expression level in HPC CSF group was even higher than that in normal CSF group (P < 0.01). Whereas the expressions of Bax in normal and HPC CSF groups were significantly lower than that in OGD group (P < 0.01), and the Bax expression in HPC CSF group was even lower than that in normal CSF group (P < 0.01). These results suggest that CSF from hypoxic-preconditioned rats could degrade apoptotic rate of OGD-injured hippocampal neurons by up-regulating expression of Bcl-2 and down-regulating expression of Bax.

Key words: hypoxia-preconditioning; cerebrospinal fluid; hippocampal neurons; oxygen glucose deprivation

收稿日期：2011-04-23　　录用日期：2011-07-06

通讯作者：牛敬忠　　E-mail: niujingzhong@tom.com

引用本文：

牛敬忠, 张颜波, 李美艺, 刘莉莉. 低氧预适应大鼠脑脊液减轻氧糖剥夺对培养新生鼠海马神经元的损伤及可能的机制[J]. 生理学报 2011; 63 (6): 491-497.

NIU Jing-Zhong, ZHANG Yan-Bo, LI Mei-Yi, LIU Li-Li. [Lessening effect of hypoxia-preconditioned rat cerebrospinal fluid on oxygen-glucose deprivation-induced injury of cultured hippocampal neurons in neonate rats and possible mechanism.] [Article in Chinese]. Acta Physiol Sin 2011; 63 (6): 491-497 (in Chinese with English abstract).