B细胞淋巴瘤/白血病基因2在颅脑外伤后抗神经元凋亡作用  

The role of B cell lymphoma leukemia-2 in anti-neuron apoptosis after traumatic brain injury in mice

作　　者：张燕飞[1] 降建新[1] 王瑛[1] 张夔鸣[1] 庄仲伟[1] 杨成[1] 孙志扬[1] 

机构地区：[1]同济大学附属东方医院神经外科,上海200120

出　　处：《上海医学》2012年第7期592-596,I0002,共6页Shanghai Medical Journal

基　　金：上海市浦东新区社会发展局科研基金资助项目(PW08-7)

摘　　要：目的通过研究B细胞淋巴瘤/白血病基因2(Bcl-2)高表达转基因小鼠和对照小鼠颅脑打击后神经元凋亡的差异,探寻Bcl-2抗凋亡及减轻颅脑损伤的作用。方法将构建的Bcl-2质粒转入C57BL/6J×CBA杂交小鼠的受精卵,培育得到携带Bcl-2的小鼠。Bcl-2转基因小鼠9只作为实验鼠,采用随机数字法分为实验1 d组、实验7 d组和实验14 d组,每组3只。同窝非转基因小鼠9只作为对照鼠,采用随机数字法分为对照1 d组、对照7 d组和对照14 d组,每组3只。采用液压打击法中侧方液压打击的方法,以中等打击力致小鼠中等颅脑外伤。余下3只同窝非转基因小鼠为假手术组,仅打开椎板,暴露硬脊膜,不予打击。分别于术后1、7、14 d处死小鼠,取脑组织切片行苏木精-伊红(H-E)染色,观察神经元变化;采用原位末端标记法(TUNEL)染色计数TUNEL标记的阳性细胞数即凋亡神经元,并计算TUNEL染色切片的积分光密度(IOD)值。结果经聚合酶链反应及Western印迹法检测证明所培育的转基因小鼠为Bcl-2高表达的小鼠。假手术组小鼠脑细胞未见明显变化。实验鼠和对照鼠的脑组织都有出血,结构被破坏,实验鼠的损伤肿胀及出现核固缩或解离的神经元数量少于对照鼠。假手术组小鼠的脑组织未见明显的凋亡神经元细胞。实验鼠和对照鼠的脑组织标本可见TUNEL染色阳性细胞,随时间的推移逐渐增多,在损伤后7 d达到高峰,然后稳定并逐渐减少。实验1 d组、实验7 d组、实验14 d组小鼠的每个光学显微镜高倍镜视野内的TUNEL阳性神经元数(P值分别为0.042、0.039、0.033)和TUNEL染色切片的IOD值(P值均为0.000)均分别显著少于对照1 d组、对照7 d组、对照14 d组。结论 Bcl-2蛋白是中枢神经系统内在的重要的抗凋亡因子,高表达Bcl-2能够抑制颅脑损伤后神经元的凋亡,从而减轻大脑的损害。Objective To compare the apoptosis of neurons in B cell lymphoma/leukemia-2 （Bcl-2） transgenic （TG） mice and control mice, and to explore the role of Bcl-2 on the anti-neuron apoptosis and functional recovery after traumatic brain injury （TBI）. Methods We transplanted the plasmid with Bcl-2 gene into the fertilized ovum of 057BL/6J x CBA mice and obtained the TG mice. Nine Bcl-2 TG mice and nine control mice were subjected to moderate TBI at the left cerebral hemisphere by hydraulic percussion method. The mice were divided into three groups according to random number （n = 3）. Another 3 non-TG mice were taken as sham operation group. The mice were sacrificed at day 1, 7 and 14 after TBI, respectively. Brain tissue sections were stained with haematoxylin-eosin staining to observe the changes of neurons. In situ end labeling （TUNEL） was applied to count the apoptosis of neurons. Integrated optical density （IOD） was also recorded. Results Polymerase chain reaction （PCR） and Western blotting proved that Bcl-2 was highly expressed in the TG mice. There was no obvious change of brain cells in the mice with sham operation. There were bleeding and tissue injury in the brain of both TG mice and control mice. The abnormal neurons （swelling, nuclear condensation or dissociation） in the TG mice weresignificantly less than those in the control mice. TUNEL positive neurons were seen in the brain tissue of TG mice and control mice. The number of the neurons reached the peak at day 7 after TBI and then decreased gradually. The number of TUNEL positive neurons （ P = 0. 042, 0. 039, 0. 033） and IOD score （all P = 0. 000） in the Bcl-2 TG mice were both significantly lower than those in the control mice 1, 7, and 14 days after TBI. Conclusion Bcl-2 protein is an important factor against apoptosis. Over-expression of Bcl-2 may suppress neuron apoptosis and relieve brain damage after TBI.

关 键 词：颅脑损伤 白血病基因2 神经元凋亡 侧方液压冲击 转基因小鼠 

分 类 号：R651.1[医药卫生—外科学]