摘要
目的 探讨创伤性脑损伤后神经细胞凋亡机制及其时序空间分布。 方法 采用末端脱氧核苷酸转移酶介导的生物素脱氧尿嘧啶核苷酸缺口末端标记法（ＴＵＮＥＬ）、琼脂糖凝胶电泳、透射电镜、流式细胞术、凋亡调节蛋白（Ｂｃｌ－２、Ｆａｓ） 免疫组织化学方法检测大鼠轻、重度脑损伤不同脑域神经细胞凋亡的动态变化。 结果 伤后６ ｈ 出现凋亡神经细胞，高峰时间为伤后２ ～３ ｄ，持续时间可达１４ ｄ。ＴＵＮＥＬ阳性染色细胞按电镜检测分为表现为坏死细胞特点的Ⅰ型细胞和表现为典型凋亡细胞特点的Ⅱ型细胞。ＴＵＮＥＬ Ⅱ型细胞主要集中于海马和齿状回区域。琼脂糖凝胶电泳显示，创伤脑组织ＤＮＡ出现特异凋亡电泳带。流式细胞检测呈现典型亚二倍体核型峰（Ａｐ 峰） 。重型脑伤后海马、齿状回Ｆａｓ 蛋白表达强度明显升高，而Ｂｃｌ－ ２ 蛋白表达强度呈下降趋势。 结论 创伤性脑损伤后存在神经细胞凋亡现象。海马、齿状回区域神经细胞死亡以细胞凋亡为主。创伤性神经细胞凋亡的发生与凋亡调节基因的非平衡表达有关。
Objective To explore the mechanism and temporal and spatial pattern of neuro cells apoptosis after traumatic brain injury(TBI). Methods Terminal deoxynucleotidyl transferease mediated biotinylated deoxyuridine triphosphate nick end labelling (TUNEL), gel electrophoresis, electromicroscopy, flow cytomehry and immunohistochemistry of apoptotic regulating gene (Bcl 2, Fas) were employed to detect the dynamic changes of neuro cells apoptosis in different regions of the rats' brains subjected to mild to severe traumatic injury. Results Apoptotic neuro cells were identified as early as 6 hours post injury,and reached maximum level 2 or 3 days later,and then declined after 14 days. The number of TUNEL positive neurons were prominent in ipsilateral hippocampal and dentate gyrus. Two types of TUNEL positive cells were demonstrated by electron microscopy, including type Ⅰ cells that displayed morphological features of necrocell and type Ⅱ cells that displayed morphological features of classic apoptotic cells. Gel electrophoresis of DNA extracted from traumatic brain tissues revealed internucleosomal fragmentation. Flow cytometry of damaged tissue showed a typical Ap wave. The strong expression of Fas protein was noted in hippocampus and dentate gyrus, where the expression of Bcl 2 protein was declined after severe TBI. Conclusions These data indicate that TBI associates with significant brain cells death. The mechanism of neuro cells apoptosis is related to the unbalanced expression of apoptotic regulating gene.
出处
《中华创伤杂志》
CAS
CSCD
北大核心
2000年第2期97-99,共3页
Chinese Journal of Trauma
