摘要
目的观察单纯缺氧损伤对体外培养海马神经元内源性神经营养因子-3(neurotrophin-3, NT-3)表达的影响及外源性NT-3转导对单纯缺氧所致神经元凋亡的保护作用。方法体外分散培养新生Wistar大鼠海马神经元,体外培养第7天通过充氮法建立单纯缺氧损伤模型;用重组腺病毒载体pAC- CMV-PLPA构建携带NT-3全长cDNA的重组腺病毒Ad-NT-3,并分别于损伤前后向体外培养的海马神经元转导外源性NT-3;采用Western Blot检测在缺氧损伤前后及有无外源性NT-3转导的情况下NT-3 及Bcl-2的表达水平;采用TUNEL法检测缺氧及外源性NT-3转导后神经元凋亡的情况。结果 (1)单纯缺氧损伤后海马神经元的凋亡细胞标记指数由15．2％上升至56．4％,内源性NT-3表达量下降至对照组水平的71％。(2)缺氧损伤前重组腺病毒转导可使损伤后NT-3表达量上升至对照组的1.88倍、损伤后重组腺病毒转导亦可使NT-3表达量上升至对照组的1．42倍,而Bcl-2的表达量相应地上升至对照组的 1．69倍和1．32倍,凋亡细胞标记指数降至32．8％和45．4％。(3)统计学分析显示,海马神经元NT-3与Bcl- 2表达量间呈显著性正相关,二者与凋亡细胞标记指数间均呈显著性负相关。结论单纯缺氧损伤可使体外培养的海马神经元内源性NT-3表达量下降;腺病毒转导的外源性NT-3可保护单纯缺氧损伤神经元免于凋亡;其保护作用部分可能是通过对Bcl-2表达的诱导实现的。
Objective To observe the influence of hypoxia on the expression of endogenous neurotrophin-3 （NT-3） in hippocampal neurons cultured in vitro and the protective effect of exogenous NT-3 against the neuron apoptosis caused by simple hypoxia. Methods The hippocampal neurons from neonatal Wistar rats were cultivated in vitro and the simple hypoxia model was established on day 7 in vitro by nitro- gen inflation; Ad-NT-3, a recombinant adenovirus constructed on the basis of the adenoviral vector pAC-CMVPLPA containing full length eDNA of NT-3 and the exogenous NT-3 was transduced into the hippoeampal neurons cultured invitro before and after hypoxia injury. Western blot technique was adopted to monitor the expression levels of NT-3 and Bcl-2 with or without exogenous NT-3 transduction before and after hypoxia. The severity of neuron apoptosis during hypoxia or after transduction with exogenous NT-3 was detected by means of TUNEL. Results 1） After hypoxia the apoptotic cell labeling index of hippocampal neuron increased from the control level of 15.2% to 56.4%, and the expression of endogenous NT-3 decreased to 71% of the control level. 2） The transduction of recombinant adenovirus before hypoxia may up regulate the post hypoxia expression level of NT-3 to 1.88 fold of the control and transduction of recombinant adenovirus after hypoxia may up regulate NT-3 expression to 1.42 fold of the control, along with the expression level of Bcl-2 increased to 1.69 fold and 1.32 fold of the control level respectively, and the apoptotic cell labeling index decreased to 32.8% and 45.4% respectively. 3） Statistic analysis revealed there was significant positive relationship between the expression levels of NT-3 and Bcl-2 in hippocampal neuron, but significant negative relationship between the expressions of these two proteins and the apoptotic cell labeling index was found. Conclusion Simple hypoxia could suppress the NT-3 expression in hippocampal neurons cultured in vitro, exogenous NT-3 transduced by adenovirus may prote
出处
《中国现代神经疾病杂志》
CAS
2006年第1期57-61,共5页
Chinese Journal of Contemporary Neurology and Neurosurgery
