脑源性神经营养因子对缺氧神经元的保护作用及其细胞内信号传递

Protective effect of brain-derived neurotrophic factor on hypoxic cultured cortical neurons and its cellular signal transduction pathway

目的:观察体外培养大鼠胚脑皮质神经元对缺氧耐受的时间窗,探讨外源性脑源性神经营养因子对缺氧神经元保护作用的时效-量效关系,及该保护作用可能的细胞内信号传递途径。方法:实验于2004-02/2005-02在四川大学华西医院移植工程及移植免疫实验室完成。体外培养孕17～19dSD大鼠皮质神经元,随机数字法分为基线对照组、单纯缺氧组及缺氧+脑源性神经营养因子组,7d后作缺氧培养,采用四氮唑盐比色法检测0～10h段各时间点单纯缺氧组和缺氧+脑源性神经营养因子组(分别在缺氧前24h及缺氧前即刻加入25～100μg/L脑源性神经营养因子)存活率的差别,并用Westernblotting检测两组神经元在Akt/磷酸化Akt及CREB/磷酸化CREB表达的差别。结果:①脑源性神经营养因子对缺氧神经元的保护作用是相对的,该保护作用具有时效-量效关系,缺氧前24h加入大剂量脑源性神经营养因子组(100μg/L)优于缺氧即刻加入小剂量(25μg/L)组,缺氧损伤到一定程度后(>10h)脑源性神经营养因子的保护作用将明显减弱犤缺氧前24h加入100μg/L脑源性神经营养因子在1,2,4,8,10h细胞活性分别为基线对照组的(133.85±3.72)%,(109.83±5.43)%,(86.15±0.68)%,(53.78±1.71)%,(33.41±1.64)%,而缺氧前即刻加入25μg/L脑源性神经营养因子组在以上时点细胞活性分别为基线对照组的(81.55±1.07)%,(92.10±4.89)%,(78.75±1.87)%,(43.58±2.42)%,(33.13±0.94)%,单纯缺氧组的细胞活性分别为基线对照组的(82.12±3.15)%,(81.79±2.61)%,(64.5±4.56)%,(45.9±1.74)%,(35.45±1.25)%犦。②加入脑源性神经营养因子可使磷酸化Akt表达增加,磷酸化CREB表达提前并维持较长时间。结论:脑源性神经营养因子可减轻缺氧对神经元的损害,而Akt表达上调可能是脑源性神经营养因子发挥其对缺氧神经元保护作用的重要途径之一。

Abstract AIM： To observe the time window of cultured cortical neurons exposed to hypoxic environment in vitro and to investigate the time and dose-effect relationship.of brain-derived neurotrophic factor （BDNF） on hypoxic-cultured neurons, and the possible signal transmission pathway of the protective effect. METHODS： The experiment was completed in Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University from February 2004 to February 2005. Embryonic cortical neurons of 17- 19 day pregnant embryo SD rat were cultured in vitro and the cultured neurons were randomly divided into baseline control group, simple hypoxic group and hypoxic ± BDNF group. Seven days later, hypoxic culture was conducted, and the difference of survival rate in the simple hypoxic group and hypoxic ± BDNF group （25-100 μg/L BDNF was added at 24 hours and immediately before hypoxia, respectively） was detected at each time point of 0-10 hours with diphenyhetrazohum bromide （MTT） assay. The difference of expression of Akt, phosphate Akt and CREB, phosphate CREB was detected with western blotting method. RESULTS： （1）The protective effect of BDNF on hypoxic-cultured neurons was relative, which had time and dose-effect relationship, and it Was better in the BDNF group receiving a large dose of BDNF （1（30 μg/L） before exposed to hypoxia than that in the BDNF group receiving a small dose of BDNF （25 μg/L）. The protective effect of BDNF after the hypoxia reaching a certain degree （〉10 h） would significantly weaken [The neuron viability at 1, 2, 4, 8 and 10 hours after exposed to hypoxic environment were （ 133.85 ±3.72）%, （ 109.83 ±5.43 ）%, （86.15 ±0.68）%, （53.78 ±1.71 ）%, （33.41±1.64）% after receiving 100 μg/L BDNF at hour 24 before hypoxia of that of the baseline control group. The neuron viability in BDNF group receiving 25 μg/L BDNF immediately before hypoxia was （81.55±1.07）%, （92.10±4.89）%, （78.75±1.87）%, （43.58±9.42）%, （33.13±0.94）% of that of the baseline control group at the above mentioned time point. The neuron viability in the simply hypoxic group was （82.12±3.15）%, （81.79±9.61）%, （64.5±4.56）%, （45.9±1.74）%, （35.45±1.25）% of that of the baseline control group, respectively]. （2）BDNF could induce phosphate Akt expression increase, and the phosphate CREB expression sooner up and sustain for much longer time.. CONCLUSION： BDNF can relieve the hypoxic-induced cortical neuron injury, while the up-regulation of Akt expression may be one of the possible mechanisms of protecting cortical neurons from hypoxia.