摘要
目的:探索重组腺病毒心肌营养素-1(Adv-CT-1)对谷氨酸(Glu)诱导神经元凋亡的保护作用及相关机制,为脑损伤提供新的治疗措施.方法:海马神经元分为3组:单纯Glu组、CT-1干预组(Adv-CT-1转染)及对照组;利用相差显微镜检查、DNA片段检测、乳酸脱氢酶(LDH)活性及流式细胞技术,对各组神经元进行凋亡检测;WesternBlot检测Caspase-3活性变化及线粒体内和胞浆内细胞色素C(CytC)水平变化.结果:Glu组神经元呈现明显形态改变,并见DNA梯状条带形成,CT-1组未见类似改变;Glu组神经元在Glu暴露后6,9及18h,LDH释放量(14.3%,24.6%及28.4%)及凋亡率(6.2%,18.8%及26.4%)均高于CT-1组(分别为9.2%,16.1%,19.7%及3.6%,11.3%,16.1%);Glu组Caspase-3活性3h即增加(25.6%),6及9h达到62.4%及46.1%,CT-1组3h时的Caspase-3表达分别为13.6%,45.2%及17.1%,均低于Glu组(P<0.05);Glu暴露30min,神经元胞质内CytC水平增加,之后进行性增高,线粒体内CytC进行性减少;Glu暴露后6h,CT-1组神经元胞质内CytC较Glu组减少了55.7%(P<0.05).结论:Glu诱导海马神经元凋亡的机制涉及线粒体CytC释放,激活Caspase级联反应;CT-1对损伤神经元的保护作用可能是经由线粒体信号转导途径、通过减少线粒体CytC释放而实现的.
AIM： To investigate the neuroprotective effects of recombinant adenovirus cardiotrophin-1 （ Adv-CT-1 ） in glutamateinduced hippocampal neurons damage model, and to determine the mechanisms by which the protective effects may be mediated. METHODS： Hippocampal neurons were divided into 3 groups： glutamate, CT-1 （transfected with Adv-CT-1 ） and control group. Apoptosis of neurons in 3 groups was determined by morphological changes, DNA fragmentation, lactate dehydrogenase （ LDH ） release assay and flow cytometry; and caspase-3 protein level and cytochrome c（ Cyt C） release from mitochondria into cytosol were determined by Western Blotting. RESULTS： Glutamate treatment alone induced cell death that was associated with obvious morphological changes and DNA fragmentation. These changes were inhibited by transfection of CT-1 into neurons. At 6,9 and 18 h after glutamate loading, LDH release（ 14.3%, 24.6% and 28.4% ） and apoptosis cells（6.2% , 18.8% and 28.4% ） in the glutamate alone group, were significantly higher than that in the CT-1 group, which were 9. 2%,16. 1%,19.7% and 3.6%, 11.3% ,16. 1%, respectively. The differences were significant （ P 〈 0.05 ）. In the neurons treated with glutamate alone, the levels of caspase-3 protein increased significantly at 3 h（25.6% ）, and reached 62.4% and 46.1% respectively at 6 h and 9 h after glutamate loading. In contrast, transfection with Adv-CT-1 significandy decreased the expression of caspase-3 protein at 3 （13.6%）,6（45.2%） and 9 h（11.7%）. For the neurons treated with glutamate alone, cytosolic Cyt C showed significant increase（ P 〈0.05 ） as early as 30 min and the level increased in a time-dependent manner. Meanwhile, the mitochondrial levels of Cyt C decreased significantly in a time-dependent fashion. At 6 h after glutamate loading, the levels of Cyt C in the cytosol from CT-1 group cells were 55.7%, lower than that in the glutamate alone treated ceils （ P 〈 0.05 ）. CONCLUSION： Glutamate treatment can induce a significant apoptosis in cultured hippocampal neurons. The apoptosis may be related to the damage of mitochondria that results in Cyt C release into cytosol, which activates caspase cascade, leading to apoptosis of the cells. The protective effects of CT-1 may be mediated via mitochondrial signal transduction pathways by decreasing release of Cyt C.
出处
《第四军医大学学报》
CAS
北大核心
2008年第17期1555-1558,共4页
Journal of the Fourth Military Medical University
基金
贵州省优秀人才省长资金(200407-4)
