实验性缺氧缺血新生猪脑线粒体DNA损伤的研究

Brain mitochondrial DNA damage of newborn piglets following hypoxia-ischemia

摘要:

目的：观察实验性缺氧缺血后新生猪不同时段脑线粒体DNA8003bp损伤,探讨缺氧缺血性脑损伤(hypoxicischemicbraindamage,HIBD)能量代谢障碍的分子生物学基础。方法：将3日龄新生猪(n=50)随机分为对照组和HIBD实验0,24,48,72h组。实验组结扎左侧颈总动脉并置于8%氧气2h制作HIBD动物模型,对照组行假手术。取各组动物左侧大脑海马区皮质提取脑线粒体DNA(mitochondrialDNA,mtDNA),LXPCR方法扩增检测200bp及8003bpmtDNA片段。PCR产物行琼脂糖凝胶电泳,结果以积分光密度(IOD)值表示。结果：8003bp片段缺氧缺血后0hIOD值22.616±2.276较对照组56.995±0.317显著降低(P<0.05),24h时IOD值为27.719±0.309和48h为49.491±3.233,有所恢复,仍低于对照组(P<0.05),72h时IOD值为55.972±2.236与对照组相比无统计学差异(P>0.05)。结论：缺氧缺血后新生猪脑海马区神经元mtDNA发生断裂性损伤,72h恢复至正常水平。缺氧缺血性mtDNA损伤可能与缺氧缺血情况下线粒体呼吸链复合物活性下降及迟发性神经元死亡有关。

Abstract:

OBJECTIVE:  This study investigated the 8003 base pair (bp) fragmentation damage of brain mitochondrial DNA in newborn piglets at different times after hypoxic-ischemic brain damage (HIBD) so as to explore the biomolecular foundation of neonatal neuronal metabolic disorders. METHODS: Fifty 3-day-old piglets were randomly assigned into Control and HIBD groups. The HIBD group was subdivided into groups sacrificed at 0, 24, 48 and 72 hrs post-HIBD (n=10). HIBD was induced by left carotid ligation and exposure to 8% oxygen for 2 hours. The Control group was exposed to air and was sham-operated. The left hippocampal cortexes of all subjects were obtained to amplify the fragments of 200 bp and 8003 bp by the LX-PCR method. The PCR products were electrophoresed on agaros gels to obtain the integral optical density (IOD). RESULTS: The IOD of 8003 bp fragment was markedly reduced in the HIBD 0 hr group (22.616±2.276) when compared with that of the Control group (56.995±0.317)( P<0.05). The IOD value remained lower at 24 hrs (27.719±0.309) and 48 hrs post-HIBD (49.491±3.233) (P<0.05). Until 72 hrs post-HIBD, the IOD (55.972±2.236) restored to the control value. CONCLUSIONS: The brain mitochondrial DNA was fragmented in newborn piglets following brain hypoxia-ischemia. It did not recover to normal until 72 hrs post-HIBD. The fragmentation damage of mitochondrial DNA may be related to the depression of mitochondrial respiratory enzymes activity and neuron apoptosis.