Neurobiological Basis of Neuronal Survival

在本财政年度，我们研究了低温治疗联合干细胞移植改善新生儿缺氧缺血性样损伤的治疗方案和作用机制。原代培养的大鼠神经元暴露在缺氧缺糖(OGD)环境中，造成体外缺氧缺血样损伤，然后分别在25℃(重度低温)、34℃(中低温)和37℃(常温)下与间充质基质细胞(MSCs)共培养。亚低温联合MSCs可显著提高OGD暴露后的细胞存活率和线粒体活性。Delta阿片类人胚胎肾细胞(HEK293)暴露于亚低温可减弱OGD介导的细胞改变，这种改变在过度表达Delta阿片受体的HEK293细胞中更为明显。此外，在34℃低温和干细胞处理的原代大鼠神经元中加入Delta阿片肽，对OGD表现出协同的神经保护作用，这种作用明显强于中低温和MSCs的双重组合，并被阿片受体拮抗剂纳曲酮显著减少，但并未完全取消，这完全意味着神经保护的配体-受体机制。对非阿片类药物治疗信号通路的进一步研究发现，生长因子调节和抗凋亡功能伴随着观察到的治疗效果。这些结果支持低温和干细胞在体外联合治疗缺氧缺血性样损伤，这可能对目前使用单独低温或干细胞治疗新生儿脑病的临床试验产生直接影响。

In this fiscal year we studied to reveal the therapeutic regimen and mechanism of action underlying hypothermia treatment in combination with stem cell transplantation for ameliorating neonatal hypoxic-ischemic-like injury. Primary rat neurons were exposed to oxygen-glucose deprivation (OGD), which produced hypoxic-ischemic-like injury in vitro, then incubated at 25C (severe hypothermia), 34C (moderate hypothermia), and 37C (normothermia) with or without subsequent co-culture with mesenchymal stromal cells (MSCs). Combination treatment of moderate hypothermia and MSCs significantly improved cell survival and mitochondrial activity after OGD exposure. The exposure of delta opioid human embryonic kidney cells (HEK293) to moderate hypothermia attenuated OGD-mediated cell alterations, which were much more pronounced in HEK293 cells overexpressing the delta opioid receptor. Further, the addition of delta opioid peptide to 34C hypothermia and stem cell treatment in primary rat neurons showed synergistic neuroprotective effects against OGD which were significantly more robust than the dual combination of moderate hypothermia and MSCs, and were significantly reduced, but not completely abolished, by the opioid receptor antagonist naltrexone altogether implicating a ligand-receptor mechanism of neuroprotection. Further investigations into non-opioid therapeutic signaling pathways revealed growth factor mediation and anti-apoptotic function accompanying the observed therapeutic benefits. These results support combination therapy of hypothermia and stem cells for hypoxic-ischemic-like injury in vitro, which may have a direct impact on current clinical trials using stand-alone hypothermia or stem cells for treating neonatal encephalopathy.