目的：细胞凋亡在新生儿缺氧缺血性脑病(HIE)的发病机制中起重要作用，亚低温治疗是HIE最有前途的治疗方法之一。通过观察缺氧缺血后凋亡通路上关键成分的变化，探讨亚低温减轻新生大鼠脑细胞凋亡的作用及机制。方法：采用7日龄SD清洁级大鼠, 建立新生大鼠HIBD标准模型。模型动物随机分为常温缺氧缺血组 (IN, 肛温=37℃)和亚低温缺氧缺血组 (IH，肛温=33℃)。采用TUNEL结合苏木素-伊红染色、神经元Nissl染色等方法检测脑细胞凋亡；Western blotting加免疫组织化学法观察线粒体及胞浆细胞色素C蛋白改变；分别用RT-PCR和显色底物法检测caspase-3 mRNA表达及其酶活性改变。结果：IN组海马CA1区TUNEL阳性锥体细胞明显增多，DNA电泳梯状条带明显；72 h亚低温治疗显著降低脑细胞凋亡发生率，与常温比较差异有显著性（6.4±1.7 vs 25.3±1.5，P<0.01）。IN组胞浆Cyt c水平6 h开始明显升高，72 h达高峰，而线粒体内Cyt c水平则出现相应的下降；亚低温治疗组胞浆Cyt c水平降低和对应线粒体Cyt c水平的升高，以24 h、48 h和72 h最为明显，与IN组比较差异有显著性（P＜0.05）。HIBD后24 h组结扎侧脑组织caspase-3 mRNA表达明显增加，亚低温治疗显著降低caspase-3 mRNA表达水平，以48 h、72 h 治疗组最明显（P＜0.05），而caspase-3酶活性却在24 h达高峰，亚低温治疗可明显降低HIBD 后24 h 的caspase-3酶活性，与常温比较差异有显著性（2.42±0.5 RFU vs 34.7±3.2 RFU ，P＜ 0.01)。结论：亚低温治疗能够显著降低HIBD后细胞凋亡发生率，其机制可能作用于凋亡通路的多个部位：减少Cyt c释放，减轻或抑制caspase-3表达及其蛋白酶活性等。［中国当代儿科杂志，2007，9（1）：37-41］

OBJECTIVE: To investigate the effects of mild hypothermia on sequential events of neuronal apoptosis following hypoxic-ischemic brain damage (HIBD) in neonatal rats. METHODS: A model of HIBD was prepared by ligating the left common carotid artery in 7-day-old rats, followed by 8% hypoxia exposure. HIBD rats were randomly assigned into a hypothermia group (rectal temperature = 33 ℃) and a normothermia group (rectal temperature = 36 ℃). TUNEL, Haematoxylin and Eosin, and Nissl staining were used to detect neuronal apoptosis. Western blotting, RT-PCR and enzyme activity measurement were used to evaluate the changes of plasma and mitochondrial cytochrome c (Cyt c), caspase-3 mRNA expression and caspase-3 enzyme activity, respectively. RESULTS: The number of apoptotic cells in the ipsilateral hemisphere of the hypothermia group was significantly reduced compared with that of the normothermia group at 72 hrs post-HI (6.4±1.7% vs 25.3 ± 1.5%) (P<0.01).Analysis of Western blotting showed that Cyt c levels increased in the cytosolic fraction, but decreased significantly in the mitochondrial fraction in the ipsilateral hemisphere of the hypothermia group at 24, 48 and 72 hrs of HI insult compared with the normothermia group (P<0.05). Caspase-3 mRNA increased significantly after 24 hrs post-HI in the normothermia group, and this change became more pronounced with time. Mild hypothermia treatment decreased significantly caspase-3 mRNA expression at 24, 48 and 72 hrs post-HI (P<0.05). Caspase-3 activity gradually increased 2 hrs after HI insult and peaked at 24 hrs in the normothermia group. Mild hypothermia treatment resulted in a significant reduction in caspase-3 activity in the ipsilateral hemisphere, with an optimal effect produced at 24 hrs post-HI (2.42 ± 0.5 RFU vs 34.7 ± 3.2 RFU; P＜0.01). CONCLUSIONS: Mild hypothermia treatment attenuates neuronal apoptosis following HIBD, possibly through a reduction in Cyt c release from mitochondria and an inhibition of caspase-3 mRNA expression and its enzyme activity. ［Chin J Contemp Pediatr, 2007, 9 (1) :37-41］