【作者】 明美秀；

【导师】 周文浩； 王来拴； 熊曼； 孙金峤；

【作者基本信息】 复旦大学 ， 儿科学， 2010， 硕士

【摘要】 【目的】亚低温(mild hypothermia)是治疗新生儿缺氧缺血性脑损伤(hypoxic ischemic brain damage,HIBD)的一种有效的方法,可通过降低脑细胞的能量代谢、减少细胞毒性产物的积聚、对抗细胞坏死等途径发挥脑保护作用。星形胶质细胞(astrocyte,AST)是脑细胞的重要组成部分,在缺氧缺血(hypoxic-ischemia, HI)后的脑损伤与修复过程中扮演重要角色。既往研究重点多集中于亚低温对神经元的保护作用,对星形胶质细胞影响的研究极少,是否存在保护作用及其机制并不明了。本研究采用在体7日龄新生大鼠脑缺氧缺血模型及体外培养海马星形胶质细胞氧糖剥夺(oxygen-glucose deprivation, OGD)模型,观察亚低温对缺氧缺血性脑损伤后新生大鼠海马星形胶质细胞增殖和凋亡的影响,探讨亚低温对未成熟脑缺氧缺血性损伤的保护机制。【方法】(一)在体实验：首先制备大鼠脑缺氧缺血模型,7日龄大鼠左侧颈总动脉结扎后置于含8%氧气的氮氧混合气的37℃缺氧箱中缺氧2 h。对照组大鼠仅实施假手术,不进行缺氧缺血。术后动物随机分为常温处理组、亚低温治疗组、常温对照组、亚低温对照组；分别在HI后24 h、72 h和7天制备脑片,通过免疫组化计数胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)阳性细胞来评价星形胶质细胞增殖的变化；通过免疫组化双标计数GFAP与caspase-3双阳性细胞检测星形胶质细胞的凋亡变化。(二)体外实验：培养1日龄大鼠海马星形胶质细胞,采用氧糖剥夺(OGD)方法以模拟在体缺氧缺血损伤过程,采用CCK-8、Tunel及DAPI染色观察亚低温处理后星形胶质细胞增殖和凋亡变化。【结果】大鼠海马区域GFAP染色结果显示,HI后72 h和7天亚低温治疗组GFAP阳性细胞低于常温组,同一时间点两组比较具有统计学意义(P<0.05)；HI后72 h常温处理组海马部位84.5%GFAP阳性细胞发生Caspase-3共表达,而亚低温治疗组仅32.3%。体外实验显示,OGD后亚低温处理组细胞肿胀较常温处理组轻；CCK-8结果显示亚低温治疗组AST增殖明显低于常温处理组(P<0.05)；亚低温治疗组较常温处理组凋亡减少,Tunel阳性细胞明显减少(P<0.05)。【结论】亚低温能抑制新生大鼠HI损伤后海马星形胶质细胞的增殖,减轻其凋亡更多还原

【Abstract】 Objective The mechanisms underlying hypoxic ischemic brain damage are only partially understood but are commonly suggested to include excitotoxicity, and induction of secondary apoptosis and inflammation. Previous studies have shown that hypoxia-ischemia (HI) results in a cascade of biochemical events and pathological reactions that lead to neuronal death in the hippocampus.Astrocytes are the most numerous cell types in the central nervous system. They provide structural, metabolic and trophic support to neurons and modulate synaptic activity. There is considerable evidence that astrocyte-conditioned media support the survival of neurons. Hypothermia is effective in reducing brain injury and improving behavioral recovery in animal experiments and is also safe and effective in neonatal clinical settings. Previous studies have shown that mild hypothermia preserves neurons and reduces astrocyte proliferation in adult animal models. Nevertheless, it is still unclear if mild hypothermia can attenuate astrocyte loss in the hippocampus of HI injured immature rat brain, and the corresponding changes of astrocyte proliferation and its apotosis response.Method(1)in vivo:Neonatal HI was induced in rats at postnatal day 7, according to the method described by Rice.The left common carotid artery was isolated and ligated, placed in a chamber perfused with a humidified gas mixture (8% oxygen in nitrogen) for 2h. The operated pups were randomly divided into the normo-group(36-37℃) and hypothermia groups(32-33℃),respectively. Control animals were anesthetized but not subjected to HI. They were then randomized to either normothermia or hypothermia.animals were killed by decapitation at at 0,3 and 7 days after HI. The brains were removed and immersion-fixed, coronal sections were cut on a freezing microtome at a thickness of 30μm for glial fibrillary acidic protein count(GFAP) and caspase-3 immunohistochemical staining. Cells were counted in the CA1 region of ipsilateral hippocampus detected the apoptosis of astrocytes.(B) In vitro:All cultured AST originated from 1 day old rat hippocampus, using oxygen and glucose deprivation (oxygen-glucose deprivation, OGD)method to simulate the HI injury, using CCK-8, Tunel and DAPI staining to assess the activation, proliferation and apoptosis of astrocyte.Results In vivo, GFAP-positive cells of hippocampus was significant decreased at 72 hour and 7 days after HI in hypothermia treatment group (P<0.05), There are significant differences between normothermia and hypothermia groups at 72hours and 7days after HI.84.5% co-expression of GFAP and Caspase-3 cells showed in hippocampus with immunofluorescence staining in HI group, but 32.3% in hypothermia treatment group. In vitro, cell activation and proliferation were observed at 24-72 hours following 1-4 hours OGD, which was significantly decreased in the hypothermia treatment group. Significant increase in cell death occurred 24-48 hours after 1-4 hours OGD, and cell death measured at 24 hours,48 hours was significantly reduced in hypothermia treatment group. Experiments showed that the Tunel positive cells decreased significantly (P<0.05).Conclusion Mild hypothermia can reduce the activation, proliferation,and inhibit apoptosis of the AST in neonatal rat hippocampus injured by HI.更多还原