【作者】 张盼盼；

【导师】 王红阳；

【作者基本信息】 河北联合大学 ， 内科学， 2011， 硕士

【摘要】 目的通过建立重度慢性间歇低氧大鼠模型,观察大鼠海马神经细胞ERK通路及凋亡的变化,探讨ERK通路对神经系统的影响。方法48只成年雄性Wistar大鼠采用随机数字法分组原则分为2组:5%间歇低氧组(5%CIH组)、空白对照组(UC组),每组按不同时间点分为4个亚组:分别为2、4、6、8w组。将5%CIH组大鼠循环交替给予不同流速的氮气和压缩空气(每一循环120s,使5%CIH组箱内最低氧浓度至5%,然后恢复至21%,8h/d);UC组大鼠不给予任何处理。分别在实验第2、4、6、8w后,采用免疫组化学法检测大鼠海马神经细胞ERK 1/2、c-fos蛋白表达;采用原位细胞凋亡方法检测大鼠海马神经细胞凋亡变化。应用Motic医学图像分析系统对免疫组化结果ERK1/2及c-fos蛋白半定量分析,结果用积分光密度(IOD)值表示。观察各组大鼠第2、4、6、8w大鼠海马神经细胞ERK 1/2、c-fos蛋白的表达和凋亡指数的变化。统计学处理采用SPSS13.0统计软件包进行数据分析,所得数据用均数±标准差(xˉ±s)表示,组间比较采用独立样本T检验,组内比较采用单因素方差分析,多重比较采用LSD法,以P<0.05为差异有统计学意义。结果1重度慢性间歇低氧对海马神经细胞ERK1/2蛋白表达的影响1.1光学显微镜下观察到大部分阳性表达细胞胞浆呈棕黄色或浅黄色。各组大鼠均有阳性表达细胞,阳性细胞的表达程度不同。UC组可见散在阳性表达细胞;5%CIH组阳性表达细胞明显增多,主要集中在海马CAl区。1.2 5%CIH组海马CAl区神经细胞ERK1/2蛋白表达IOD值在2、4、6、8w均显著高于UC组(P <0.05),UC组海马CAl区神经细胞ERK1/2蛋白表达IOD值不同时间水平无显著差异(P﹥0.05),5%CIH组海马CAl区神经细胞ERK1/2蛋白表达IOD值不同时间水平有显著差异(P <0.05),表现为:ERK1/2蛋白表达IOD值随时间的延长呈先上升后下降趋势,于第6周达到高峰后下降。2重度慢性间歇低氧对海马神经细胞c-fos蛋白表达的影响2.1光学显微镜下观察到大部分阳性表达细胞胞核呈棕黄色或浅黄色,各组大鼠均有阳性表达细胞,阳性细胞的表达程度不同。UC组可见散在阳性表达细胞;5%CIH组阳性表达细胞明显增多,主要集中在海马CAl区。2.2 5%CIH组海马CAl区神经细胞c-fos蛋白表达IOD值在2、4、6、8w显著高于UC组(P <0.05),UC组海马CAl区神经细胞c-fos蛋白表达IOD值不同时间水平无显著差异(P﹥0.05),5%CIH组海马CAl区神经细胞c-fos蛋白表达IOD值不同时间水平有显著差异(P<0.05),表现为:c-fos蛋白表达IOD值随时间的延长呈先上升后下降趋势,于第6周达到高峰后下降。3重度慢性间歇低氧对海马神经细胞凋亡的影响3.1光学显微镜下观察到凋亡细胞形态呈圆形或椭圆形,胞核呈棕黄色或棕褐色,形态呈碎点状,不规整,大小不一致,非凋亡细胞胞核被苏木素复染呈蓝色,核相对较大,形态大小较为一致。光学显微镜下观察到各组大鼠神经细胞均有凋亡, UC组可见散在凋亡细胞, 5%CIH组凋亡细胞明显增多,主要分布在海马CAl区。3.2 5%CIH组凋亡指数在2、4、6、8w显著高于UC组(P<0.05),UC组海马CAl区神经细胞凋亡指数不同时间水平无显著差异(P﹥0.05),5%CIH组海马CAl区神经细胞凋亡指数不同时间水平有显著差异(P<0.05),表现为:神经细胞凋亡指数随时间的延长呈先上升后下降趋势,于第6周达到高峰后下降。结论1重度慢性间歇低氧可激活大鼠海马神经细胞ERK通路,同时诱导大鼠海马神经细胞凋亡。2 ERK通路可通过介导大鼠海马神经细胞凋亡的发生,进而导致重度慢性间歇低氧早期脑损害的形成。更多还原

【Abstract】 Objective a rat model of serious chronic intermittent hypoxia was established, the changes of ERK pathway and apoptosis in hippocampal neurocyte was observed, and effects of ERK pathway on nervous injury were explored.Method Adult male Wistar rats (n=48) were randomly divided into 5% intermittent hypoxia group (5%CIH group), control group (UC group), According to different time points each group was divided into four sub-groups:respectively 2、4、6、8w. 5%CIH group were given alternating cycle of different nitrogen and compressed air flow rate (each cycle of 120s, so that 5%CIH group inside the minimum oxygen concentration to 5%, and then recovered to 21%, 8h/d); UC group were not given any treatment. Respectively after 2、4、6、8w of intermittent hypoxia,By immunohistochemical method to detect expression of c-fos and ERK1/2 protein in hippocampal neurocyte;By TUNEL method to detect serious chronic intermittent hypoxia induced neuronal apoptosis in hippocampal neurocyte. To analyze c-fos and ERK1/2 protein semi-quantitatively by Motic Medical image analysis system. The results with the 40 millimeter test optical density (IOD) value indicate. Changes of ERK1/2、c-fos protein and apoptotic index at 2、4、6、8w were observed in hippocampal neurocyte.Statistical analysis SPSS13.0 statistical package used for data analysis, the data with mean±standard deviation(xˉ±s). Independent-Sample T test was used to the comparison between groups. One-Way ANOVA was used to the comparison within group, LSD method was used on multiple comparisons, P <0.05 was considered statistically significant. Result1 Effects on ERK1/2 protein in hippocampal neurocyte after serious chronic intermittent hypoxia.1.1 Most of the cytoplasm of positive cells stained in brown or pale yellow was observed under Optical microscope. Rats of each group have positive cells. The degrees of positive cells are different. UC group shows scattered positive cells; 5%CIH group of positive cells were significantly increased, most of positive cells were in CA1 of hippocampus.1.2 In CA1 of hippocampus,expression of ERK1/2 protein in IOD values in CIH 5% Group is more than in UC Group at 2、4、6、8w (P <0.05); Expression of ERK1/2 protein in IOD values in UC Group at different times there was no significant difference in CA1 of hippocampus (P > 0.05); expression of ERK1/2 protein in IOD values in 5%CIH group at different time levels were significantly different in CA1 of hippocampus (P <0.05), as follows: it roses to the peak at 6w and then decreased.2 Effects on c-fos protein in hippocampal neurocyte after serious chronic intermittent hypoxia.2.1 Most of the nuclei of positive cells stained in brown or pale yellow were observed under Optical microscope. Rats of each group have positive cells. The degrees of positive cells are different. UC group shows scattered positive cells; 5%CIH group of positive cells were significantly increased, most of positive cells were in CA1 of hippocampus.2.2 In CA1 of hippocampus,expression of ERK1/2 protein in IOD values in CIH 5% Group is more than in UC Group at 2、4、6、8w (P <0.05); Expression of ERK1/2 protein in IOD values in UC Group at different times there was no significant difference in CA1 of hippocampus (P > 0.05); expression of ERK1/2 protein in IOD values in 5%CIH group at different time levels were significantly different in CA1 of hippocampus (P <0.05), as follows: it roses to the peak at 6w and then decreased.3. Effects on neuronal apoptosis in hippocampal neurocyte after serious chronic intermittent hypoxia. 3.1 Under Optical microscope , apoptotic cells stained in brown or tan color were round or oval nuclei, shape was broken form, which is not regular, size inconsistency, non-apoptotic cell nuclei were stained with hematoxylin blue, nuclei were relatively large, more consistent shape and size. Rats of each group were apoptotic neurons, UC group showed scattered apoptotic cells, Apoptotic cell of 5%CIH group was significantly increased; Apoptotic cells of 5%CIHare widely distributed, mainly in CAl of hippocampus.3.2 In CA1 of hippocampus,apoptotic index in 5%CIH Group is more than in UC Group at 2、4、6、8w (P <0.05); Apoptotic index in UC Group at different times there was no significant difference in CA1 of hippocampus (P > 0.05); Apoptotic index in 5%CIH group at different time levels were significantly different in CA1 of hippocampus (P <0.05), as follows: it roses to the peak at 6w and then decreased.Conclusionl Serious chronic intermittent hypoxia activated ERK pathway and induced apoptosis in hippocampal cells of rats.2 ERK pathway lead to the formation of early brain damage in serious intermittent hypoxia through which mediated apoptosis in hippocampal cells of rats.更多还原