【作者】 肖琴；

【导师】 陈娟； 熊英； 陈大鹏； 唐军；

【作者基本信息】 四川大学 ， 儿科学， 2007， 硕士

【摘要】 目的1．建立新生大鼠脑室周围白质软化(periventricular leukomalacia，PVL)模型，观察新生大鼠PVL时脑组织病理学改变。2．研究正常新生大鼠和PVL大鼠不同时间点脑组织ephrin-B3mRNA的表达变化，以及神经细胞凋亡的变化。3．探讨ephrin-B3与神经细胞凋亡的关系。4．探讨ephrin-B3在PVL损伤及CNS神经再生抑制机制中的意义。方法160只2日龄新生SD大鼠，随机分为2组，实验组(PVL组)86只，对照组(假手术组)74只，缺氧缺血(Hypoxic-ischemic，HI)后实验组死亡14只，对照组死亡2只，故进入实验的两组均为72只。采用Back等的方法建立PVL模型，分别于缺氧后0h、8h、24h、48h、72h、7d处死，HE染色观察新生大鼠PVL时脑组织病理学改变；DAPI染色检测缺氧缺血后不同时间点神经细胞凋亡；荧光定量RT-PCR检测缺氧缺血后不同时间点脑组织ephrin-B3mRNA表达变化。SPSS11．0进行统计分析，P＜0．05为差异有统计学意义。结果1．脑组织HE染色光镜观察：对照组细胞排列有序、胞核大、左右侧脑室对称。PVL组缺氧缺血后24h即见胼胝体细胞稀疏、排列紊乱；48小时纹状体内细胞稀疏、出现核固缩；7d可见侧脑室旁白质呈筛网状坏死。2．ephrin-B3mRNA的表达变化：对照组大鼠脑白质中ephrin-B3mRNA表达各时间点无明显变化，差异无统计学意义(P＞0．05)；实验组大鼠脑白质中ephrin-B3mRNA表达在缺氧缺血后8h开始升高，72小时达到峰值，7天开始下降，组内不同时间点两两比较，差异有统计学意义(P＜0．05)，与对照组比较，在8h、24h、48h、72h、7d差异有统计学意义(P＜0．05)。3．细胞凋亡检测：对照组大鼠脑室周围白质纹状体、胼胝体区在各个时间点均有少量凋亡细胞，实验组大鼠纹状体和胼胝体区细胞凋亡指数(apoptosis index，AI)在缺氧缺血后8小时开始升高，48小时达到高峰，72小时开始降低，与对照组相比，在8h、24h、48h、72h差异有统计学意义(P＜0．05)，7天时与对照组相比差异无统计学意义(P＞0．05)。结论1．脑组织HE染色光镜观察发现实验组大鼠缺氧缺血后出现细胞稀疏、排列紊乱、核固缩、侧脑室旁白质筛网状坏死，符合PVL病理改变，且出现生长发育不良、神经行为异常，证实PVL模型制作成功。2．新生大鼠PVL时，脑白质ephrin-B3mRNA表达显著增加，而对照组ephrin-B3mRNA表达几乎没有变化，提示ephrin-B3参与了PVL缺氧缺血性脑损伤，可能与CNS神经元再生抑制机制有关。3．新生大鼠PVL时，脑组织细胞凋亡指数较对照组明显增加，证实细胞凋亡仍为未成熟鼠脑缺氧缺血(Hypoxic-ischemic，HI)损伤的主要病理形式，其参与了PVL的损伤过程。4．新生大鼠PVL时，脑组织细胞凋亡指数和ephrin-B3mRNA表达均呈先升高后降低的趋势，提示两者存在一定的相关性，但二者高峰时间并不重合，推测除了ephrin-B3还存在其它因素参与神经细胞凋亡损伤。更多还原

【Abstract】 Objective1.To set up the neonatal rat PVL ( Periventricular leukomalacia) model, to detect the pathological changes of brain in those with PVL ;2.To study the expression of ephrin-B3mRNA and change of apoptosis in the brain tissues of neonatal rats with PVL;3.To investigate the correlations between ephrin-B3 and apoptosis.4.To explore the functions of ephrin-B3 in mechanism during PVL and CNS regrowth inhibition.MethodsA total of 160 2-d SD rats were randomly divided into experimental group (86) and control group(74). After establishment of PVL model ,the rats were executed at Oh, 8h,24h,48h,72h, 7d after hypoxic-ischemia. HE stain and light microscope were used to observe the pathological changes of brain tissues.Real time RT-PCR method was applied to detect the changes in the expressions of ephrin-B3mRNA,DAPI was applied to detect apoptotic cells in the brain tissues. SPSS 11.0 software was used to analyze the data, a =0.05 was considered the level of significance. Results1. Observed by light microscope: In the control group, brains cells were arrayed in an orderly manner with big nucleus, less cytoplasm and symmetrical vertical on both left and right sides; while in the experimental group, the pathological changes of brain tissues included cellular swelling, pyknosis, reticular necrosis and so on in early period of hypoxic-ischemic(HI). 7d after HI, sieve-like necrosis was observed in the preventricular white matter.2. Expression of ephrin-B3mRNA : the expression of ephrin-B3mRNA in the experimental group reached the peak value at 72h, demonstrating significant differences at 8h,24h,48h,72h, 7d compared with the control group(P<0.05).3. changes of apoptosis index: AI reached the peak value at 48h, demonstrating significant differences at 8h,24h,48h,72h compared with the control group(P<0.05).Conclusions1.the pathological changes of brain tissues from the 2-d SD rats with PVL have been proved to accord with PVL by the observation under light microscope ,and also the experimental group expressed lower weight and abnormal behavior,Which means that the establishment of PVL model is successful.2.In the PVL neonatal rat, ephrin-B3mRNA increased significantly compared with the control group, which suggests that ephrin-B3 may participate in the pathogenesis of PVL,it may play important functions in CNS regrowth inhibition. 3. the AI increased significantly compared with the control group, which suggests that apoptosis is the major pathogenic pattern of HI for immature rats.4.In the PVL neonatal rat,the Expression of ephrin-B3mRNA and the apoptosis index have similar tendency ,which suggests that there are some correlations between ephrin-B3 and apoptosis in the brain tissues.更多还原