神经生长因子对新生大鼠脑神经元细胞缺氧缺血损伤的保护作用

Protective effect of nerve growth factor on hypoxic-ischemic neuronal cells in brain of neonatal rats

目的:观察神经生长因子对新生大鼠脑神经元细胞缺氧缺血性损伤的保护作用。方法:实验于2004-01/06在东南大学临床医学院中心实验室完成。①体外培养新生脑皮质神经元细胞后,改良Goldberg方法建立神经元细胞“缺氧缺血”损伤模型。②应用光学显微镜、透射电镜观察不同组之间(正常组、缺氧缺血组、缺氧缺血后即刻给以神经生长因子-80μg/L治疗组)神经元细胞形态、超微结构改变。③应用细胞免疫化学方法检测神经生长因子治疗组细胞色素C基因在转录及翻译水平的表达,与正常组及缺氧缺血组进行比较。结果:①光镜测量缺氧缺血组细胞突起长度明显低于正常组和治疗组[(7.591±1.354),(23.7595±0.945),(23.466±1.005)μm],而其细胞色素C阳性率及AIFmRNA表达相对含量明显高于另两组[(69.318±6.646)%,(41.193±4.472)%,(41.817±4.364)%,P<0.05]。上述测值正常组与治疗组差异无显著性(P>0.05)。②透射电镜观察细胞:缺氧缺血对照组细胞核形状不规则,染色质凝聚成块,核周间隙明显增厚,线粒体肿胀空泡化;正常组神经元细胞及治疗组神经元细胞核大呈圆形或椭圆形,常染色质均匀分布,可见明显的核仁,胞浆内线粒体、粗面内质网结构清晰,核糖体丰富。结论:①体外培养新生大鼠脑皮质神经元细胞,剥夺氧及糖6h后能够模拟缺氧缺血性神经元细胞损伤。与正常组相比,缺氧缺血模型组光镜及电镜示凋亡小体存在,线粒体结构异常;细胞色素C细胞免疫化学示阳性率明显增加。②适宜浓度(80μg/L)的外源性神经生长因子能够促进缺新生大鼠脑缺氧缺血性神经元细胞修复,保护线粒体。

AIM： To observe the protective effect of nerve growth factor on hypoxicischemic neuronal cells in brain of neonatal rats. METHODS： The experiment was carried out in the central laboratory of Clinical Medical School of Southeast University between January and June 2004. （1） After the neuronal cells in neonatal cortex were cultured in vitro, models of hypoxic-ischemic injury were induced by the improved Goldberg＇s method. （2）The changes of form and uhrastructure of neuronal ceils among difference groups （normal group, hypoxie-isehemic injury group, 80μg/L nerve growth factor treated group immediately after hypoxic-ischemic injury） were observed under light microscope and transmission microscope. （2） The expression of eytachrome C at the levels of transcription and translation in the nerve growth factor treated group was detected with cellular immunohistochemical method, and then compared with those in the normal group and hypoxic-ischemic injury group. RESULTS： （1） The neurite length measured under light microscope in the hypoxic-ischemic injury group was obviously shorter than those in the normal group and nerve growth factor treated group [（7.591±1.354）, （23.759 5±0.945）, （23.466±1.005） μm], the the positive rate of cytcghrome C and the relative contents of AIF mRNA expression were obviously higher than those in the other two groups [（69.318±6.646）%, （41.193±4.472）%, （41.817±4.364）%, P 〈 0.05], which had no significant differences between the normal group and nerve growth factor treated group （P 〉 0.05）. （1） Cells observed under transmission microscope： In the hypoxic-ischemic injury group, the shape of nucleus was irregular, chromatin agglutinated into blocks, the perinuclear space was obviously thickened, the swelling of mitochondrion turned to vacuole. The neuronal cells in the normal group and the neuronal nucleus in the treated group were big and round or oval-shape, euchromatin was evenly distributed, obvious nucleolus could observed, the mltochondrion in cytoplasm and the structure of rough endoplasmic reticulum was clear, and ribosome was rich. CONCLUSION： （1） The in vitro cultured neuronal cells of neonatal brain can imitate the hypoxic-ischemic injury of neuronal cells after deprivation of oxygen and glucose for 6 hours. Compared with the normal group, the light microscope and electron microscope in the hypoxic-ischemic injury group showed that the apoptotic body existed, the structure of mitochondrion was abnormal. The cellular immunohistochemial results indicated that the positive rate of cytochrome C was obviulsy increased. （2） Ectogenous nerve growth factor of proper concentration （80μg/L） can accelerate the repair of hypoxic-ischemic neuronal cells, and protect mitochondrion.