神经生长因子对儿童难治性癫(癎)脑神经细胞及早期生长反应基因1表达的影响

摘要

目的 探讨外源性神经生长因子(NGF)对儿童难治性癫(癎)脑(癎)性放电区神经细胞及早期生长反应基因1(Egr-1)表达的影响.方法 采用解离细胞即刻培养技术,将术中取下脑海马(癎)性放电区组织块解离后进行培养,设立对照组和NGF 12.5、50、100 ng/ml 3个不同浓度的加药组,孵育4 h后将其固定;通过免疫荧光细胞化学法,以双苯酰亚胺(Bb)染核,联合细胞特异性标志蛋白GFAP、MAP2标记星形胶质细胞、神经元,计算各组神经细胞总数,并利用RT-PCR技术半定量分析细胞中Egr-1mRNA表达情况.结果 外源性NGF作用4 h后,海马区存活的神经细胞总数及GFAP(+)的星形胶质细胞、MAP2(+)的神经元数较空白对照组升高,且与NGF剂量呈量效关系,随NGF浓度增高而增加(P<0.05);与此同时,神经细胞表达Egr-1mRNA也随NGF浓度增高而表达量增加,即A_(Egr-1)/A_(β-actin)值与NGF剂量呈量效关系(P<0.01);且存活的神经细胞数与其表达的Egr-1mRNA量呈正相关(r=0.780,P<0.01).结论 外源性NGF对(癎)性放电区损伤神经细胞有保护作用,其作用可能通过促进Egr-1基因的表达来实现.%Objective To investigate the effect of nerve growth factor (NGF) on the neural cells and expression of early growth response gene-1 (Egr-1 gene) in the hippocampus discharge zone of childhood intractable epileptics. Methods Acutely dissociated cell suspensions of childhood hippocampus discharge zone with non specific pathological changes (n = 16) were exposed to NGF group and control group respectively. There were three subgroups exposed to NGF at the levels of 12.5, 50, 100 ng/ml in NGF group. After 4 hr culturing, the astrocytes and neurons were lablled by Bb immunostain with the specific markers such as GFAP and MAP2. The total number of neural cells was counted under inversion fluorescence microscope and the Egr-1mRNA expression was detected by semiquantitative RT-PCR analysis. Results There were significant differences of the numbers of neural cells survived in hippoeampus region between the NGF group and the non-added NGF group (P < 0.01). Among the different levels of NGF 12.5, 50, 100 ng/ml, the number of the total cultural neurons and GFAP(+) astrocytes, MAP_2(+) neurons were increased with ascending levels of NGF (P < 0.05). At the same time, the expression of Egr-1mRNA (A_(Egr-1)/A_(β-actin)) in the NGF groups was also increased (P < 0.01). There was positive correlation between the number of the survivable neural cells and Egr-1mRNA quantity (r = 0.780, P < 0.01) among the three NGF groups. Conclusions NGF can protect the neural cells of epileptic discharge zone by promoting the expression of Egr-1 gene.