Slit2N与丝素蛋白混合物诱导大鼠海马神经元迁移的体外模型建立

摘要

目的:利用Slit排斥导向迁移和丝素蛋白,探索建立简便可行、经济实惠、作用持久的神经元导向迁移模型新方法.方法:提取SD新生鼠海马组织,以专用细胞培养片体外培养神经元,分为空白对照组、单纯丝素蛋白、单纯Slit 2N和Slit 2N与丝素蛋白混合物组(以下简称混合物组),分别随机选择不同视野下50个神经元,用显微镜拍照记录胞体坐标及突起状态,除空白对照组外,其他3组均距每个神经元100 μm处添加相应诱导物,共观察30 min,再次记录后,用免疫荧光染色法鉴定细胞性质及其阳性率.结果:单纯Slit 2N组和混合物组均可见突起向浓度低处迁移或弯曲,且长度有所缩短,空白对照组和单纯丝素蛋白组未见明显变化.突起变化的平均持续时间及平均长度差从大到小依次为混合物组、单纯Slit 2N组、单纯丝素蛋白组(P＜0.05),单纯丝素蛋白组和空白对照组间无明显变化(P＞0.05).四组神经元MAP-2阳性率均达到90％以上.结论:丝素蛋白对Slit 2N诱导大鼠海马神经元迁移作用无明显影响,可有效减缓Slit 2N扩散速度,使作用时间延长,为治疗中枢神经系统疾病建立三维神经定向修复提供有利的体外实验构建基础.%Objective:To explore a simply feasible and affordable method to establish neuritis migration model induced by Slit 2N and silk fibroin mixture in the rat hippocampal neurons in vitro.Methods:Neurons were derived from SD rat hippocampal tissues and cultured with a special cell culture-plate in vitro.The cultured neurons were divided into four groups,named as control group,pure silk fibroin,pure Slit 2N and Slit 2N mixture with silk fibroin (mixture group),and 50 different neurons were randomly selected in each group.Moreover,we photographed and recorded the soma coordinate and added the silk fibroin,Slit 2N and mixture to each neurite with a distance of 100 μm,except control group.Record again after 30 ain.Property and positive rate of cells were identified by immunofluorescence staining.Results:Neurites of the pure Slit 2N group and the mixture group became shorter,and there was no significant change in the pure silk fibroin and control groups.The results showed that the average duration and length difference before and after changed were in descending order of mixture group,Slit 2N group,silk fibroin group (P ＜ 0.05),and the silk fibroin and control groups were no significant change (P ＞ 0.05).The positive rate of MAP-2 in four groups was more than 90％.Conclusion:There were no significant effects of Silk fibroin on induced by Slit 2N in rat hippocampal neuron migration.It had an effect on reducing Slit 2N diffusion rate and extending its working time.It provides an advantageous construction method in vitro on based 3D directed neural repair for the treatment of central nervous system diseases.