两种荧光标记物对中枢神经系统内源性神经干细胞的标记效果比较

目的 观察DIL和DAPI两种不同的标记物对中枢神经系统内源性神经干细胞的标记效果.方法 清洁级健康成年SD大鼠36只,随机分为实验组(DIL组,DAPI组)和对照组(DMSO组,PBS组).使用立体定位仪,向实验组大鼠侧脑室注入0.2％ DIL或10μg/ml DAPI各10μl,对照组大鼠侧脑室相应注入DMSO或PBS各10μl.注射后2h和24h对动物进行神经功能损伤评分(NSS).采用激光共聚焦显微镜观察注射后1、3、7d脑室及脊髓颈、胸、腰段室管膜细胞的荧光标记情况,对目标区域荧光强度进行半定量分析,并对不同组别和时间点的脑室及脊髓标本测定结果进行比较.结果 注射DIL及DAPI后2h,大鼠NSS评分很低,与相应对照组相比差异无统计学意义(P＞0.05),24h后基本恢复正常.DIL注射后1d,侧脑室及各节段的脊髓室管膜细胞胞质均显示红色荧光,一直持续到注射后7d.DAPI注射后1d,侧脑室内标记的细胞核形态清晰,呈蓝色,脑室脉络丛大量细胞亦可见蓝色荧光标记,但脊髓各节段室管膜区的细胞均未见蓝色荧光,注射后3、7d的情况与1d类似.两种试剂标记后,目标区域内各时间点的荧光强度差异无统计学意义(P＞0.05).结论 DIL可对内源性神经干细胞的细胞质进行标记,适合作为侧脑室和脊髓室管膜内源性神经干细胞的标记物.DAPI可标记细胞核,适合用于侧脑室内源性神经干细胞的标记.脑室注射荧光染料对动物影响小,是一项相对安全的操作.

Comparison of the marker effects of two different fluorescent dyes in labeling endogenous neural stem cells in the central nervous system

Objective To observe the marker effects of two different fluorescent dyes,DIL and DAPI,in labeling endogenous neural stem cells(ENSCs) in rat central nervous system.Methods Thirty-six Sprague-Dawley rats were randomized into staining groups,comprising DIL group and DAPI group,and the corresponding control groups,including DMSO group for DIL group and PBS group for DAPI group.0.2% DIL 10μl or 10μg/ml DAPI 10μl was stereotactically injected into the lateral ventricle of rats of DIL group or DAPI group,while DMSO or PBS 10μl was introduced into that of DMSO group or PBS group.Neurological severity score(NSS) was determined 2 hours and 24 hours respectively after the operation.Rats were sacrificed at day 1,3,7 after the injection.Serial coronal sections of the brain and spinal cord were carried out on a cryostat,and then they were observed under a confocal microscope.The fluorescence intensity of the targeted area,which highlighted by labeled ependymal cells,in the brain and spinal cord of cervical vertebrae,thoracic vertebrae and lumbar vertebrae were semi-quantified.Fluorescence intensity of each section was measured in triplicate,and a mean value was obtained.Statistical analysis was performed on 3 data sets,randomly selected from sections of brain and spinal cord obtained at day 1,3,7.Results Two hours after DIL injection,the rats showed no evident neurological defect.NSS value was very low,and there was no significant difference compared with the DMSO group(P>0.05).Twenty-four hours later,normal neurological function recovered in all the rats.Red fluorescence could be seen in the cytoplasm of ependymal cells in the lateral ventricle and each spinal cord segment at day 1 after the DIL injection,and it did not disappear until the 7th day.Nuclei of DAPI-labeled lateral ventricle cells were blue,with clear nuclear morphology.Choroid plexus cells of the ventricle were also labeled.However,there was no blue fluorescence in the medulla oblongata or any segment of the spinal cord.The picture at day 3 and day 7 was similar to that of day 1.No significant difference was found between fluorescence intensity in DIL or DAPI stained cells(P>0.05) at any time point.Conclusions DIL may serve as a marker of the cytoplasm of ependymal cells in the brain ventricle and spinal central canal.DAPI,which is often used in the nuclear staining,can label the ENSCs in the brain ventricle.Intraventricular injection of fluorescent dye is a relatively safety procedure.