一种鸡胚发育过程脊髓神经纤维投射研究方法的建立  

A new method to study nerve fiber projecting in the spinal cord during chicken embryo development

作　　者：胡阿珍 杨慈清[1,2] 付苏雷[2] 贾阳阳 李晗[2] 郭志坤 林俊堂[1,2] 

机构地区：[1]河南省医用组织再生重点实验室,河南新乡453003 [2]新乡医学院生命科学技术学院,河南新乡453003

出　　处：《解剖学报》2014年第5期724-728,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(31000475);河南省教育厅科学技术研究重点资助项目(13A320866);新乡医学院重点研究领域招标课题资助项目(ZD2011-26)

摘　　要：目的建立一种鸡胚发育过程脊髓神经纤维投射的研究方法。方法采用鸡胚带壳开窗培养技术,在鸡胚胚龄3d(E3),通过活体电转基因技术将携带有报告基因绿色荧光蛋白(GFP)的质粒(pCAGGS-GFP)准确注射到脊髓腔进行定时定位活体电转,转染后3d在体视荧光显微镜下进行观察;取出GFP阳性表达的胚胎,剥离出脊髓,从顶板处破开之后将脊髓展开,用4%多聚甲醛固定1h后,对神经钙黏蛋白(N-cadherin)进行免疫荧光染色,用4’6-二脒基-2-苯基吲哚(DAPI)染细胞核;封片后在荧光显微镜下观察神经纤维投射情况。结果对比横向切片和脊髓展开标本,两者均观察到GFP阳性转染侧的神经元纤维穿过底板沿对侧脊髓白质区边缘投射到神经结节,在脊髓展开标本中还可观察到神经纤维穿过底板再纵向向脑部投射;而N-cadherin免疫荧光染色结果表明,GFP基因的转染对机体正常的发育无明显影响。结论本实验建立了一种鸡胚发育过程脊髓神经纤维投射的研究方法。Objective To develop a method of studying fiber projecting in the spinal cord duiring chicken embryo development.Methods At embryonic incubation 3 day （E3）, pCAGGS-green fluorescent protein （GFP） plasmid was injected into the spinal cord using in vivo electroporation.Three days after transfection （E6）, GFP-positive embryos were collected under a stereo fluorescence microscope .Subsequently , the spinal cord was separated from the embryos and cut from the roof plate as an open book .After fixed with 4%paraformaldehyde （ PFA） for one hour , the opened spinal cords were used for immunohistochemistry with N-cadherin antibody and with DAPI for nuclei .Finally, the nerve fiber projecting was photographed and analyzed under a fluorescence microscope . Results Based on the opened spinal cord and immunostaining in the cryosection , we observed that the nerve fibers projected across the midline of the floor plate and reached to the sulcus terminalis along the white matter of the contra side .The immunoreaction against N-cadherin indicated that overexpression of GFP has no significant effect on chicken embryonic development .Conclusion A new method to study fiber projecting in the developing chicken spinal cord is established successfully in this study .

关 键 词：脊髓 神经纤维 投射 活体电转 鸡胚 

分 类 号：R322.85[医药卫生—人体解剖和组织胚胎学]