双色荧光示踪鸡胚脊髓两侧连合纤维投射研究方法的建立  

A method of simultaneously tracing the trajectory of bilateral commissural axons in developing chick spinal cord using dual-color fluorescence

作　　者：于亚楠[1] 刘彦礼[1,2] 杨慈清[1,2] 张必超[1] 徐振平[1,2] 林俊堂[1,2] 

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

出　　处：《解剖学报》2016年第2期284-288,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金(31440049);新乡医学院研究生科研创新支持计划(YJSCX20426Y)

摘　　要：目的建立1种双色荧光示踪鸡胚脊髓两侧连合纤维投射的实验方法。方法鸡胚孵育至胚龄2.5-3d,通过鸡胚活体原位电转基因技术将携带有报告基因绿色荧光蛋白（GFP）的质粒（p CAGGS-GFP）准确注射到鸡胚脊髓腔,实现定时、定位活体电转基因。转染后继续孵育至6d,取GFP阳性表达的胚胎,部分做脊髓横向切片,部分利用open-book技术将脊髓展开观察连合纤维的发育情况,每组至少取3个标本。其后在脊髓非转染侧连合神经元所在之处,点状注射Di I乙醇溶液,封片后于4℃避光孵育3d,在荧光显微镜下观察脊髓连合纤维投射情况。结果脊髓横切及open-book结果显示,鸡胚脊髓GFP阳性转染侧的神经元轴突穿过底板投射到脊髓对侧;同时在open-book结果中还可观察到,转染侧轴突穿过底板后分别沿腹索和外侧索向头尾部投射;Di I标记的非转染侧连合神经元轴突也同样穿过底板投射到对侧,并在侧索白质内延伸。结论本实验成功建立了1种双色荧光示踪鸡胚脊髓两侧连合纤维投射的研究方法,为研究脊髓神经发育提供技术保障。Objective To develop a method of simultaneously tracing the development of commissural axons from bilateral chick spinal cord using dual-color fluorescent. Methods The p CAGGS-GFP plasmid was transfected into the chick spinal cord using ovo electroporation after incubation of fertilized eggs for HH stage17-18. GFP-positive chick embryos were obtained at day 6. At least three samples were collected for each group. The collected specimens were undergone either transverse section or ＂open-book＂processing. In ＂open-book＂processing,the spinal cord was unfolded and placed on the glass slide. After injecting Di I solution into the contralateral side of transfected region,the samples were incubated for 3d at 4℃,and commissural axons were observed under a fluorescent microscopy. Results The results of GFP labeling from both transverse section and open-book processing showed that commissural axons crossed the midline region through the floor plate to the contralateral side of the spinal cord,then they projected rostrally and caudally along the antero-caudal（ AP） axis within ventral fasciculus and lateral fasciculus. Di I tracing showed similar trajectory of commissural axons. Conclusion Dual-color fluorescent tracing method is successfully established,which may provide a new method for the study of development of commissural axons of the spinal cord.

关 键 词：胚胎 脊髓 连合纤维 投射 活体电转 DI I示踪 鸡 

分 类 号：Q954.48[生物学—动物学]