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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
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（pCAGGS-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DiI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；DiI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 pCAGGS-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 DiI 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. DiI 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.
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国家自然科学基金项目;新乡医学院研究生科研创新支持计划资助项目
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