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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 establish a method of spinal cord nerve fiber projection during chicken embryo development. Methods The chick embryo cultured in vitro was stained with plasmid pCAGGS-GFP (pCAGGS-GFP) carrying the reporter gene by electroporation in the spinal cord cavity at the embryo age of 3d (E3) Tissue electrotransportation was performed at regular intervals. The cells were observed under stereomicroscope three days after transfection. The GFP positive embryos were removed and the spinal cord was dissected. The spinal cord was unfolded from the top plate and fixed with 4% paraformaldehyde for 1 hour. N-cadherin was stained with 4’6-diamidino-2-phenylindole (DAPI), and the nuclei were stained with a fluorescence microscope. Results Comparing transverse sections and spine expanded specimens, both neuronal fibers on the GFP-positive transfection side were observed projecting into the nodules along the margin of the floor along the margin of the white matter of the contralateral spinal cord, and nerve fibers were also observed in the unfolded spinal cord specimens Through the floor and then projected to the brain longitudinally; and N-cadherin immunofluorescence staining results showed that GFP gene transfection on the normal development of the body had no significant effect. Conclusions This study established a method for the projection of spinal nerve fibers during chicken embryonic development.