解剖学报 ›› 2014, Vol. 45 ›› Issue (5): 724-728.doi: 10.3969/j.issn.0529-1356.2014.05.025

• 神经生物学 • 上一篇    

一种鸡胚发育过程脊髓神经纤维投射研究方法的建立

胡阿珍1 杨慈清1,2 付苏雷2 贾阳阳1 李晗2 郭志坤1 林俊堂1,2*   

  1. 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
  • 收稿日期:2013-11-21 修回日期:2014-01-06 出版日期:2014-10-06 发布日期:2013-10-06
  • 通讯作者: 林俊堂 E-mail:linjuntang2002@yahoo.co.uk
  • 基金资助:

    Delta原钙黏蛋白(delta-protocadherin)在脊髓发育过程中的表达和功能调控研究;ADAM17过表达促进血管生成及其与肿瘤发生关系的研究;ADAM17过表达促进血管生成及其与肿瘤发生关系的研究

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

HU A-zhen1 YANG Ci-qing 1,2 FU SU-lei2 JIA Yang-yang1 LI Han2 GUO Zhi-kun1 LIN Jun-tang 1,2*   

  1. 1. Key Laboratory of He’nan Province for Medical Tissue Regeneration;2. College of Life Science and Technology, Xinxiang Medical University, He’nan Xinxiang 453003, China
  • Received:2013-11-21 Revised:2014-01-06 Online:2014-10-06 Published:2013-10-06
  • Contact: LIN Jun-tang E-mail:linjuntang2002@yahoo.co.uk

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。

关键词: 脊髓, 神经纤维, 投射, 活体电转, 鸡胚

Abstract:

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.

Key words: Spinal cord, Nerve fiber, Projecting, Electroporationin vivo, Chicken embryo