解剖学报

解剖学报 ›› 2015, Vol. 46 ›› Issue (5): 667-672.doi: 10.16098/j.issn.0529-1356.2015.05.016

• 神经生物学 • 上一篇    下一篇

鸡胚发育过程中音猬因子超表达对Pax家族基因表达的影响

李晗1杨慈清1,2乔梁1,2张必超1林俊堂1,2*   

  1. 1.新乡医学院生命科学技术学院，河南 新乡 453003; 2.新乡医学院河南省医用组织再生重点实验室，河南 新乡 453003
  • 收稿日期:2015-02-02 修回日期:2015-04-23 出版日期:2015-10-06 发布日期:2015-10-06
  • 通讯作者: 林俊堂 E-mail:juntang.lin@googlemail.com
  • 基金资助:

    国家自然科学基金资助项目;新乡医学院研究生科研创新支持计划资助项目

Influence of sonic hedgehog overexpression on Pax family in the spinal cord during chicken embryonic development

LI Han1 YANG Ci-qing 1,2 QIAO Liang 1,2 ZHANG Bi-chao1 LIN Jun-tang 1,2*   

  1. 1.College of Life Science and Technology, Xinxiang Medical University，He’nan Xinxiang 453003, China; 2.Key Laboratory of He’nan Province for Medical Tissue Regeneration, Xinxiang Medical University, He’ nan Xinxiang 453003, China
  • Received:2015-02-02 Revised:2015-04-23 Online:2015-10-06 Published:2015-10-06
  • Contact: Lin Juntang E-mail:juntang.lin@googlemail.com

摘要：

目的 探讨鸡胚发育过程中，音猬因子（Shh）基因在脊髓中超表达后对配对框（Pax）基因家族Pax3、Pax6以及Pax7蛋白表达的影响。 方法 在鸡胚龄2.5～3.0d（E2.5～E3）时，实验组利用鸡胚脊髓活体电转基因技术将携带Shh基因的质粒和携带有绿色荧光蛋白（GFP）报告基因的质粒共转入鸡胚脊髓，对照组则只转入报告基因质粒。E4时取材，每组至少取3例，冷冻切片后采用原位杂交技术检测Shh基因的mRNA表达水平，利用荧光免疫技术检测Shh在鸡胚脊髓中超表达后对Pax3，Pax6以及Pax7蛋白表达水平的影响，并将对照组及实验组中3种基因在脊髓两侧的表达量进行统计学分析。 结果 原位杂交结果表明，E4时Shh的mRNA主要表达在脊髓底板和脊索，实验组结果显示，利用鸡胚脊髓活体电转技术成功建立了Shh超表达模型。对照组中荧光免疫结果表明，Pax3主要表达于脊髓翼板、生肌节、背根以及背根神经节；Pax6主要表达于跨越翼板和基板的脊髓中间部位；Pax7则主要均匀表达于翼板、顶板和生肌节。Shh超表达后，脊髓转染侧与未转染侧相比Pax3、Pax6以及Pax7的表达量差异极显著（P<0.01），这3种基因在脊髓中的表达均被抑制。 结论 在鸡胚发育过程中，Pax家族Pax3、Pax6以及Pax7 3种基因在鸡胚脊髓中具有不同的表达模式，Shh超表达后对其均具有明显的抑制作用，显示这3种Pax基因受Shh信号通路调控。

关键词: 音猬因子, 配对框, 鸡胚脊髓活体电转, 原位杂交, 鸡

Abstract:

Objective To study the effect of sonic hedgehog (Shh) gene overexpression on Pax family in the spinal cord during chicken embryonic development.
Methods At E2.5-E3, Shh expression plasmids and green fluorescence protein (GFP) reporter gene plasmids were co-transfected into chicken embryonic spinal cord by in vivo electroporation. Only GFP reporter gene plasmids were transfected by the same way as control. Samples were collected from three or more embryos from the experimental and control groups and sliced at E4. Thenin situhybridization was used to detect whether Shh was really overexpressed. Immunofluorescence histochemistry was used to study whether Pax3, Pax6 and Pax7 were regulated by the overexpressed Shh.Statistical software was utilized to analyze the expression quantity of Pax3, Pax6 and Pax7 between two sides of the chicken spinal cords in the control and experimental group. Results The results ofin situhybridization showed that Shh was mainly expressed in the floor plate and notochord and the overexpression model of Shh in chicken spinal cord was built successfully in the experimental group. The immunohistochemical positive staining for Pax3 was distributed in alar plate,dermatome, dorsal root and dorsal root ganglion in the E4 chicken embryo spinal cord. The immunofluorescence positive signal for Pax6 was in the middle of spinal cord across the alar plate and the basal plate. The signal of Pax7 mainly existed evenly in the alar plate roof plate and dermatome. There was highly significant difference between electroplated side and normal side (P<0.01). The expressions of Pax3, Pax6 and Pax7 were all depressed by Shh overexpression. Conclusion There are different expression patterns in Pax3,Pax6,and Pax7 in the developmental chicken spinal cord, and the intensities of immunofluorescence positive signal for these genes are decreased by Shh overexpression, which indicates that they are regulated by Shh signal pathway.

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