鸡胚发育早期Sonic Hedgehog在脊髓中的异位表达对形态结构及相关蛋白表达的影响

Sonic Hedgehog Ectopic Expression Affects the Structure and Protein Expression of the Spinal Cord During Chicken Early Embryonic Development

该文主要分析音猬因子（Sonic Hedgehog,Shh）在鸡胚发育过程中对脊髓形态结构的形成和相关蛋白表达的影响。实验过程采用鸡胚带壳开窗培养技术,待胚胎发育至第3 d,将2μg/μL pCAGGS-Shh和0.25μg/μL pCAGGS-GFP质粒以1：8浓度混合,将0.1~0.5μL混合液准确地注射到神经管,在电压18 V、每次脉冲60 ms、间隔100 ms、电脉冲6次的条件下进行定时定位活体电转基因,电转后6 h开始到5 d分别收集胚胎,冰冻切片,采用荧光免疫组化和DAPI染色观察组织形态结构及相关蛋白的变化。结果表明,电转后6 h便可以观察到GFP的表达,24 h时Shh在脊髓中的异位表达能够诱导转录因子Nkx2.2（NK2 homeobox 2）的表达,并且能够抑制Pax7（paired-type homeobox 7）的表达,而Shh异位表达时脊髓的结构发生了明显的改变;说明Shh作为脊髓发育过程重要的信号分子,其异位表达能够诱导和抑制相关蛋白的表达,影响脊髓正常发育。

This research mainly analyzes the effect of Sonic Hedgehog （Shh） on the formation of the morphological structure of spinal cord and expression of related proteins during the chicken embryonic development. Using in ovo electroporation, 2 μg/μL pCAGGS-Shh and 0.25 μg/μL pCAGGS-GFP plasmid were mixed by 1：8 and then with 0.1-0.5 μL accurately injected into the spinal cord on E3 embryo. The condition of in ovo electropo- ration was voltage 18 V, each pulse 60 ms, interval 100 ms and total 6 times pulses. The positive embryos were col- lected after electroporation from 6 h to 5 d, for frozen sections. Fluorescent immunohistochemistry and DAPI stain- ing were carried out to observe the change of the morphological structure and the related proteins expression. The results show that GFP expression was observed in 6 h after in ovo electroporation. After 24 h of electroporation, it was detected that the Shh ectopic expression induces the expression of Nkx2.2 （NK2 homeobox 2） in the spinal cord, however Pax7 （paired-type homeobox） expression was inhibited, furthermore, Shh ectopic expression leadsto a change of the spinal cord structure. As an important signaling molecule, ectopic expression of Shh induces or inhibits the expression of related proteins, and leads to the spinal cord malformations