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目的利用Sprouty2基因阻断成纤维细胞生长因子(FGF)信号,探讨FGF在早期鸡胚胎发育过程中对神经嵴细胞迁移的影响及其机制。方法通过体内培养的方法孵育鸡胚至HH9期,通过显微注射的方法将Sprouty2-绿色荧光蛋白(GFP)质粒注射入神经管腔内。实验侧使用电穿孔转染的方法转染胚胎半侧神经管,另一侧正常神经管设为对照侧。采用神经嵴细胞特异标记物HNK1免疫荧光的方法检测Sprouty2基因阻断FGF信号后是否影响胚胎头部和躯干部神经嵴细胞的迁移过程。随后,进一步通过检测神经细胞钙黏分子N-Cadherin的表达来观察细胞之间黏附作用的改变。结果 HNK1免疫荧光检测结果显示,Sprouty2转染侧即阻断FGF信号通路后,HNK1在早期鸡胚胎的头部和躯干部的表达量均比对照侧的表达量增多;而神经细胞钙黏分子N-Cadherin检测结果表明,Sprouty2转染侧和正常对照侧N-Cadherin在头部和躯干部神经管上表达量的差异均无显著性。结论 Sprouty2基因阻断FGF信号后,促进了早期鸡胚胎神经嵴细胞的迁移,但是FGF信号对此过程的影响可能不是由神经钙黏分子N-Cadherin介导的。
OBJECTIVE: To use Sprouty2 gene to block the signal of fibroblast growth factor (FGF) and to investigate the effect of FGF on migration of neural crest cells in early embryo development and its mechanism. Methods The chicken embryos were incubated with HUVECs in vivo and the Sprouty2-green fluorescent protein (GFP) plasmid was injected into the neural lumen by microinjection. Experimental side using electroporation transfection transfected embryonic half of the neural tube, the other side of the normal neural tube as the control side. The neural crest-specific marker HNK1 immunofluorescence was used to detect whether Sprouty2 blockade of FGF signaling affected the migratory process of neural crest cells in the head and trunk of embryos. Subsequently, the change of adhesion between cells was further observed by detecting the expression of N-Cadherin, a neuronal cell calcium-binding molecule. Results The results of HNK1 immunofluorescence showed that the expression of HNK1 in the head and trunk of early chicken embryos increased more than that in the control side after blocking the FGF signaling pathway by Sprouty2 transfection; however, the expression of Ca2 + Cadherin results showed that there was no significant difference in the expression of N-Cadherin between the Sprouty2 transfection side and the normal control side in the neural tube of the head and trunk. Conclusion Sprouty2 gene can block the migration of neural crest cells of chicken embryos after blocking FGF signaling. However, the effect of FGF signaling on this process may not be mediated by N-Cadherin.