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目的构建鸡源Wnt3a融合蛋白真核表达载体(pCAG-MCs-Wnt3a-EGFP),并探讨其在鸡胚脊髓发育过程中超表达后对神经前体细胞增殖及轴突形成的影响。方法利用分子生物学手段,提取鸡胚脊髓总RNA并获得Wnt3a片段,将其克隆到pCAG-MCs-EGFP载体中构建pCAG-MCs-Wnt3a-EGFP表达载体。在鸡胚发育至2.5~3d(E2.5~E3)时,利用鸡胚活体电转技术将pCAG-MCs-Wnt3a-EGFP(实验组)和pCAG-MCs-EGFP(对照组)质粒分别转入鸡胚脊髓,E4时取材切片,每组5个胚胎组织,采用免疫荧光染色技术检测Wnt3a和增殖细胞核抗原(PCNA)蛋白表达变化分析Wnt3a与细胞增殖间的关系,根据载体自发绿色荧光蛋白(GFP)观察脊髓神经前体细胞轴突生成情况。结果 pCAG-MCs-Wnt3a-EGFP表达载体基因测序结果与Gene bank中基因序列一致,将pCAGMCs-Wnt3a-EGFP导入鸡胚脊髓中发现绿色荧光。在脊髓组织切片水平上,免疫荧光染色结果表明,Wnt3a在鸡胚脊髓中能够超表达。Wnt3a超表达后,与对照组比较,含有轴突的神经元数量明显减少(n=3,P<0.01),而PCNA的表达量显著增加(n=3,P<0.01)。结论成功构建了鸡源性Wnt3a融合蛋白真核表达载体,并证实Wnt3a在鸡胚发育过程中促进神经前体细胞的增殖并抑制轴突的形成。
Objective To construct the eukaryotic expression vector (pCAG-MCs-Wnt3a-EGFP) of chicken Wnt3a fusion protein and investigate its effect on proliferation and axon formation of neural precursor cells after overexpression of chicken embryonic spinal cord. Methods The total RNA of chicken spinal cord was extracted by molecular biology and the Wnt3a fragment was obtained. The fragment was cloned into pCAG-MCs-EGFP vector to construct pCAG-MCs-Wnt3a-EGFP expression vector. The pCAG-MCs-Wnt3a-EGFP (experimental group) and pCAG-MCs-EGFP (control group) plasmids were transfected into chickens at 2.5-3 days (E2.5 ~ E3) The embryonic spinal cord was harvested at E4 for 5 days. The expression of Wnt3a and proliferating cell nuclear antigen (PCNA) were detected by immunofluorescence staining. The relationship between Wnt3a and cell proliferation was analyzed. According to the results of GFP, To observe the formation of axonal neurons in spinal cord. Results The gene sequence of pCAG-MCs-Wnt3a-EGFP expression vector was identical with that of Genebank. The green fluorescent protein was found when pCAGMCs-Wnt3a-EGFP was introduced into chicken spinal cord. At the level of spinal cord section, immunofluorescence staining showed that Wnt3a was overexpressed in chicken spinal cord. After Wnt3a overexpression, the number of axon-containing neurons was significantly decreased (n = 3, P <0.01), while the expression of PCNA was significantly increased (n = 3, P <0.01) compared with the control group. Conclusion The eukaryotic expression vector of chicken Wnt3a fusion protein was successfully constructed and proved that Wnt3a promoted the proliferation of neural precursor cells and inhibited the formation of axons during the development of chicken embryos.