鸡源Wnt3a融合蛋白表达载体的构建及其对鸡胚脊髓神经前体细胞增殖和轴突形成的影响

李秋玲 杨慈清* 张必超 朱少义 林俊堂*

doi:10.16098/j.issn.0529-1356.2017.01.002

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解剖学报 ›› 2017, Vol. 48 ›› Issue (1) : 7-13. DOI: 10.16098/j.issn.0529-1356.2017.01.002

神经生物学

鸡源Wnt3a融合蛋白表达载体的构建及其对鸡胚脊髓神经前体细胞增殖和轴突形成的影响

李秋玲¹ 杨慈清^1,2* 张必超¹朱少义¹林俊堂^1,2*

作者信息 +

Construction of Wnt3a fusion protein vector and its effect on the proliferation and axon formation of neural precursor cells during the chick embryonic spinal cord development

LI Qiu-ling¹ YANG Ci-qing ^1,2* ZHANG Bi-chao¹ ZHU Shao-yi¹ LIN Jun-tang ^1,2*

Author information +

文章历史 +

摘要

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pCAG-MCs-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。

Abstract

Objective To construct a eukaryotic vector of chicken-derived Wnt3a tagged with EGFP (pCAG-MCs-Wnt3a-EGFP) and investigate the influence to the proliferation and axonal formation of neural precursor cells when Wnt3a was overexpressed during the development of chick embryonic spinal cord. Methods Wnt3a gene was amplified from the total RNA obtained from chick embryonic spinal cord using molecular techniques, then connected with pCAG-MCs-EGFP to construct pCAG-MCs-Wnt3a-EGFP, which was identifled by digestion and genetic sequencing. At embryonic day (E) 2.5-3.0, pCAG-MCs0-Wnt3a-EGFP (experimental group) and pCAG-MCs-EGFP (control group) were transfected into the chick embryonic spinal cord using in vivo electroporation, respectively. Samples were collected at E4 (5 simples of each groups ) and then conducted frozen section. The immunofluorescent staining was performed to detect the expression of Wnt3a and proliferating cell nuclear actigen (PCNA) for analyzing the relationship between Wnt3a and cell proliferation, and observe the axonal formation of neural precursor according to the green fluorescence of Wnt3a protein. Results pCAG-MCs-Wnt3a-EGFP was obtained and its gene sequencing was identical with the Gene bank. Green fluorescence was observed at E4 after pCAG-MCs-Wnt3a-EGFP transformed to chick spinal cord. In transversal section of chick embryonic spinal cord, the results of immunofluorescent staining showed Wnt3a was successfully overexpressed. Meanwhile, the amount of neurons projecting axons was dramatically decreased (n=3, P<0.01), compared to the control group, concomitant with the significant elevation of PCNA level (n=3, P<0.01). Conclusion pCAG-MCs-Wnt3a-EGFP is successfully constructed and our study confirmed that Wnt3a plays a vital role in the proliferation and axonal formation of neural precursor cells in the developing chick spinal cord.

导出引用

李秋玲杨慈清张必超朱少义林俊堂. 鸡源Wnt3a融合蛋白表达载体的构建及其对鸡胚脊髓神经前体细胞增殖和轴突形成的影响[J]. 解剖学报. 2017, 48(1): 7-13 https://doi.org/10.16098/j.issn.0529-1356.2017.01.002

LI Qiu-ling YANG Ci-qing ZHANG Bi-chao ZHU Shao-yi LIN Jun-tang. Construction of Wnt3a fusion protein vector and its effect on the proliferation and axon formation of neural precursor cells during the chick embryonic spinal cord development[J]. Acta Anatomica Sinica. 2017, 48(1): 7-13 https://doi.org/10.16098/j.issn.0529-1356.2017.01.002

参考文献

［1］Li XY, Zhai WJ, Teng CB. Notch signaling in pancreatic development ［J］. Int J MolSci, 2015, 17(1):E48.

［2］Larue L, Delmas V. The WNT/beta-catenin pathway in melanoma ［J］. Front Biosci, 2006, 11:733-742.

［3］Nelson WJ, Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways ［J］. Science, 2004, 303(5663):1483-1487.

［4］Holstein TW. The evolution of the Wnt pathway ［J］. Cold Spring Harb Perspect Biol, 2012, 4(7):a007922.

［5］Bilir B, Kucuk O, Moreno CS. Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells ［J］. J Transl Med, 2013, 11(1):280.

［6］Peng X, Yang L, Chang H, et al. Wnt/β-catenin signaling regulates the proliferation and differentiation of mesenchymal progenitor cells through the p53 pathway ［J］. PLoS One, 2014, 9(5): e97283.

［7］Patapoutian A, Reichardt LF. Roles of Wnt proteins in neural development and maintenance ［J］. Curr Opin Neurobiol, 2000, 10(3):392-399.

［8］Kondoh H, Takada S, Takemoto T. Axial level-dependent molecular and cellular mechanisms underlying the genesis of the embryonic neural plate ［J］. Dev Growth Differ, 2016, 58(5):427-436.

［9］Yang CQ, Mao HL, Lin JT. Establishment of chicken embryo culture system and in vivo electroporation methods ［J］. Acta Anatomica Sinica, 2012, 43(4):564-568.(in Chinese)

杨慈清, 毛会丽, 林俊堂. 鸡胚培养及活体电转基因方法的建立［J］. 解剖学报, 2012, 43(4): 564-568.

［10］Li H,Yang CQ, Wang CR, et al. Spinal cord nerve fiber projection is repressed by sonic hedgehog overexpression during chicken embryonic development ［J］. Acta Anatomica Sinica, 2015, 46(1):1-5. (in Chinese)

李晗, 杨慈清, 王聪睿, 等. 鸡胚发育过程中Shh超表达对脊髓神经纤维投射的抑制作用［J］. 解剖学报, 2015, 46(1):1-5.

［11］Mihara E, Hirai H, Yamamoto H, et al. Active and water-soluble form of lapidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin ［J］. Elife, 2016, 5:e11621.［12］Michaelidis TM, Lie DC. Wnt signaling and neural stem cells: caught in the Wntweb ［J］. Cell Tissue Res, 2008, 331(1):193-210.

［13］Bello JO, Nieva LO, Paredes AC, et al. Regulation of the Wnt/β-catenin signaling pathway by human papillomavirus E6 and E7 oncoproteins ［J］. Viruses, 2015, 7(8):4734-4755.

［14］Wang YZh, Chen ET, Feng DF, et al. The effect of Wnt3a on the proliferation of neuralstem cells in vitro ［J］. Journal of Bengbu Medical College, 2012, 37(1):1-4.(in Chinese)

王永志, 陈二涛, 冯东福,等. Wnt3a影响大鼠海马神经干细胞增殖的体外研究［J］. 蚌埠医学院学报, 2012, 37(1):1-4.

［15］Lyu J, Costantini F, Jho EH, et al. Ectopic expression of Axin blocks neuronal differentiation of embryonic carcinoma p19 cells ［J］. J Biol Chem, 2003, 278(15):13487-13495.

［16］Muroyama Y, Fujihara M, Ikeya M, et al. Wnt signaling plays an essential role in neuronal specification of the dorsal spinal cord ［J］. Genes Dev, 2002, 16(5):548-553.

［17］Misteli T, Spector DL. Applications of the green fluorescent protein in cell biology and biotechnology ［J］. Nat Biotechnol, 1997, 15(10):961-964.

［18］Yang CQ, Mao HL, Guo ZhK, et al. Ouality assessment of report gene green fluorescence protein in chicken embryo electroporation ［J］. Acta Anatomica Sinica, 2012,43(4):468-472. (in Chinese)

杨慈清, 毛会丽, 郭志坤, 等. 鸡胚活体原位电转基因技术报告基因绿色荧光蛋白质量评估［J］. 解剖学报, 2012, 43(4):468-472.

［19］Yu YN, Liu YL, Yang CQ, et al. A method of simultaneously tracing the trajectory of bilateral commissural axons in developing chick spinal cord using dual-color fluorescence ［J］.Acta Anatomica Sinica, 2015, 47(2):284-288. (in Chinese)

于亚楠, 刘彦礼, 杨慈清, 等. 双色荧光示踪鸡胚脊髓两侧连合纤维投射研究方法的建立［J］. 解剖学报, 2015, 47(2):284-288.

［20］Yin ZS, Zu B, Chang J, et al. Repair effect of Wnt3a protein on the contused adult rat spinal cord ［J］. Neurol Res, 2008, 30(5): 480-486.