小鼠神经母细胞瘤细胞中神经型钙黏蛋白超表达对突起形成及连环蛋白表达的影响

doi:10.16098/j.issn.0529-1356.2018.05.006

解剖学报 ›› 2018, Vol. 49 ›› Issue (5): 598-604.doi: 10.16098/j.issn.0529-1356.2018.05.006

小鼠神经母细胞瘤细胞中神经型钙黏蛋白超表达对突起形成及连环蛋白表达的影响

朱少义^1,2 管丽红^1,2* 杨慈清^1,2 张必超^1,2 李秋玲^1,2 林俊堂^1,2,3*

1. 新乡医学院生命科学技术学院，干细胞与生物治疗技术研究中心，河南新乡 453003; 2. 河南省医用组织再生重点实验室，河南新乡 453003; 3. 新乡医学院生物医学工程学院，河南新乡 453003

收稿日期:2017-11-16 修回日期:2018-03-21 出版日期:2018-10-06 发布日期:2018-10-06
通讯作者: 管丽红;林俊堂 E-mail:linjtlin@126.com
基金资助:
国家自然科学基金;河南省高校科技创新人才计划;新乡医学院科研创新课题资助项目;河南省自然科学基金

Effect of N-cadherin overexpression on protrusion formation and catenins expression in mouse neuroblastoma N2a cells

ZHU Shao-yi ^1,2 GUAN Li-hong ^1,2* YANG Ci-qing ^1,2 ZHANG Bi-chao ^1,2 LI Qiu-ling ^1,2 LIN Jun-tang ^1,2,3*

1. College of Life Science and Technology, Research Center of Stem Cell and Biotherapy, Xinxiang Medical University，He’nan Xinxiang 453003, China; 2. He’nan Key Laboratory of Medical Tissue Regeneration，He’nan Xinxiang 453003, China; 3. College of Biomedical Engineering, Xinxiang Medical University, He’nan Xinxiang 453003, China

Received:2017-11-16 Revised:2018-03-21 Online:2018-10-06 Published:2018-10-06
Contact: GUAN Li-hong ; LIN Jun-tang E-mail:linjtlin@126.com

摘要/Abstract

摘要：

目的探讨神经型钙黏蛋白（N-cadherin）调控连环蛋白（catenin）对小鼠神经母细胞瘤细胞（N2a）形态的影响。方法构建鼠源的N-cadherin融合蛋白超表达载体，利用脂质体转染的方法将空载体pCAG-MCS-EGFP和超表达载体pCAG-N-Cad-EGFP分别转染至N2a细胞，作为对照组和实验组；细胞划痕和黏附实验检测N-cadherin超表达对N2a细胞迁移和黏附的影响，免疫荧光检测N-cadherin超表达对N2a细胞形态的影响，并结合Western blotting检测连环蛋白家族的表达情况。结果与对照组相比，N-cadherin超表达促进细胞突起的形成，使得N2a细胞突起增多和伸长；N-cadherin超表达增强了黏附性而抑制了细胞的迁移能力；N-cadherin超表达使得细胞内连环蛋白家族中α-catenin、β-catenin和p120蛋白表达均显著上升（P<0.05），而γ-catenin表达下降但差异不显著（P>0.05）；另外，N-cadherin超表达的细胞有明显的聚集现象。结论 N-cadherin超表达促进N2a细胞突起形成，增强细胞的黏附性从而抑制细胞的迁移能力，调控连环蛋白家族成员的表达并促使细胞聚集。

关键词: 神经型钙黏蛋白, 小鼠神经母细胞瘤细胞, 细胞突起, 连环蛋白, 细胞黏附, 免疫荧光, 小鼠

Abstract:

Objective To analyse the effects of N-cadherin overexpression on cell morphology and catenins expression by using mouse neuroblastoma N2a cells. Methods The mouse N-cadherin gene was cloned to pCAG-MCS-EGFP plasmid and transfected into N2a cells by Lipofectamine 3000. The empty vector pCAG-MCS-EGFP and the overexpression vector pCAG-N-Cad-EGFP transfected group were used as control group and experimental group, respectively. Cell scratche and cell adhesion experiments were used to detect the effects of N-cadherin overexpression on the migration and adhesion of N2a cells. Immunofluorescence was used to detect the effect of N-cadherin overexpression on the morphology of N2a cells. The expression of catenin family was detected by Western blotting. Results Compared with the control group, overexpressing N-cadherin promoted the protrusion formation of N2a cells, which led to the increase and elongation of protrusion in N2a cells. N-cadherin overexpression enhancea cell adhesion and inhibited migration. N-cadherin overexpression resulted in the expression level of α-catenin, β-catenin and p120 protein significantly increased (P<0.05), while γ-catenin expression decreased but the difference was not significant (P>0.05). In addition, the cell aggregation phenomenon was observed when N-cadherin overexpression. Conclusion N-cadherin overexpression promotes protrusion formation and adhesion ability in N2a cells, but inhibits cell migration, regulates the expression of the family of catenin and promotes cell aggregation.

Key words: N-cadherin, Mouse neuroblastoma N2a cell, Cell protrusion, Catenin, Cell adhesion, Immunofluorescence, Mouse

朱少义管丽红杨慈清张必超李秋玲林俊堂 . 小鼠神经母细胞瘤细胞中神经型钙黏蛋白超表达对突起形成及连环蛋白表达的影响[J]. 解剖学报, 2018, 49(5): 598-604.

ZHU Shao-yi GUAN Li-hong YANG Ci-qing ZHANG Bi-chao LI Qiu-ling LIN Jun-tang. Effect of N-cadherin overexpression on protrusion formation and catenins expression in mouse neuroblastoma N2a cells[J]. Acta Anatomica Sinica, 2018, 49(5): 598-604.

参考文献

［1］ Zhang Y, Zhang JSh, Chen JE, et al. Expression of oligodendrocyte transcription factor and neurological cadherin in the developing spinal cord of the human embryo ［J］. Acat Anatomica Sinica, 2015, 46(5):655-659. (in Chinese)

张泳，张居适，陈键尔, 等. 少突胶质细胞转录因子和神经钙黏附蛋白在人胚胎脊髓发育阶段的表达［J］. 解剖学报, 2015, 46(5):655-659.

［2］ Logan CM, Rajakaruna S, Bowen C, et al. N-cadherin regulates signaling mechanisms required for lens fiber cell elongation and lens morphogenesis ［J］. Dev Biol, 2017, 428(1):118-134.

［3］ Cai Y, Shashikanth N, Leckband DE, et al. Cadherin diffusion in supported lipid bilayers exhibits calcium-dependent dynamic heterogeneity ［J］. Biophys J, 2016, 111(12):2658-2665.

［4］ Takeichi M. The cadherins: cell-cell adhesion molecules controlling animal morphogenesis ［J］. Development, 1988, 102(4):639-655.

［5］ Shih W, Yamada S. N-cadherin as a key regulator of collective cell migration in a 3D environment ［J］. Cell Adh Migr, 2012, 6(6): 513-517.

［6］ Nagafuchi A, Takeichi M, Tsukita S. The 102 kd cadherinassociated protein: similarity to vinculin and posttranscriptional regulation of expression ［J］. Cell, 1991, 65(5):849-857.

［7］ Jiang J, Dean D, Burghardt RC, et al. Disruption of cadherin/catenin expression, localization, and interactions during HgCl2-induced nephrotoxicity ［J］. Toxicol Sci, 2004, 80(1):170-182.

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

［9］ Chiang MC, Cheng YC, Chen HM, et al. Rosiglitazone promotes neurite outgrowth and mitochondrial function in N2a cells via PPAR gamma pathway ［J］. Mitochondrion, 2014, 14(1):7-17.

［10］Koike S, Shibuya N, Kimura H, et al. Polysulfide promotes neuroblastoma cell differentiation by accelerating calcium influx ［J］. Biochem Biophys Res Commun, 2015, 459(3):488-492.

［11］Qiao P, Zhao F, Liu M, et al. Hydrogen sulfide inhibits mitochondrial fission in neuroblastoma N2a cells through the Drp1/ERK1/2 signaling pathway［J］. Mol Med Rep, 2017, 16(1):971-977.

［12］Tong S, Xiong NX, Shen JY. Comparison of two different methods for culture of mouse neuroblastoma N2a cells ［J］. Acta Medicinae Universitatis Scientiae et Technology Huazhong, 2014, 43(1): 28-31. (in Chinese)

童松, 熊南翔, 沈建英. 两种培养小鼠神经母细胞瘤N2a细胞方法的比较［J］. 华中科技大学学报(医学版), 2014, 43(1): 28-31.

［13］Yagi T, Takeichi M. Cadherin superfamily genes: functions, genomic organization, and neurologic diversity ［J］. Genes Dev, 2000, 14(10): 1169-1180.

［14］Shi HJ, Zhang Y, Tao QH. C-cadherin controls cortical F-actin assembly in the early xenopus embryo ［J］. Chinese Journal of Cell Biology, 2012, 34(12):1207-1214. (in Chinese)

史慧娟, 张艳, 陶庆华. C-cadherin控制非洲爪蛙早期胚胎中微丝骨架的合成［J］. 中国细胞生物学学报, 2012, 34(12):1207-1214.

［15］Kang H, Bang Ⅰ, Jin KS, et al. Structural and functional characterization of Caenorhabditis elegansα-catenin reveals constitutive binding to β-catenin and F-actin ［J］. J Biol Chem, 2017, 292(17):7077-7086.

［16］Smith ME, Pignatelli M. The molecular histology of neoplasia: the role of the cadherin/catenin complex ［J］. Histopathology, 1997, 31(2): 107-111.

［17］Li XY, Yang CQ, Fan YF, et al. The effect of chicken source N-cadherin abnormal expression on the 293T cell morphology and catenin expression ［J］. Chinese Journal of Immunology, 2013, 29(9):914-917. (in Chinese)

李小英, 杨慈清, 范义峰, 等. N-cadherin 异常表达对293T细胞形态结构及连环蛋白表达的影响［J］. 中国免疫学杂志, 2013, 29(9):914-917.

［18］Li XH, Dou HR, Ma Y, et al. Construction and functional analysis of N-cadherin protein expression vector ［J］. Journal of Jilin University (Medical Sciences), 2002, 28(1): 36-36. (in Chinese)

李贤惠, 窦贺荣, 马勇,等. N-cadherin蛋白表达载体构建及功能分析［J］.吉林大学学报(医学版), 2002, 28(1): 36-36.

［19］Pokutta S, Choi HJ, Ahlsen G, et al. Structural and thermodynamic characterization of cadherin. beta-catenin.alpha-catenin complex formation ［J］. J Biol Chem, 2014, 289(19): 13589-13601.

［20］Yang C, Li X, Wang C, et al. N-cadherin regulates beta-catenin signal and its misexpression perturbs commissural axon projection in the developing chicken spinal cord ［J］. J Mol Histol, 2016, 47(6):541-554.

［21］Dang XM, Sun ZhM, Shang D, Yang L, et al. Expressions of α-catenin and β-catenin in human non-small cell lung cancer and their clincal signficance ［J］. Journal of Xi’an Jiaotong University (Medical Sciences), 2009, 30(1): 89-92. (in Chinese)

党晓敏, 孙忠民，尚东，等. α-连环素和β-连环素在非小细胞肺癌中的表达及其临床意义［J］. 西安交通大学学报（医学版）, 2009, 30(1): 89-92.

［22］Huelsken J, Vogel R, Brinkmann Ⅴ, et al. Requirement for beta-catenin in anterior-posterior axis formation in mice ［J］. J Cell Biol, 2000, 148(3): 567-578.

［23］Ozaki C, Yoshioka M, Tominaga S, et al. p120-Catenin is essential for N-cadherinmediated formation of proper junctional structure, thereby establishing cell polarity in epithelial cells ［J］. Cell Struct Funct, 2010, 35(2):81-94.

［24］Alema S, Salvatore AM. p120 catenin and phosphorylation: Mechanisms and traits of an unresolved issue ［J］. Biochim Biophys Acta, 2007, 1773(1):47-58.

［25］McCrea PD, Park JI. Developmental functions of the P120-catenin sub-family ［J］. Biochim Biophys Acta, 2007, 1773(1):17-33.

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小鼠神经母细胞瘤细胞中神经型钙黏蛋白超表达对突起形成及连环蛋白表达的影响

Effect of N-cadherin overexpression on protrusion formation and catenins expression in mouse neuroblastoma N2a cells

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