小鼠Dishevelled 2表达质粒的构建及其在RAW264.7细胞中的表达

doi:10.3969/j.issn.1000-1182.2011.03.022

华西口腔医学杂志

小鼠Dishevelled 2表达质粒的构建及其在RAW264.7细胞中的表达

黄旭1 赵鹃2 毛英杰3 谷志远3

1.浙江大学医学院附属第一医院口腔科，杭州310003；2.浙江大学医学院附属口腔医院修复科； 3.颌面外科，杭州310006

收稿日期:2011-06-25 修回日期:2011-06-25 出版日期:2011-06-20 发布日期:2011-06-20
通讯作者: 赵鹃，Tel：0571-87217225
作者简介:黄旭（1977—），男，浙江人，主治医师，硕士
基金资助:
国家自然科学基金资助项目（30700958）；浙江省自然科学基金资助项目（Y2100333）

Construction of vectors encoding mouse Dishevelled 2 and its expression in RAW264.7 cells

HUANG Xu1, ZHAO Juan2, MAO Ying-jie3, GU Zhi-yuan3

1. Dept. of Stomatology, The First Affiliated Hospital of Medical College of Zhejiang University, Hangzhou 310003, China; 2. Dept. of Prosthodontics, The Affiliated Hospital of Stomatology, Medical College of Zhejiang University, Hangzhou 310006, China; 3. Dept. of Oral and Maxillofacial Surgery, The Affiliated Hospital of Stomatology, Medical College of Zhejiang University, Hangzhou 310006, China

Received:2011-06-25 Revised:2011-06-25 Online:2011-06-20 Published:2011-06-20
Contact: ZHAO Juan，Tel：0571-87217225

摘要/Abstract

摘要：

目的构建小鼠Dishevelled 2（Dvl2）表达质粒，并检测其转染小鼠破骨前体细胞RAW264.7后的表达，为进一步研究Dvl2在破骨细胞分化和骨重建中的作用、筛选调节骨重建潜在靶点奠定基础。方法根据GenBank小鼠Dvl2 cDNA序列设计两条特异性引物，提取RAW264.7细胞总RNA，以RT-PCR获得Dvl2的开放阅读框（ORF），并将其克隆到真核表达质粒pEZ-M29上，酶切电泳，测序鉴定构建的pEZ-M29/Dvl2质粒。用脂质体介导瞬时转染RAW264.7细胞，通过荧光显微镜观察转染效果，实时定量RT-PCR检测Dvl2基因表达情况。结果成功构建pEZ-M29/Dvl2表达载体，酶切电泳和测序结果与目的基因相符；转染RAW264.7细胞后48 h，荧光显微镜下可见绿色荧光蛋白表达；实时定量RT-PCR可见实验组Dvl2基因较对照组强烈过表达。结论成功构建小鼠Dvl2真核表达质粒pEZ-M29/Dvl2，瞬时转染小鼠破骨前体细胞RAW264.7可显著提高Dvl2的mRNA水平。

关键词: Dishevelled 2, 表达质粒, RAW264.7细胞, 破骨细胞

Abstract:

Objective To construct the recombinant vectors that express mouse Dishevelled 2（Dvl2）, and to evaluate its expression level in transfected RAW264.7 cells. Methods A pair of specific primers were designed according to the mouse Dvl2 cDNA sequence published in GenBank. Total RNA of RAW264.7 cells was extracted, and open reading frame of Dvl2 was obtained by RT-PCR, which was then cloned into pEZ-M29 plasmid. Electrophoresis after macrorestriction and DNA sequence analysis were used to identify the reconstructed plasmids. After transient transfection via liposome, the transfection of RAW264.7 cells was confirmed under a fluorescence microscope, and the expression level of Dvl2 was evaluated by real-time RT-PCR. Results The recombinant plasmid containing mouse Dvl2, namely pEZ-M29/ Dvl2, was successfully constructed, and confirmed by DNA sequence analysis. After 48 h of tranfection, the expression of enhanced green fluorescene protein was observed under a fluorescence microscope, and real-time RT-PCR analysis revealed that the Dvl2 mRNA level was prominantly elevated in the transient transfected RAW264.7 cells. Conclusion The recombinant plasmids pEZ-M29/Dvl2 are successfully constructed and can elevate the Dvl2 mRNA level in the transient transfected RAW264.7 cells, which can be used in further studies aiming at revealing the functional significance of Dvl2 in the osteoclastogenesis.

Key words: Dishevelled 2, expression plasmid, RAW264.7 cells, osteoclasts

黄旭赵鹃毛英杰谷志远. 小鼠Dishevelled 2表达质粒的构建及其在RAW264.7细胞中的表达[J]. 华西口腔医学杂志, doi: 10.3969/j.issn.1000-1182.2011.03.022.

HUANG Xu1, ZHAO Juan2, MAO Ying-jie3, GU Zhi-yuan3. Construction of vectors encoding mouse Dishevelled 2 and its expression in RAW264.7 cells[J]. West China Journal of Stomatology, doi: 10.3969/j.issn.1000-1182.2011.03.022.

[1]	戴振宁, 郑蔚晗, 利时雨. 核因子κB受体活化因子配体和肿瘤坏死因子α经炎性牙周膜干细胞外泌体促进破骨细胞分化[J]. 华西口腔医学杂志, 2022, 40(4): 377-385.
[2]	石玉. 能量代谢在成骨和破骨细胞中的研究[J]. 华西口腔医学杂志, 2021, 39(5): 501-509.
[3]	肖勉, 胡智慧, 江恒华, 房维, 龙星. 破骨细胞分化在颞下颌关节骨关节炎发生中的作用[J]. 华西口腔医学杂志, 2021, 39(4): 398-404.
[4]	刘伟, 李春洁, 李龙江. 口腔癌颌骨侵犯的分子机制研究进展[J]. 华西口腔医学杂志, 2021, 39(2): 221-226.
[5]	吴湘楠, 马媛媛, 浩志超, 王航. 溶血磷脂酸对骨组织细胞生物学调控功能的研究进展[J]. 华西口腔医学杂志, 2020, 38(3): 324-329.
[6]	仉红,王丽娜,左美娜,董明,史东梅,徐慧君,牛卫东. 布鲁顿酪氨酸激酶对破骨细胞增殖及分化作用的实验研究[J]. 华西口腔医学杂志, 2019, 37(4): 361-365.
[7]	陈筑, 宿凌恺. 丹皮酚对牙龈卟啉单胞菌诱导骨髓来源巨噬细胞功能的影响[J]. 华西口腔医学杂志, 2017, 35(2): 139-144.
[8]	王丽萍李伯琦王琪铁晓敏刘奕杉. 无翅型MMTV整合位点家族成员5A/受体酪氨酸激酶样孤儿受体2在大鼠牙囊细胞中的表达及其意义[J]. 华西口腔医学杂志, 2016, 34(4): 341-345.
[9]	舒林径伍颖颖谭震宫苹. 叉头转录因子-1与骨代谢关系的研究进展[J]. 华西口腔医学杂志, 2016, 34(4): 429-432.
[10]	平依林娄锋杨肖张平. Notch1蛋白高表达抑制破骨细胞增殖和分化[J]. 华西口腔医学杂志, 2016, 34(2): 121-124.
[11]	连俊翔杜玮孟姝. 微小RNAs在破骨细胞分化调控中的研究进展[J]. 华西口腔医学杂志, 2015, 33(5): 543-547.
[12]	何飞吴勇周艳. Notch信号促进核因子κB受体活化因子配体诱导的破骨细胞分化的体外研究[J]. 华西口腔医学杂志, 2015, 33(1): 25-28.
[13]	林珏杉董伟徐纯峰孙红冯晓洁戚孟春. 唑来膦酸对破骨细胞黏附及整合素α_v、β₃基因表达的影响[J]. 华西口腔医学杂志, 2014, 32(6): 547-551.
[14]	张静李纾吕琳琳. 失神经支配对牙周组织中P物质及破骨细胞的影响[J]. 华西口腔医学杂志, 2014, 32(2): 162-165.
[15]	黄生高凌天牖钟孝欢刘云峰. 压应力对小鼠单核细胞RAW264.7 DNAX活化蛋白12、抗酒石酸酸性磷酸酶表达的影响[J]. 华西口腔医学杂志, 2013, 31(4): 360-363.

小鼠Dishevelled 2表达质粒的构建及其在RAW264.7细胞中的表达

Construction of vectors encoding mouse Dishevelled 2 and its expression in RAW264.7 cells

RichHTML

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价