华西口腔医学杂志 ›› 2020, Vol. 38 ›› Issue (1): 6-10.doi: 10.7518/hxkq.2020.01.002

• 基础研究 • 上一篇    下一篇

口腔鳞状细胞癌稳定过表达蛋白酶体激活因子PA28γ细胞株的构建

辛川,王冏珂,李敬,曾昕()   

  1. 口腔疾病研究国家重点实验室 国家口腔疾病临床医学研究中心 四川大学华西口腔医院黏膜病科,成都 610041
  • 收稿日期:2019-04-16 修回日期:2019-11-05 出版日期:2020-02-01 发布日期:2020-02-06
  • 通讯作者: 曾昕 E-mail:zengxin22@163.com
  • 作者简介:辛川,博士,E-mail: xinchuanr@163.com
  • 基金资助:
    国家重点研发计划重点专科课题(2017YFC0840100);国家重点研发计划重点专科课题(2017YFC0840107);国家自然科学基金(81472533);国家自然科学基金(81672675);国家自然科学基金(281771081)

Construction of an oral squamous cell carcinoma cell line for stable PA28γ overexpression

Xin Chuan,Wang Jiongke,Li Jing,Zeng Xin()   

  1. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Dept. of Oral Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
  • Received:2019-04-16 Revised:2019-11-05 Online:2020-02-01 Published:2020-02-06
  • Contact: Xin Zeng E-mail:zengxin22@163.com
  • Supported by:
    The National Key Research Program of China(2017YFC0840100);The National Key Research Program of China(2017YFC0840107);The National Natural Science Foundation of China(81472533);The National Natural Science Foundation of China(81672675);The National Natural Science Foundation of China(281771081)

摘要:

目的 构建蛋白酶体激活因子PA28γ过表达的口腔鳞状细胞癌(OSCC)细胞稳转株,并验证其过表达PA28γ的效率。方法 首先采用聚合酶链反应(PCR)克隆PA28γ基因至pLOV.CMV.cherry.2A.EF1a.PuroR慢病毒载体中,采用PCR和DNA测序比对分析进行鉴定;其次采用293T细胞包装病毒;然后采用病毒感染OSCC细胞构建PA28γ稳定过表达细胞株。最后利用免疫印迹技术检测OSCC细胞稳转株中PA28γ表达的水平。结果 DNA测序结果显示成功构建PA28γ过表达慢病毒载体构建;荧光结果显示,PA28γ过表达慢病毒成功感染OSCC细胞,并呈现樱桃红色荧光;免疫印迹结果显示,构建的PA28γ稳定过表达细胞显著提高细胞中PA28γ表达水平。结论 PA28γ过表达慢病毒载体能显著提高细胞中PA28γ蛋白表达水平,为进一步研究PA28γ对OSCC发生发展的影响及其机制提供了稳定的细胞转染载体。

关键词: 口腔鳞状细胞癌, PA28γ, 慢病毒, 过表达载体, 过表达稳转株

Abstract:

Objective To construct a PA28γ overexpression cell line and determine its effects after infecting an oral squamous cell carcinoma (OSCC) cell line. Methods The PA28γ gene was cloned into the pLOV.CMV.cherry.2A.EF1a.PuroR lentiviral vector by polymerase chain reaction (PCR), and PCR and DNA sequencing alignment analysis were used for identification. Then, 293T cells were used to package viral diseases. Infected OSCC cells were used to construct a cell line with stable PA28γ overexpression. Finally, the level of PA28γ expression in the OSCC cell line was detected through Western blot. Results The successful construction of PA28γ recombinant lentiviral vectors was confirmed by DNA sequencing. The results of immunofluorescence showed that the PA28γ overexpression lentivirus successfully infected the OSCC cells and showed cherry red fluorescence. The results of Western blot demonstrated that the constructed cells with stable PA28γ overexpression significantly increased the expression of PA28γ. Conclusion The PA28γ overexpression lentiviral vector can significantly increase its protein expression in OSCC cells. We provide a stable OSCC cell line for further study on the effect of PA28γ in OSCC.

Key words: oral squamous cell carcinoma, PA28γ, lentivirus, overexpression vector, stable overexpression cell line

中图分类号: