中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (20): 3755-3758.doi: 10.3969/j.issn.1673-8225.2012.20.033

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

piggyBac转座子调节pPiggyBac-Rb质粒的构建*★○

潘雪珂1，陈  朝1，田思佳1，Joseph M. Kaminski2○，余克明1，庄  菁1   

  1. 1中山大学中山眼科中心，广东省广州市  510060；2Manoa Biosciences, Inc. Honolulu, Hawaii, 96822, USA
  • 收稿日期:2012-01-18 修回日期:2012-02-22 出版日期:2012-05-13 发布日期:2012-05-13
  • 通讯作者: 余克明，博士，主任医师，中山大学中山眼科中心，广东省广州市 510060 yukeming66@126.com 并列通讯作者：庄菁，博士，副教授，中山大学中山眼科中心，广东省广州市 510060
  • 作者简介:潘雪珂★，女，1987年生，安徽省黄山市人，汉族，中山大学在读硕士，主要从事视网膜母细胞瘤基因治疗的研究。xuekepan@163.com
  • 基金资助:

    国家自然科学基金面上项目(30970923)。

Expression of exogenous Rb gene mediated by piggyBac transposon in retinoblastoma cells   

Pan Xue-ke1, Chen Zhao1, Tian Si-jia1, Joseph M. Kaminski2○, Yu Ke-ming1, Zhuang Jing1   

  1. 1State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou  510060, Guangdong Province, China; 2Manoa Biosciences, Inc. Honolulu, Hawaii, 96822, USA
  • Received:2012-01-18 Revised:2012-02-22 Online:2012-05-13 Published:2012-05-13
  • Contact: Corresponding author: Yu Ke-ming, Doctor, Chief physician, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, Guangdong Province, China yukeming66@126.com Corresponding author: Zhuang Jing, Doctor, Associate professor, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, Guangdong Province, China zhuangj@mail.sysu.edu.cn
  • About author:Pan Xue-ke★, Studying for master’s degree, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, Guangdong Province, China xuekepan@163.com
  • Supported by:

     the National Natural Science Foundation of China, No. 30970923*

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摘要：

背景：外源性Rb基因的导入是抑制视网膜母细胞瘤细胞增殖的有效途径，piggyBac转座子介导的外源基因表达载体高效安全，在基因治疗方面具有广阔的应用前景。
目的：探讨piggyBac转座子介导的外源性Rb基因的转染效率及其对视网膜母细胞瘤细胞的抑制作用。
方法：采用RT-PCR技术扩增Rb基因编码区Rb cDNA，定向插入到载体pPiggyBac，获得PiggyBac调节的Rb表达质粒pPiggyBac-Rb；通过单独转染或合并pPiggyBac-helper转染人视网膜母细胞瘤细胞SO-RB50。
结果与结论：考马斯亮蓝染色证明piggyBac转座子系统的转染效率最高，荧光定量PCR以及免疫荧光实验证明其介导的目的基因Rb与宿主SO-RB50细胞基因组整合效率最高且可在细胞中长期稳定表达，MTT实验证明经其所介导的外源性Rb基因表达对细胞活性影响最为显著。结果提示piggyBac转座子有可能成为视网膜母细胞瘤基因治疗的安全有效载体。
关键词：piggyBac转座子；SO-RB50细胞；转染；Rb基因；基因治疗
doi:10.3969/j.issn.1673-8225.2012.20.033

关键词: piggyBac转座子, SO-RB50细胞, 转染, Rb基因, 基因治疗

Abstract:

BACKGROUND: The introduction of exogenous Rb gene into retinoblastoma is an effective way to inhibit growth of the tumor. piggyBac transposon as a vector is safe and highly efficient, which is a potential vector on gene therapy.
OBJECTIVE: To explore the transfection efficiency and its effect on inhibiting retinoblastoma by transfecting human retinoblastoma cell line SO-RB50 with an exogenous Rb gene expression system mediated by piggyBac transposon.
METHODS: pPiggyBac-Rb plasmid that contains a piggyBac transposon sequence and express normal RB gene was constructed and used to transfect SO-RB50 cells alone or with pPiggyBac-helper. Coomassie brilliant blue staining, quantitative real-time PCR and immunofluorescence were adopted to evaluate the transfect efficiency of exogenous Rb gene mediated by piggyBac transposon. The viability of retinoblastoma cells was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
RESULTS AND CONCLUSION: Transfection efficiency was the highest with help of piggyBac transposon. piggyBac transposon-mediated exogenous Rb gene integrated into genome and obtained a long-term and stable expression．It also had a remarkable inhibition effect on the viability of SO-RB50. All these indicate that piggyBac transposon is a potential vector for retinoblastoma gene therapy.
 

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