中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (41): 7738-7741.doi: 10.3969/j.issn.1673-8225.2011.41.034

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

构建survivin shRNA重组载体及靶向下调PC3细胞survivin mRNA的表达

徐建华1，陈丹娜1，何  敏1，黄宪章1，庄俊华1，黎美贤1，金小宝2，卢雪梅2，朱家勇2     

  1. 1广东省中医院检验医学部，广东省广州市 510120
    2广东药学院药用生物活性物质研究所，广东省生物活性药物研究重点实验室，广东省广州市 510006
  • 收稿日期:2011-04-20 修回日期:2011-08-04 出版日期:2011-10-08 发布日期:2011-10-08
  • 通讯作者: 朱家勇，教授，博士生导师，广东药学院药用生物活性物质研究所，广东省生物活性药物研究重点实验室, 广东省广州市 510006 zhujy@gdpu. edu.cn
  • 作者简介:徐建华★，男，1976年生，湖南省衡阳市人，汉族，南方医科大学2010级在读博士，硕士生导师，副主任技师，主要从事生化与分子生物学研究。 jhxu1976@126. com
  • 基金资助:

    广东省科技计划项目(2011B031800207)；广东省医学科学技术研究基金项目(A2008240)；广东省中医院青年后备人才科研项目(E206001)。

Construction of survivin shRNA recombinant expression vector and downregulation of survivin mRNA expression in PC3 cells

Xu Jian-hua1, Chen Dan-na1, He Min1, Huang Xian-zhang1, Zhuang Jun-hua1, Li Mei-xian1, Jin Xiao-bao2, Lu Xue-mei2, Zhu Jia-yong2   

  1. 1Department of Clinical Laboratory, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou  510120, Guangdong Province, China
    2Institute of Pharmaceutical Bioactive Substances of Guangdong University of Pharmacy, Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangzhou  510006, Guangdong Province, China
  • Received:2011-04-20 Revised:2011-08-04 Online:2011-10-08 Published:2011-10-08
  • Contact: Zhu Jia-yong, Professor, Doctoral supervisor, Institute of Pharmaceutical Bioactive Substances of Guangdong University of Pharmacy, Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangzhou 510006, Guangdong Province, China zhujy@gdpu.edu.cn
  • About author:Xu Jian-hua☆, Studying for doctorate, Master’s supervisor, Associate chief technician, Department of Clinical Laboratory, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 510120, Guangdong Province, China jhxu1976@126.com
  • Supported by:

    Guangdong Provincial Science and Technology Development Program, No. 2011B031800207*; a grant from Medical Science and Technology Research Program of Guangdong Province, No. A2008240*；Research Foundation for Young Talented Scholars of Guangdong Provincial Hospital of Chinese Medicine, No. E206001* 

PDF

393

被引次数

6

摘要：

背景：survivin基因特异性表达于肿瘤和胚胎组织，与肿瘤细胞的分化增殖、浸润转移以及多药耐药密切相关。
目的：构建靶向survivin基因的shRNA重组质粒表达载体，转染前列腺癌细胞PC3，验证该载体能否下调细胞survivin基因mRNA水平。
方法：以survivin基因为靶点设计具有短发夹结构的shRNA序列，经退火成互补双链后克隆入pENTR/U6建立重组表达载体pENTR/U6-SUR；转化E.coli TOP10菌株，挑取阳性菌落进行菌落PCR和测序鉴定；将重组质粒转染前列腺癌细胞株PC3细胞，RT-PCR检测重组质粒对细胞survivin基因mRNA水平的抑制效果。
结果与结论：将设计合成的shRNA序列经退火后克隆至pENTR/U6载体中，菌落PCR可扩增出目的条带，测序结果证实插入片段为所需序列；pENTR/U6-SUR重组质粒转染后PC3细胞survivin基因mRNA表达水平显著下降，且24 h比48 h作用更明显。成功构建了靶向survivin基因的shRNA质粒表达载体，并证实该载体显著下调了PC3细胞中survivin基因mRNA水平。

关键词: 短发夹RNA, survivin基因, 前列腺癌细胞, 载体, 凋亡, 组织构建

Abstract:

BACKGROUND: survivin mRNA is specifically expressed in tumor and embryonic tissue and is closely related to differentiation, proliferation, infiltration and metastasis of tumor cells as well as multidrug resistance.
OBJECTIVE: To construct the expression vector of small hairpin RNA (shRNA) targeting human survivin gene and detect the effectiveness of gene silencing in PC3 cells (human prostate cancer cell line).
METHODS: Two single-stranded DNA oligonucleotides for shRNA expression targeted survivin gene were chemically synthesized. The top and bottom strand oligos were annealed to generate a double-stranded oligonucleotide (ds oligo). The the oligo was cloned into pENTR/U6 expression vector (pENTR/U6-SUR), and then PCR and sequencing analyses were conducted to verify the constructs. After transfecting the verified plasmids into PC3 cells, RT-PCR was performed to determine the mRNA level of survivin gene.
RESULTS AND CONCLUSION: PCR and sequencing analyses demonstrated that shRNA template targeting survivin gene had been inserted at the expected site and the insertion sequence was perfectly corrected. The RT-PCR results showed that survivin expression in PC3 was downregulated at mRNA level. The recombinant plasmid had a better affect at 24 hours than 48 hours. The shRNA expression vector targeting survivin gene has been constructed successfully, and it would be a useful method to develop specific survivin-silencing therapeutics in further gene therapy study.

中图分类号: