中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (15): 2737-2740.doi: 10.3969/j.issn.1673-8225.2012.15.017

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇    下一篇

靶向血管内皮生长因子基因的微小RNA真核表达载体的构建***★

熊建宁1, 2，孙  建2，区应亮2，简志祥2   

  1. 1南方医科大学，广东省广州市  510515；2广东省人民医院肝胆外科，广东省广州市  510080
  • 收稿日期:2011-11-03 修回日期:2012-03-08 出版日期:2012-04-08 发布日期:2012-04-08
  • 通讯作者: 孙建，博士，副主任医师、副教授，广东省人民医院肝胆外科，广东省广州市 510080 akeyman@yahoo.com.cn
  • 作者简介:熊建宁★：男，1970年生，甘肃省兰州市人，汉族，南方医科大学在读硕士，主治医师。neilxjn@163.com
  • 基金资助:

    广东省科技计划项目(2009B080701021，2010B080701021)，广东省医学科研基金(A2010002)。

Construction of a microRNA expressing eukaryotic vector targeting vascular endothelial growth factor  

Xiong Jian-ning1, 2, Sun Jian2, Ou Ying-liang2, Jian Zhi-xiang2   

  1. 1Southern Medical University, Guangzhou  510080, Guangdong Province, China; 2Department of Hepatobiliary Surgery, Guangdong Provincial People’s Hospital, Guangzhou  510080, Guangdong Province, China
  • Received:2011-11-03 Revised:2012-03-08 Online:2012-04-08 Published:2012-04-08
  • Contact: author: Sun Jian, Doctor, Associate chief physician, Associate professor, Department of Hepatobiliary Surgery, Guangdong Provincial People’s Hospital, Guangzhou 510080, Guangdong Province, China akeyman@yahoo.com.cn
  • About author:Xiong Jian-ning★, Studying for master’s degree, Attending physician, Southern Medical University, Guangzhou 510080, Guangdong Province, China; Department of Hepatobiliary Surgery, Guangdong Provincial People’s Hospital, Guangzhou 510080, Guangdong Province, China neilxjn@163.com
  • Supported by:

    the Science and Technology Program of Guangdong Province, No. 2009B080701021*, 2010B080701021*; the Medical Science Research Foundation of Guangdong Province, No. A2010002*

PDF

257

被引次数

3

摘要：

背景：有研究表明微小RNA及潜在靶基因在肝癌中起重要作用，但具体机制仍不清楚。
目的：构建靶向血管内皮生长因子基因微小RNA真核表达载体，评估其转染人肝癌细胞株HepG2后血管内皮生长因子基因的干扰效果。
方法：根据血管内皮生长因子序列设计合成4对微小RNA不同干扰片段，克隆到pcDNA6.2-GW/EmGFP-微小RNA真核表达载体上，测序分析鉴定插入序列的完整性；并将其转染至HepG-2细胞株中。采用实时荧光定量聚合酶链式反应分析血管内皮生长因子微小RNA干扰效果，蛋白印迹技术测定重组体对血管内皮生长因子基因蛋白表达情况。
结果与结论：构建的4组重组体插入片段的碱基序列完全正确，重组体能干扰肝癌细胞HepG2细胞血管内皮因子基因的表达，4组重组体基因的mRNA的表达水平和蛋白表达水平与阴性对照组相比明显降低(P < 0.05)，其中血管内皮生长因子微小RNA-3表达水平最低，抑制率87%。结果证实，实验成功构建血管内皮生长因子微小RNA表达载体，在体外能有效抑制HepG2细胞血管内皮生长因子基因表达。
关键词：血管内皮生长因子；微小RNA；肝癌；肝细胞；抑制；基因表达；组织工程
缩略语注释：VEGF: vascular endothelialcell growth factor，血管内皮生长因子
doi:10.3969/j.issn.1673-8225.2012.15.017

关键词: 血管内皮生长因子, 微小RNA, 肝癌, 肝细胞, 抑制, 基因表达, 组织工程

Abstract:

BACKGROUND: Studies have shown that the microRNA (miRNA) and its potential target gene play an important role in hepatocellular carcinoma, but the exact mechanism is still unclear.
OBJECTIVE: To construct a vascular endothelial growth factor (VEGF) miRNA expressing eukaryotic vector and to identify biological activity of VEGF miRNA transfected into HepG-2 cells.
METHODS: According to the sequence of VEGF mRNA, the VEGF miRNA was designed and synthesized, and then cloned into the pcDNA6.2-GW/EmGFP-miRNA vector and transfected into HepG-2 cell lines. The integrity of the insert fragment was detected using colony PCR and sequencing analysis. The biological activity of VEGF miRNA by way of real-time PCR and Western blot was determined. 
RESULTS AND CONCLUSION: Sequences of the inset fragment in the four miRNA expressing recombinants were correct. VEGF mRNA expression of the four miRNA recombinants were significantly decreased (P < 0.05), especially in the VEGF-miRNA-3 with an inhibitory rate of 87%. Four VEGF-targeting miRNA expressing recombinants were successfully constructed which can significantly inhibit VEGF gene expression in HepG2 cells.
Xiong JN, Sun J, Ou YL, Jian ZX. Construction of a microRNA expressing eukaryotic vector targeting vascular endothelial growth factor. Zhongguo Zuzhi Gongcheng Yanjiu. 2012;16(15): 2737-2740.     [http://www.crter.cn  http://en.zglckf.com]
 

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