中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (24): 4553-4556.doi: 10.3969/j.issn.1673-8225.2011.24.045

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

富含亮氨酸重复序列免疫球蛋白样蛋白1基因特异性RNA干扰表达载体的构建、鉴定和稳定株筛选

刘红朝1，王宝峰2，谢蕊繁2，蔡明俊2，郭东生2，雷  霆2   

  1. 1华中科技大学同济医学院附属梨园医院神经外科，湖北省武汉市 430077
    2华中科技大学同济医学院附属同济医院神经外科，湖北省武汉市  430030
  • 收稿日期:2011-01-06 修回日期:2011-04-16 出版日期:2011-06-11 发布日期:2011-06-11
  • 通讯作者: 王宝峰，博士，主治医师，华中科技大学同济医学院附属同济医院神经外科，湖北省武汉市 430030
  • 作者简介:刘红朝☆，男，1966年生，博士，2009年华中科技大学毕业，湖北省大悟县人，汉族，副教授，主要从事神经外科基础与临床研究。
  • 基金资助:

    课题受国家自然科学基金(30500521，81001116)资助。

Construction and identification of RNAi expressing vector specific for leucine-rich repeats and immunoglobulin-like domains 1 gene and selection of stably transfected cell clone

Liu Hong-chao1, Wang Bao-feng2, Xie Rui-fan2, Cai Ming-jun2, Guo Dong-sheng2, Lei Ting2   

  1. 1Department of Neurosurgery, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan  430077, Hubei Province, China
    2Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan  430030, Hubei Province, China
  • Received:2011-01-06 Revised:2011-04-16 Online:2011-06-11 Published:2011-06-11
  • Contact: Wang Bao-feng, Ph.D., Attending physician, Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China wangbaofeng@yahoo.cn
  • About author:Liu Hong-chao☆, Ph.D., Associate professor, Department of Neurosurgery, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430077, Hubei Province, China liuhongchao838@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30500521*,81001116*,

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摘要：

背景：有研究表明富含亮氨酸重复序列免疫球蛋白样蛋白1(leucine-rich repeats and immunoglobulin-like domains 1，LRIG1)基因在神经胶质瘤细胞中呈低表达，LRIG1基因过表达后对神经胶质瘤细胞的 LRIG1 mRNA和蛋白表达均明显增强和抑制其生物学行为，但却很少有研究从阻断LRIG1基因的表达的角度进行论证。
目的：构建针对LRIG1基因的特异性RNA干扰质粒，稳定转染人脑胶质瘤GL15细胞系，观察其对目的基因LRIG1表达的影响。
方法：根据GenBank提供的LRIG1基因序列设计2条RNA干扰序列，命名为LRIG1-shRNA1和LRIG1-shRNA2，并设计1条非特异性序列作为阴性对照，命名为pGenesil2-negative shRNA。合成各自的寡核苷酸链，退火后与pGenesil2质粒载体连接，转化扩增后测定序列。用不同浓度的G418作用于GL15细胞确定G418对GL15细胞的筛选浓度。将3种重组表达载体转染GL15细胞，G418筛选后挑单克隆并扩增获得稳定株。Western blot检测LRIG1蛋白的表达。
结果与结论：构建的重组pGenesil2-LRIG1- shRNA质粒经限制性酶切及DNA测序分析证明其序列插入正确。转染pGenesil2- LRIG1-shRNA1(LRIG11)细胞和pGenesil2- LRIG1-shRNA2(LRIG12)细胞LRIG1蛋白表达水平较阴性对照组pGenesil2-negative shRNA分别下降47.9%(P < 0.01)和32.8% (P > 0.05)。结果证实，实验成功构建了针对LRIG1基因的特异性shRNA表达载体pGenesil2-LRIG1-shRNA1，其转染GL15细胞后可抑制LRIG1的表达。

关键词: 富含亮氨酸重复序列免疫球蛋白样蛋白1, RNA干扰, 胶质母细胞瘤, 转染, 组织工程

Abstract:

BACKGROUND: Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) gene showed low expression in glioma cells. LRIG1 gene overexpression significantly enhanced LRIG1 mRNA and protein expression and inhibited its biological behavior. However, very few researchs are reported from the stand-point of inhibition of LRIG1 gene expression.
OBJECTIVE: To construct specific RNA interference plasmids for LRIG1, establish stably transfected human glioma GL15 cell line, and observe its effect on expression of target gene LRIG1.
METHODS: Designed and synthesized two shRNAs (named LRIG1-shRNA1 and LRIG1-shRNA2) specific for LRIG1 mRNA according to the GenBank, and one scrambled shRNA sequence as negative control, named pGenesil2-negative shRNA. The shRNA was inserted into pGenesil2 vector and sequenced. The recombinant vectors were transformed into E. coli. Picked up the positive clones and extracted the plasmids, which were transfected into GL15 cells by Metafectine. G418 was applied to select the stably transfected cell clones. Western Blotting was performed to examine the LRIG1 protein level.
RESULTS AND CONCLUSION: The recombinant plasmids which contain shRNA were analyzed by restriction endonuclease digestion and DNA sequence, and it was proved that the fragment was inserted into the expected sites. Compared with the negative control group, the level of LRIG1 protein expression in pGenesil2-LRIG1-shRNA1(LRIG11) transfected cells and in pGenesil2-LRIG1-shRNA2(LRIG12) transfected cells was decreased by 47.9% (P < 0.01) and 32.8% (P > 0.05). The results confirmed that RNAi expressing vector specific for LRIG1 gene (pGenesil2-LRIG1-shRNA1) was successfully constructed, and the stable cell clones transfected with the shRNA expression vector showed inhibition of the expression of LRIG1 in glioma cell line GL15.

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