中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (28): 5255-5259.doi: 10.3969/j.issn.2095-4344.2012.28.026

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

SIRT1基因shRNA慢病毒载体构建及干扰效应检测

邓昭玲1，王 彦2，杜利清2，刘 强2，赵 辉3，陈乃耀1   

  1. 1河北联合大学附属医院血液科，河北省唐山市 063000；
    2中国医学科学院放射医学研究所，天津市分子核医学重点实验室，天津市 300192；
    3天津商业大学生物技术与食品科学学院，天津市食品生物技术重点实验室，天津市 300314
  • 收稿日期:2011-12-01 修回日期:2011-12-22 出版日期:2012-07-08 发布日期:2012-07-08
  • 通讯作者: 陈乃耀，主任医师，教授，硕士生导师，河北联合大学附属医院血液科，河北省唐山市 063000 nychenncmc@yahoo.com.cn
  • 作者简介:邓昭玲★，女，1985年生，湖南省武冈市人，汉族，河北联合大学在读硕士，主要从事分子生物学研究。 ling112711@163.com

Construction of a short hairpin RNA interference lentiviral vector of SIRT1 gene and detection of interference effect

Deng Zhao-ling1, Wang Yan2, Du Li-qing2, Liu Qiang2, Zhao Hui3, Chen Nai-yao1   

  1. 1Department of Hematology, Affiliated Hospital of Hebei United University, Tangshan 063000, Hebei Province, China;
    2Institute of Radiation Medicine, Chinese Academy of Medical Sciences, Tianjin Key Laboratory of Molecular Nuclear Medicine, Tianjin 300192, China;
    3Biochemistry and Food Technology School, Tianjin University of Commerce, Tianjin Key Laboratory of Food Science and Biotechnology, Tianjin 300134, China
  • Received:2011-12-01 Revised:2011-12-22 Online:2012-07-08 Published:2012-07-08
  • Contact: Chen Nai-yao, Chief physician, Professor, Master’s supervisor, Department of Hematology, Affiliated Hospital of Hebei United University, Tangshan 063000, Hebei Province, China nychenncmc@yahoo.com.cn
  • About author:Deng Zhao-ling★, Studying for master‘s degree, Department of Hematology, Affiliated Hospital of Hebei United University, Tangshan 063000, Hebei Province, China ling112711@163.com

PDF

1025

被引次数

4

摘要：

背景：SIRT1基因在细胞能量代谢、凋亡及衰老过程中发挥极重要的作用，另有研究发现SIRT1可能在炎症反应方面起着调控作用。
目的：构建SIRT1特异性的shRNA慢病毒载体，并初步检测其对THP-1细胞SIRT1基因的抑制效应。
方法：设计SIRT1靶点特异性的寡核苷酸序列，连接到经Age Ⅰ和EcoR Ⅰ酶切线性化的pGCSIL-GFP载体，包装293T细胞产生慢病毒，再转染THP-1细胞，通过实时荧光定量PCR实验及Western Blot检测对SIRT1靶基因的抑制情况。
结果与结论：阳性克隆PCR及测序证实SIRT1基因shRNA慢病毒载体构建成功，实时荧光定量PCR及Western Blot检测该载体在mRNA和蛋白水平能抑制THP-1细胞的SIRT1表达。

关键词: RNA干扰, SIRT1, 转染, 炎症, 慢病毒载体

Abstract:

BACKGROUND: SIRT1 gene plays a very important role in cell energy metabolism, apoptosis and ageing, while other studies have found that SIRT1 may play a regulatory role in the inflammatory response.
OBJECTIVE: To construct short hairpin RNA interference lentiviral vector of SIRT1 gene and to evaluate their inhibitory effect in THP-1 cells.
METHODS: The SIRI1 target specific oligonucleotide sequence was designed, synthesized and connected into the pGCSIL-GFP vector digested by Age Ⅰ and EcoRⅠ 293T cells were packaged to produce the lentivirus, and then transfected with THP-1 cells. The inhibitory effect on SIRT1 gene was tested by the real-time quantitative PCR and Western Blot.
RESULTS AND CONCLUSION: The PCR and DNA sequencing of positive clones demonstrated that the lentivirus shRNA vector of SIRT1 gene was constructed successfully. The real-time quantitative PCR and Western Blot confirmed that the vector could suppress the expression level of SIRT1 at mRNA and protein levels.

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