PTTG靶向siRNA表达载体构建及其沉默效率评价

doi:高等学校博士学科点专项科研基金资助课题（

PTTG靶向siRNA表达载体构建及其沉默效率评价

徐松柏¹，赵刚¹，赵红光²，许侃¹，于洪泉¹，夏常丽³，康淑红²

1.吉林大学第一医院神经外科，吉林长春 130021；2. 吉林大学第一医院核医学科，吉林长春 130021；3. 吉林大学基础医学院人体解剖学教研室，吉林长春 130021

收稿日期:2008-01-18 修回日期:1900-01-01 出版日期:2008-05-28 发布日期:2008-05-28
通讯作者: 赵刚

Construction of PTTG siRNA expressing vector and evaluation of its gene silencing efficiency

XU Song-bai¹, ZHAO Gang¹, ZHAO Hong-guang², XU Kan¹, YU Hong-quan¹, XIA Chang-li³, KANG Shu-hong²

1. Department of Neurosurgery, First Hospital, Jilin University, Changchun 130021, China; 2. Department of Nuclear Medicine, First Hospital, Jilin University, Changchun 130021, China; 3. Department of Anatomy, School of Basic Medical Sciences, Jilin University, Changchun 130021, China

Received:2008-01-18 Revised:1900-01-01 Online:2008-05-28 Published:2008-05-28
Contact: ZHAO Gang

摘要/Abstract

摘要： 目的：构建针对人脑胶质瘤细胞系U251的高效率沉默垂体瘤转化基因(PTTG)的小分子干扰RNA(siRNA)表达载体。方法：合成特异性干扰PTTG 的siRNA片段，并与pGenesil2 载体连接，构建PTTG干扰载体（pGenesil2-PTTG siRNA）。利用脂质体将其转染U251细胞，分为正常细胞对照组、HK阴性对照组和3个siRNA干扰组（pGenesil2-PTTG siRNA1、pGenesil2-PTTG siRNA2和pGenesil2-PTTG siRNA3），应用半定量逆转录聚合酶链式反应(RT-PCR)法和流式细胞术对转染后U251细胞中PTTG 的mRNA和蛋白表达水平进行分析。结果：酶切证实PTTG-siRNA表达载体构建成功，插入片段测序结果与合成的siRNA结果一致；转染pGenesil2-PTTG siRNA后，3个干扰组的U251 细胞中PTTG基因和蛋白表达水平均较正常对照组显著降低（P<0.01）。结论：成功构建了能高效率沉默PTTG 的RNAi表达载体；pGenesil2-PTTG siRNA高效地抑制了胶质瘤U251细胞中PTTG基因的表达。

关键词: 垂体肿瘤转化基因, 神经胶质瘤, siRNA

Abstract: Abstract:Objective To construct the specific high efficiency small interfering RNA (siRNA) expression vector that can silence PTTG gene. Methods Using vector based RNA interference technique, vectors were constructed to transcribe functional siRNA specially targeting PTTG. The vectors were used to transfect U251 cells by lipofectmine2000 reagent. And the cells were dividedinto five groups: normal control, HK negative group and three siRNA interfering groups (pGenesil2-PTTG siRNA1, pGenesil2-PTTG siRNA2 and pGenesil2-PTTG siRNA3). The expression levels of mRNA and protein of PTTG were analyzed by RT-PCR and flow cytometry methods. Results By restriction endonuclease and DNA sequencing analyzing, eukyaryotic expression plasmid of PTTG was successfully constructed. PTTG mRNA and its protein level in transfected U251 cells with PTTG RNAi plasmid decreased significantly compared with the normal control group (P<0.01). Conclusion pGenesil2-PTTG siRNA vectors were successfully constructed, and they can silence the PTTG gene in U251 cells with high efficiency.

Key words: PTTG, glioma, siRNA

中图分类号:

R739.4

徐松柏，赵刚，赵红光，许侃，于洪泉，夏常丽，康淑红. PTTG靶向siRNA表达载体构建及其沉默效率评价[J]. J4, 2008, 34(3): 411-414.

XU Song-bai, ZHAO Gang, ZHAO Hong-guang, XU Kan, YU Hong-quan, XIA Chang-li, KANG Shu-hong. Construction of PTTG siRNA expressing vector and evaluation of its gene silencing efficiency[J]. J4, 2008, 34(3): 411-414.

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