[1]张立英),刘绍霞)#,赵国强),等.TNFα基因siRNA真核表达载体的构建及其对A549细胞TNFα和TGFβ1表达的影响*[J].郑州大学学报(医学版),2012,(05):629.
 ZHANG Liying),LIU Shaoxia),ZHAO Guoqiang),et al.Construction of eukaryotic expression vectors of siRNA against TNFα gene and their silencing effects on TNFα and TGFβ1 in A549 cells[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2012,(05):629.
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TNFα基因siRNA真核表达载体的构建及其对A549细胞TNFα和TGFβ1表达的影响*
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2012年05期
页码:
629
栏目:
论著
出版日期:
2012-09-20

文章信息/Info

Title:
Construction of eukaryotic expression vectors of siRNA against TNFα gene and their silencing effects on TNFα and TGFβ1 in A549 cells
作者:
张立英1)刘绍霞1)#赵国强2)张国俊1)
1)郑州大学第一附属医院呼吸内科 郑州 450052 2)郑州大学基础医学院微生物学与免疫学教研室 郑州 450001
Author(s):
ZHANG Liying1)LIU Shaoxia1)ZHAO Guoqiang2)ZHANG Guojun1)
1)Department of Respiratory Medicine,the First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052 2)Department of Microbiology and Immunology,College of Basic Medical Sciences,Zhengzhou University,Zhengzhou 450001
关键词:
肿瘤坏死因子α转化生长因子β1RNA干扰肺腺癌A549细胞
Keywords:
TNFαTGFβ1RNA interferencelung adenocarcinomaA549 cell
分类号:
R33
摘要:
目的:构建靶向肿瘤坏死因子α(TNFα)基因的小干扰RNA(siRNA)真核表达载体,观察其对人肺腺癌A549细胞中TNFα和转化生长因子β1(TGFβ1)表达的影响。方法:依据GenBank中人TNFα(NM_000594)序列和siRNA靶序列设计原则,设计并合成一对针对TNFα的特异性序列(siTNFα)和一对无关序列(siCon)。退火合成DNA双链,再分别重组入pRNATU6.1载体,经过PCR和DNA测序鉴定。将构建好的重组载体pRNATU6.1siTNFα、pRNATU6.1siCon及空载体pRNATU6.1用脂质体介导转染A549细胞株,分别采用Real time PCR和Western blot方法检测细胞中TNFα和TGFβ1 mRNA和蛋白的表达水平。结果:PCR和DNA测序鉴定证实载体构建成功;转染pRNATU6.1siTNFα的A549细胞中TNFα和TGFβ1 mRNA和蛋白表达水平均低于其他3组(FmRNA=478.663,4.081;F蛋白=123.420,6.312,P均<0.05)。结论:成功构建了靶向TNFα基因的siRNA真核表达载体,该载体能够有效沉默A549细胞中TNFα的表达,TGFβ1基因表达亦受到抑制。
Abstract:
Aim: To clone the eukaryotic expression vectors of small interfering RNA (siRNA) against TNFα gene and to evaluate their silencing effects on TNFα and TGFβ1 in A549 cells.Methods:According to TNFα gene (NM_000594) sequence of GenBank, using RNAi Designer software to design the siRNA specific target sequence (siTNFα) and the unrelated control sequence (siCon) for TNFα. The annealing products were recombined into pRNATU6.1, PCR and DNA sequencing confirmed the insertion sequence. The recombinant plasmids (pRNATU6.1siTNFα,pRNATU6.1siCon) were transfected to the A549 cells with lipofectamine,and the expressions of TNFα, TGFβ1 mRNA and protein in cells detected by Real time PCR and Western blot methods.Results:PCR and DNA sequencing confirmed the vectors were consistent;the expressions of TNFα, TGFβ1 mRNA and protein in the cells transfected pRNATU6.1siTNFα were markedly lower than the other three controls(FmRNA=478.663,4.081;Fprotein=123.420,6.312,all P<0.05).Conclusion:The TNFα siRNA eukaryotic expression vector is constructed successfully,which could silence the expression of TNFα in A549,as well as the expression of TGFβ1.

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备注/Memo

备注/Memo:
*河南省杰出青年基金资助项目094100510014 #通讯作者,女,1970年11月生,博士,副教授,副主任医师,研究方向:肺间质病变的发病机制与治疗,Email:liushaoxiavip@yahoo.cn
更新日期/Last Update: 1900-01-01