[1]王露,翟玮玮,杨向荣,等.慢病毒介导RNA干扰沉默Fas 基因在脐带间充质干细胞中的表达[J].南方医科大学学报,2014,(10):1475.
点击复制

慢病毒介导RNA干扰沉默Fas 基因在脐带间充质干细胞中的表达()
分享到：

《南方医科大学学报》[ISSN:/CN:]

卷:
期数:
2014年10期
页码:
1475
栏目:
出版日期:
2014-10-15

文章信息/Info

Title:
Lentivirus-mediated stable Fas gene silencing in human umbilical cord-derived
mesenchymal stem cells
作者:
王露; 翟玮玮; 杨向荣; 王芳; 李见; 张强; 李玉云
关键词:
慢病毒; siRNA; Fas; 脐带间充质干细胞
Keywords:
lentiviral vector;  small interferiing RNA;  Fas;  umbilical cord mesenchymal stem cells
摘要:
目的应用慢病毒介导的RNA干扰技术，构建Fas基因的siRNA慢病毒表达载体，建立Fas基因稳定干扰的人脐带间充质
干细胞（UC-MSCs）株，为下一步使用低表达Fas 基因的UC-MSCs治疗再生障碍性贫血奠定实验基础。方法以人Fas 基因
mRNA序列作为干扰靶点，设计4组靶向Fas的shRNA序列，合成寡核苷酸，与经过BamHI、EcoRI双酶切后的LV3载体连接获
得重组慢病毒，通过感染293T 细胞对慢病毒进行包装和滴度测定。体外培养人UC-MSCs，使用包装好的慢病毒感染
UC-MSCs，应用Real time- PCR和Western blotting检测感染后细胞中Fas mRNA及蛋白的表达情况。结果经酶切以及基因测
序鉴定证明慢病毒载体构建成功，病毒悬液滴度为3×108 TU/ml。Real time-PCR和Western blotting结果显示干扰组细胞的Fas
表达水平较对照组显著降低。结论慢病毒介导的SiRNA能有效沉默UC-MSCs中Fas基因的表达。
Abstract:
Objective To construct a lentivirus-mediated vector for RNA interference (RNAi) of Fas and establish a human
umbilical cord-derived mesenchymal stem cells (UC-MSCs) line with stable Fas gene silencing. Methods Four short hairpin
RNA sequences targeting the coding region of human Fas mRNA were designed. The synthesized oligonucleotides were
ligated with the lentivirus vectors harvested from BamHI and EcoRI double digestion of LV3 recombinant vector. The
recombinant lentivirus vectors were transfected into the packaging cells 293T, and the lentivirus titers were determined.
Cultured UC-MSCs were infected with the lentivirus, and real-time PCR and Western blotting were used to detect the
expressions of Fas mRNA and protein in the transfected cells. Results Restriction digestion and DNA sequencing showed that
the lentiviral vectors were successfully constructed, and the titer of lentivirus reached 3 × 108 TU/ml in the packaging cells.
Real-time PCR and Western blot demonstrated significantly suppressed Fas gene expression in UC-MSCs after infection with
the recombinant lentivirus. Conclusion Lentivirus-mediated RNAi can effectively inhibit Fas gene expression in cultured
UC-MSCs.

相似文献/References:

[1]李川,马纪,张越,等.慢病毒介导的shRNA靶向干扰nestin鼻咽癌稳定细胞株的建立[J].南方医科大学学报,2011,(04):604.
[2]方雷,吴海滨,陈晓岗.Survivin基因沉默抑制胃癌MGC-803细胞增殖并增强其对塞来昔布的敏感性[J].南方医科大学学报,2011,(11):1944.
[3]耿海涛,肖乾,徐邓勇,等.HNF6 重组慢病毒载体的构建及对大肠癌SW620细胞侵袭转移能力的影响[J].南方医科大学学报,2012,(01):66.
[4]刘展,张明亮,李毅妮,等.SiRNA抑制p63基因表达对胆管癌细胞增殖和侵袭的影响[J].南方医科大学学报,2012,(02):207.
[5]杨慧,张超,陆滟霞,等.Has-mir-335慢病毒表达载体的构建及其靶基因鉴定[J].南方医科大学学报,2012,(03):306.
[6]荆玉明,罗杰,张艳玲,等.p38丝裂原激活蛋白激酶基因重组慢病毒载体的构建及其在建立人前列腺癌稳定细胞株中的应用[J].南方医科大学学报,2012,(03):317.
[7]郑磊,包杰,王前,等.利用小干扰RNA表达框鉴定小鼠骨髓树突状细胞有效沉默RelB基因的实验研究[J].南方医科大学学报,2006,(03):301.
　ZHENG Lei,BAO Jie,WANG Qian,et al.Identification of effective siRNA sequence for RelB silencing in murine dendritic cells with siRNA cassette[J].,2006,(10):301.
[8]蒋鸿超,孙强明,赵丹,等.靶向survivin的siRNA表达载体的构建和表达及对HeLa细胞的影响[J].南方医科大学学报,2006,(12):1806.
　JIANG Hong-chao,SUN Qiang-ming,ZHAO Dan,et al.RNA interference-mediated inhibition of survivin expression in Hela cell line by siRNA expression vector targeting survivin gene[J].,2006,(10):1806.
[9]刘悦,郝玉娥,詹轶群,等.人甘油激酶慢病毒干涉载体的构建和表达[J].南方医科大学学报,2012,(05):614.
[10]岳枫,马文丽,宋艳斌,等.RNAi技术沉默K562细胞中c-myc基因的初步研究[J].南方医科大学学报,2005,(06):647.
　YUE Feng,MA Wen-li,SONG Yan-bin,et al.c-myc gene silencing in K562 cells with RNA interference[J].,2005,(10):647.

更新日期/Last Update: 1900-01-01