| 慢病毒介导的RNAi对大鼠SOCS3基因表达的抑制作用 |
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| 引用本文： | 张旭,刘正娟,吴鸣,滕懿群,骆秋龙,陈俊国,陆燕,章爱莲.慢病毒介导的RNAi对大鼠SOCS3基因表达的抑制作用[J].中国微生态学杂志,2011,23(10):877-880. |
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| 作者姓名： | 张旭 刘正娟 吴鸣 滕懿群 骆秋龙 陈俊国 陆燕 章爱莲 |
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| 作者单位： | 1. 嘉兴市第二医院儿科,浙江嘉兴,314000
2. 大连医科大学附属第二医院儿科,辽宁大连,116027 |
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| 基金项目： | 国家自然科学基金(30771798); 辽宁省自然科学基金(20072165) |
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| 摘 要： | 目的研究慢病毒表达载体介导的RNA于扰（RNAi）对大鼠下丘脑细胞中细胞因子信号转导抑制因子3（SOCSB）的抑制作用。方法根据大鼠SOCS3基因（NM053565），用Ambion在线软件选择3个靶序列，设计并合成包含各正反义靶序列的互补单链寡核苷酸，与经BamHI和XhoI酶切后的慢病毒载体质粒pRNA-1enti-GFP连接产生pRNA-Lenti-SOCS3-GFP慢病毒重组质粒，与慢病毒包装混合物共转染293T细胞，包装产生慢病毒，收集病毒上清，采取逐孔稀释滴度法测定病毒滴度，然后转染大鼠下丘脑细胞，通过荧光显微镜观察细胞转染情况，利用荧光实时定量PCR方法检测RNAi组（siRNAl，siRNA2，siRNA3）、空白细胞组和阴性序列组（siRNA—Negtive）中SOCS3的表达情况。结果将目的序列成功连接到载体上，并经测序分析证实载体构建成功。系列稀释法检测慢病毒悬液的滴度为1．0×10^10TU／L。荧光实时定量PCR检测显示慢病毒感染大鼠下丘脑细胞后，与空白细胞组相比，3个RNAi组都有不同程度的抑制SOCS3表达，其中siRNAI组的抑制效果最好，可使SOCS3mRNA表达下调达80％。结论构建的pRNA-Lemi-SOCS3-GFP慢病毒载体可有效地抑制大鼠SOCS3的表达，为以SOCS3基因为靶点的相关疾病的基因治疗研究奠定基础。
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| 关 键 词： | 细胞因子信号转导抑制因子3 核糖核酸干扰 慢病毒载体 下丘脑细胞 |
Inhibition effect of lentiviral vector-mediated RNAi on expression of SOCS3 gene in rat hypothalamic cells |
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| ZHANG Xu,LIU Zheng-juan,WU Ming,TENG Yi-qun,LUO Qiu-long,CHEN Jun-guo,LU Yan,ZHANG Ai-lian.Inhibition effect of lentiviral vector-mediated RNAi on expression of SOCS3 gene in rat hypothalamic cells[J].Chinese Journal of Microecology,2011,23(10):877-880. |
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| Authors: | ZHANG Xu LIU Zheng-juan WU Ming TENG Yi-qun LUO Qiu-long CHEN Jun-guo LU Yan ZHANG Ai-lian |
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| Institution: | ZHANG Xu1,LIU Zheng-juan2,WU Ming1,TENG Yi-qun1,LUO Qiu-long1,CHEN Jun-guo1,LU Yan1,ZHANG Ai-lian1 (1.Department of Pediatric,the Second Hospital of Jiaxing,Jiaxing 314000,China,2.Department of Pediatrics,the Second Affiliated Hospital of Dalian Medical University,Dalian 116027,China) |
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| Abstract: | Objective To construct a lentiviral-mediated RNAi vector targeting rat suppressors of cytokine signaling 3(SOCS3) gene and assess its gene silencing effect in rat hypothalamic cells. Method Three target sequences were selected by on-line designer software on Ambion according to rat SOCS3 mRNA sequence.The complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized.After annealing,these double strands DNA were cloned to pRNA-lenti-GFP which contained U6 promoter and gr... |
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| Keywords: | Suppressors of cytokine signaling Ribonucleic acid interference Lentiviral vector Hypothalamic cells |
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