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大鼠VR1基因siRNA表达载体的构建及体内研究

Construction of VR1 siRNA expression vectors and their silencing effects in the DRG neurons of rats
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摘要 目的探讨辣椒素受体(vanilloid receptor subtype1,VR1)在慢性疼痛发生机制中的作用,构建携带VR1siRNA的慢病毒载体并检测其对大鼠DRG神经元VR1基因的干扰作用。方法设计靶向大鼠VR1基因的发夹状siRNA,合成两对互补的寡核苷酸序列,退火后克隆到酶切的pRNAT-U6.2/Lenti siRNA载体,通过PCR和DNA测序鉴定重组质粒。空载体及重组质粒分别与慢病毒包装质粒共转染293T细胞生成慢病毒载体。通过蛛网膜下腔将慢病毒载体注射到大鼠体内,采用RT-PCR方法检测L4-L6DRG神经元内VR1的沉默效果。结果测序鉴定证实目的寡核苷酸片段被克隆到pRNAT-U6.2/Lenti载体中;经蛛网膜下腔注射后,pRNAT-U6.2/Lenti-siVR1可下调正常大鼠L4-L6DRG神经元内VR1mRNA的表达。结论成功构建了靶向VR1基因的siRNA载体,可有效抑制VR1mRNA的表达,为深入研究和治疗慢性痛提供了有力的工具。 Aim To study the function of VR1 in chronic pain,to construct VR1 siRNA expression vectors and to study their silencing effect in the DRG neurons of rats were detected.Methods The hairpin sequences of siRNAs targeting VR1 gene of rat were designed,and two pairs of oligonucleotide sequence were synthesized.The annealed oligonucleotide fragments were cloned into linearized pRNAT-U6.2/Lenti expression vector and identified by PCR and DNA sequencing.Then,they were co-transfected by lipofectamine into 293T cells.The silencing effects of the lentivector-mediated VR1 siRNAs on the expression of VR1 mRNA were determined by RT-PCR after intrathecal injection in rats.Results DNA sequencing showed that the oligonucleotide fragments were correctly cloned into linearized pRNAT-U6.2/Lenti expression vector and the expression of VR1 mRNA in L4-L6 DRG neurons was inhibited significantly by pRNAT-U6.2/Lenti-siVR1 after intrathecal injection in rats.Conclusion The lentivector-mediated siRNAs are successfully constructed and they inhibit the expression of VR1 mRNA in the DRG neurons of rats,which may provide a potential tool for the further study and treatment of chronic pain.
作者 张宏伟 方东 任鹏飞 察雪湘 聂雅莉 胡香杰 赵国强
机构地区 郑州大学基础医学院药理学教研室 郑州大学基础医学院博士后科研流动站 郑州大学基础医学院微生物学与免疫学教研室
出处 《中国药理学通报》 CAS CSCD 北大核心 2010年第3期379-382,共4页 Chinese Pharmacological Bulletin
基金 河南省杰出青年基金资助项目(No200612001000) 河南省教育厅自然科学研究计划项目(No2009A310014)
关键词 VR1 SIRNA RNA干扰 病毒载体 DRG 大鼠 VR1 siRNA RNA interference Lentiviral vector DRG rat
分类号 R332 [医药卫生—人体生理学] R392.11 [医药卫生—免疫学]
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参考文献11

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二级参考文献51

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中国药理学通报
2010年 第3期

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