天津医药

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靶向血管平滑肌细胞TLR4基因小干扰RNA载体构建及效果评价

崔丽1,李广平2,富华颖2,王兴华1,焦占全3   

  1. 1. 天津医科大学第二医院心内科
    2. 天津医科大学第二医院心脏科
    3. 医科大学第二医院心脏科
  • 收稿日期:2013-05-02 修回日期:2013-10-22 出版日期:2014-02-15 发布日期:2014-02-15
  • 通讯作者: 崔丽

Construction of siRNA expression vector targeting TLR4 gene of Vascular Smooth muscle cells and evaluation the inhibitory efficacy

  • Received:2013-05-02 Revised:2013-10-22 Published:2014-02-15 Online:2014-02-15

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摘要： 目的：设计合成有效的靶向Toll样受体4（TLR4）基因的小发夹RNA（siRNA）表达载体，并筛选出TLR4沉默的SMC稳定细胞系。方法：通过RNAi序列设计原则，以TLR4基因为靶基因合成3对siRNA寡核苷酸链，经退火、与线性化pSilence 2.1-U6 neo质粒连接，用双酶切及测序进行鉴定。脂质体法转染血管平滑肌细胞，G418加压筛选并收集稳定表达质粒的SMC细胞，RT-PCR及Weston blot法检测TLR4 mRNA和蛋白表达。结果：测序鉴定插入的发夹样序列正确，成功构建TLR4基因RNA干扰表达载体；并获得稳定沉默TLR4的SMC细胞株。RT-PCR及Weston blot证实干扰TLR4后，SMC细胞株中TLR4 mRNA和蛋白表达均明显减低，其中pSilence2.1-siTLR4-1的抑制效果最好。结论：成功构建了能有效抑制TLR4基因的siRNA表达载体及TLR4稳定沉默的SMC细胞系。

关键词: Toll样受体, RNA干扰, 发夹样RNAi, 血管平滑肌细胞系

Abstract: Objective: to construct a vector expressing a short hairpin small interfering RNA which targets the Toll like receptor-4 gene and to construct a TLR4 knock-downed SMC cell line with this TLR4 siRNA expressing vector. Methods: three double-strands shRNA targeting the TLR4 gene were designed, synthesized and clone into the pSilence 2.1-U6 neo vector. Positive clones were verified with double enzyme digestion and sequencing. Then the recombinants were transfected to vascular smooth muscle cells by the cationic lipid method respectively. SMCs expressing plasmid stably were screened by G418 and collected. TLR4 mRNA and protein expression was detected by RT-PCR and Weston blot. Results: the pSilence2.1-siTLR4 expression vectors were successfully constructed and a TLR4 stable knock-downed SMC cell line was establishing. RT-PCR and Weston blot analyze confirmed that the expression of TLR4 was significantly down-regulated in these infected SMC cell lines and pSilence2.1-siTLR4-1was the most efficacious recombinant vector. Conclusion: recombinant vectors carrying a shRNA targeting the TLR4 gene were successfully constructed, and a SMC cell line stably expressing TLR4 shRNA was established with this recombinant vector.

Key words: Toll like receptor-4, RNA interference, Short hairpin small interfering RNA, SMC cell lines