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