title: [Construction and identification of siRNA eukaryotic expression vectors targeting on TGFβ1, TIMP-1 and TIMP-2 genes in vitro].
abstract: OBJECTIVE
  To construct the siRNA eukaryotic expression vectors targeting on TGFβ1, TIMP-1 and TIMP-2 and to investigate the inhibitory efficiency of target genes expression on rat hepatic stellate cell in vitro.
  
  
  METHODS
  The siRNA cDNA sequences of TGFβ1, TIMP-1 and TIMP-2 were designed, synthesized and inserted into plasmid pGenesil-1 respectively to generate eukaryotic expression plasmids. The plasmids were transfected into HSC T6 cells in vitro and the inhibitory efficiency of target genes expression was observed with real-time PCR and Western blot.
  
  
  RESULTS
  The eukaryotic expression vectors were constructed successfully. The expressions of TGFβ1 mRNA, TIMP-1 mRNA and TIMP-2mRNA in siRNA-transfected groups were decreased by 63.4% ± 8.0%, 64.5% ± 9.0% and 55.0% ± 17.0% respectively and the expressions of TGFβ1 protein, TIMP-1 protein and TIMP-2 protein were decreased by 57.8% ± 3.0%, 55.1% ± 5.0%, 49.3% ± 1.0% respectively as compared to the control groups.
  
  
  CONCLUSIONS
  The siRNA eukaryotic expression vectors constructed targeting on TGFβ1, TIMP-1 and TIMP-2 could reduce the expressions of target genes and they might be able to used for the exploration of new anti-fibrosis drugs genetically.
authors.affiliation: Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education and Department of Infectious Diseases, the Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.
authors.forename: Ke-li
authors.initials: KL
authors.lastname: Qian
authors.forename: Ning
authors.initials: N
authors.lastname: Xu
authors.forename: Qing
authors.initials: Q
authors.lastname: Lang
authors.forename: Jing-hu
authors.initials: JH
authors.lastname: Qi
authors.forename: Yin-chun
authors.initials: YC
authors.lastname: Sun
authors.forename: Lang
authors.initials: L
authors.lastname: Xiao
authors.forename: Qi
authors.initials: Q
authors.lastname: Liu
authors.forename: Xiao-feng
authors.initials: XF
authors.lastname: Shi
mesh_terms: D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D055166:Hepatic Stellate Cells / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics*; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics*; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019715:Tissue Inhibitor of Metalloproteinase-1; D053773:Transforming Growth Factor beta1; D019716:Tissue Inhibitor of Metalloproteinase-2