Construction and identification of siRNA eukaryotic expression vectors targeting on TGFβ1, TIMP-1 and TIMP-2 genes in vitro.
- Author:
Ke-li QIAN
1
;
Ning XU
;
Qing LANG
;
Jing-hu QI
;
Yin-chun SUN
;
Lang XIAO
;
Qi LIU
;
Xiao-feng SHI
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Cell Line; Gene Expression; Genetic Vectors; Hepatic Stellate Cells; metabolism; Plasmids; RNA, Messenger; genetics; RNA, Small Interfering; genetics; Rats; Tissue Inhibitor of Metalloproteinase-1; genetics; Tissue Inhibitor of Metalloproteinase-2; genetics; Transfection; Transforming Growth Factor beta1; genetics
- From: Chinese Journal of Hepatology 2011;19(4):291-296
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSThe 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.
RESULTSThe 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.
CONCLUSIONSThe 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.
