Effect of small interfering RNA targeting transforming growth factor β receptor Ⅰ gene on the collagen synthesis of hepatic stellate cells in vitro
10.3760/cma.j.issn.1000-6680.2010.01.002
- VernacularTitle:转化生长因子βⅠ型受体基因的靶向小干扰RNA抑制肝星状细胞合成胶原的体外研究
- Author:
Fujun YU
;
Didong LOU
;
Zhuo LIN
;
Peihong DONG
;
Yongping CHEN
- Publication Type:Journal Article
- Keywords:
Receptors;
transforming growth factor beta;
RNA;
small interfering;
Liver cirrhosis;
Astrocytes;
Plasmids;
Collagen
- From:
Chinese Journal of Infectious Diseases
2010;28(1):5-9
- CountryChina
- Language:Chinese
-
Abstract:
Objective To observe the effect of small interfering RNA(siRNA)expression plasmids targeting transforming growth factor p receptor(TαR)Ⅰ gene on the collagen synthesis of hepatic stellate cells(HSCs).Methods Three siRNA expression plasmids were designed and constructed according to TBR Ⅰ sequence.Then the plasmids were transfected into HSC-T6 using 1ipofectamine2000 reagent. The mRNA and protein expressions of TβR Ⅰ were analyzed by reverse transcription polymerase chain reaction(RT-PCR)and Western blot technique, respectively. The cell proliferation was detected using methylthiazo-lyldiphenyl-tetrazolium bromide(MTT)methods. Concentrations of haluronic acid and type Ⅲ pro-collagen in the supernatants were determined by radioimmunoassay. The data were analyzed using least significant difference(LSD).Results Three recombinant plasmids expressing siRNAs were successfully constructed and confirmed by restriction enzyme assay. Compared with the blank control,all the three recombinant plasmids could inhibit the expressions of TβR Ⅰ mRNA,of which plasmid expressing siRNA2 exhibited the strongest inhibitory effect(psiRNA1 group:t=7.354,P＜0.01;psiRNA2 group:t=9.214,P＜0.01;psiRNA3 group:t=5.967,P＜0.01).The expressions of TβR Ⅰ protein were also reduced by all the three recombinant plasmids,of which the plasmid expressing siRNA2 showed the strongest inhibitory effect(psiRNA1 group: t=6.324,P＜0.01;psiRNA2 group:t=8.741,P＜0.01;psiRNA3 group:t=4.128,P＜0.01).The proliferation activity and collagen synthesis of HSCs also decreased in all three HSC groups treated with recombinant plasmids, of which, again, plasmid expressing siRNA2 exhibited the strongest inhibitory effect. However, no significant change was observed in HSCs transfected with non-related siRNA. Conclusion Recombinant plasmids targeting TβR I can inhibit collagen synthesis, which suggests a novel target for gene therapy of liver fibrosis.
