Effects Of The Proliferation Of Rat Hepatic Stellate Cells And Collagen-Ⅰ Production By Antisense Oligodeoxynucleotides Against Platelet-Derived Growth Factor B-chain

Objective:Hepatic fibrosis is a kind of severe disease with high morbidity and mortality, and its common pathological feature is over-aggradation of extracellular matrix (ECM). The activation of hepatic stellate cell (HSC) is critical step in the development of liver fibrosis. Proliferation and fibrogenesis of activated HSC are attributed to over-aggradation of ECM. There have been no satisfactory drugs so far in the treatment of hepatic fibrosis. Therefore the strategy for terminating the proliferation of activated HSC might be an excting therapy for patients with liver fibrosis. Platelet-derived growth factor B-chain (PDGF-B) is a potent mitogen for HSC. In this study, we investigated the effect of antisense oligodeoxynucleotides (ASODN) against PDGF-B in the proliferation of HSC, the expression of endogenous PDGF-B and collagen-â…  production targeting at HSC-T₆.Methods:We synthesized phosphorothioate ASODN and missense oligodeoxynucleotides (MSODN) against PDGF-B mRNA, and then treated the rat hepatic stellate cells at different concentrations respectively. We assay the effect of PDGF-B ASODN on HSC-T₆ cells. The growing curves of HSC-T₆ cell line had been drawn. We assay the inhibition of the proliferating of cultured rat hepatic stellate cells by MTT method. The expressions of PDGF-B and collagen- â…  mRNA were detected by means of xeversetranscription-polymerase chain reaction (RT-PCR). The expression of endogenous PDGF-B was determined by flow cytometry (FCM) and SABC. Collagen-1 in the supernatants was determined by Enzyme-linked Immunosorbent Assay (ELISA).Results:(1) The results showed the growth of cultured rat hepatic stellate cells (HSC) was significantly inhibited by using 2.5-10 jimol/L PDGF-B ASODN. The activity of HSC was more than 95% by typan-blue method. The inhibition was dose-dependent, but 2.5-10 n.mol/L MSODN shows no effect. However, no growth was observed for 20nmol/L ASODN group and MSODN group, the activity of HSC was about 45%, these was the non-specificity toxicity effect of cell. (2) PDGF-B ASODN could inhibit proliferation of HSC with the final concentration of 10 p.mol/L. The inhibition peaked at 48 hour after transfection by 10 \imolfL PDGF-B ASODN and the inhibition rate was 44.68% (P<0.05). By use of RT-PCR , PDGF-B ASODN could downregulate the expression of PDGF-B and collagen-1 mRNA in HSC-T6 cells by 10 nmol/L PDGF-B ASODN. The inhibition rate was 48.7%, 53.9% respectively. The expression of endogenous PDGF-B was significantly inhibited in HSC-T6 cells by SABC. Flow cytometry analysis demonstrated, the positive rate and MFI of endogenous PDGF-B showed a significant decreased, and the positive rate from 98.9% to 63.8% and MFI from 10.2 to 3.37. PDGF-B ASODN could greatly inhibit the production of collagen-1 by ELISA, the content of collagen-1 from 392.256 ng/ml to 157.397 ng/ml (P<0.001), whereas the controls (MSODN) had no effect at the same concentration.Conclusion:1. PDGF-B ASODN was able to inhibit the growth and proliferation of cultured rat hepatic stellate cells (HSC) in vitro obviously.2. PDGF-B ASODN could downregulate the expression of PDGF-B mRNA and the endogenous PDGF-B in HSC-T6 cells.3. PDGF-B ASON could inhibition the expression of collagen- I mRNA and the production of collagen-I of HSC-T6 cells. .4. PDGF-B ASON might be useful for gene therapy in liver fibrosis.