Effects of ribozyme targeting platelet-derived growth factor receptor β subunit gene on the proliferation and apoptosis of hepatic stellate cells in vitro
- Author:
- No author available
- Journal Title:
- CHINESE MEDICAL JOURNAL
- Issue:
- 12
- DOI:
- No doi available
- Key Word:
- ribozyme;receptor;platelet-derived growth factor;hepatic stellate cell;liver fibrosis
Abstract： Background Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor β subunit (PDGFR-β) is the predominant signal transduction pathyway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-β mRNA in HSC and the effect on biological characteristics of HSC.Methods Expression vector of anti-PDGFR-β ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-β, α-smooth muscle actin, and typeⅠand type Ⅲ collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.Results The expression of PDGFR-β at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49%-57% (P<0.05-0.01). The proliferation and α-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased (P<0.05-0.01), and the type Ⅰ and type Ⅲ collagen synthesis were also reduced (P<0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC (P<0.01), and typical apoptotic cells could be found under transmission electron microscopy.Conclusions The anti-PDGFR-β ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-β expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-β expression.
- [1]陈岳祥,谢渭芬.肝纤维化治疗现状与对策[J].2002,(11).
- [2]郑燕芳,张积仁,屈良鹄.抗HPV16E6核酶对宫颈癌细胞表型和基因表达影响的研究[J].2002,(10).
- [3]展玉涛,王宇,魏来,等.Effects of vitamin E on the proliferation and collagen synthesis of rat hepatic stellate cells treated with IL-2 or TNF-α[J].2003,(3).
- [4]Bagheri S,Kashani Sabet M.Ribozymes in the age of molecular therapeutics.[J].2004,4(5).
- [5]S L, Friedman.Closing in on the signals of hepatic fibrosis.[J].1997,112(4).
- [6]Shin KS,Lee SW,Sullenger BA.Ribozyme-Mediated Induction of Apoptosis in Human Cancer Cells by Targeted Repair of Mutant p53 RNA.[J].2004,10(2).
- [7]Brenner DA,Bataller R.Hepatic stellate cells as a target for the treatment of liver fibrosis.[J].2001,21(3).
- [8]Pan WH,Xin P,Morrey JD,etc.A self-processing ribozyme cassette: utility against human papillomavirus 11 E6/E7 mrna and hepatitis B virus.[J].2004,9(4).
- [9]Pinzani M,Gesualdo L,Sabbah G M,etc.Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells.[J].1989.
- [10]Sakamoto M,Sata M,Nakamura T,etc.Green tea polyphenol epigallocatechin-3-gallate inhibits platelet-derived growth factor-induced proliferation of human hepatic stellate cell line LI90.[J].2004,40(1).
- [11]Cales, P..Apoptosis and liver fibrosis: antifibrotic strategies[J].1998,52(6).
- [12]M, Pinzani,K, Rombouts.Liver fibrosis: from the bench to clinical targets.[J].2004,36(4).
- [13]Hua, Li,Jinsheng, Zhang,Guangcun, Huang,etc.Effect of retinoid kappa receptor alpha (RXRalpha) transfection on the proliferation and phenotype of rat hepatic stellate cells in vitro.[J].2002,115.
- [14]S L, Friedman.Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies.[J].1993,328.
- [15]Ikura, Y.,Morimoto, H.,Ogami, M.,etc.Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease[J].1997,32(4).
- [16]M C, Wright,R, Issa,D E, Smart,etc.Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats.[J].2001,121(3).
- [17]Iredale JP,Benyon RC,Pickering J,etc.Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.[J].1998,102(3).
- [18]Bendia E,Benedetti A,Ugili L,etc.Intracellular pH regulation and Na+/H+ exchange activity in human hepatic stellate cells: effect of platelet-derived growth factor, insulin-like growth factor 1 and insulin.[J].2001,34(3).
- [19]M, Pinzani,S, Milani,H, Herbst,etc.Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis.[J].1996,148.
- [20]C, Lu,Y, Chen,Z, Zhang,etc.[Preparation and identification of activity of antiplatelet-derived growth factor receptor beta subunit hammerhead ribozyme in vitro].[J].2001,9(5).