Effects of ribozyme targeting platelet-derived growth factor receptor beta subunit gene on the proliferation and apoptosis of hepatic stellate cells in vitro.
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 beta subunit (PDGFR-beta) is the predominant signal transduction pathway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-beta mRNA in HSC and the effect on biological characteristics of HSC.


METHODS
Expression vector of anti-PDGFR-beta ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-beta, alpha-smooth muscle actin, and typeI and 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-beta at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49% - 57% (P < 0.05 - 0.01). The proliferation and alpha-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-beta 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-beta expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-beta expression.
Chen
Yue-xiang
YX
Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China. chenyuexiang@hotmail.com
Lu
Cui-hua
CH
Xie
Wei-fen
WF
Zhang
Xing-rong
XR
Zhang
Zhong-bing
ZB
Wei
Li-xin
LX
Jin
You-xin
YX
Guo
Ya-jun
YJ
D000199:Actins / Q000096:biosynthesis; D000818:Animals; D017209:Apoptosis; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D008099:Liver / Q000166:cytology*; D008103:Liver Cirrhosis / Q000188:drug therapy / Q000473:pathology*; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics*
D000199:Actins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D003094:Collagen; D020797:Receptor, Platelet-Derived Growth Factor beta