[Effect of ribozyme against platelet-derived growth factor receptor beta subunit mRNA on the biological characters of hepatic stellate cells].
OBJECTIVE
To study the cleavage activity of hammerhead ribozyme targeting at platelet-derived growth factor receptor beta subunit (PDGFR- beta) mRNA in hepatic stellate cells (HSCs) and its effect on the biological characters of HSCs.


METHODS
Expression vector of anti-PDGFR- beta ribozyme was constructed and transfected into rat-derived HSC-T6 cells with lipofectin. The positive cell clones were gained by G418 selection. The expression of PDGFR- beta, alpha-smooth muscle actin (alpha-SMA), and type I and type III collagen was detected by means of northern blot, Western blot and immunocytochemical staining respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was demonstrated with 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 HSCs only 43% to 51% of that in control cells (t > or = 3.957, P < 0.05), and alpha-SMA expression level, type I and type III collagen synthesis ability were also reduced (t > or = 6.790, P < 0.01). The proliferation of ribozyme-transfected HSCs was significantly decreased (t > or = 3.858, P < 0.05), and the proliferation response to PDGF BB was markedly inhibited. However the apoptotic rate was significantly increased in ribozyme-transfected HSCs (chi2 > or = 14.157, P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy.


CONCLUSIONS
The anti-PDGFR- beta ribozyme can be expressed stably in HSCs, cleave the target RNA effectively, inhibit HSCs proliferation and collagen synthesis, and induce HSC apoptosis. The results suggest that inhibiting PDGFR- beta expression in HSCs may be a new therapy for liver fibrosis.
Chen
Yue-xiang
YX
Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
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
D017209:Apoptosis / Q000187:drug effects; D002455:Cell Division; D002478:Cells, Cultured; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D008099:Liver / Q000473:pathology*; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism
D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; D020797:Receptor, Platelet-Derived Growth Factor beta