[Hammerhead ribozyme-mediated cleavage of transforming growth factor beta1 RNA in a cell-free system and in hepatic stellate cells].
OBJECTIVE
To identify the activity of hammerhead ribozyme against transforming growth factor beta1 (TGFbeta1) in a cell-free system and in activated hepatic stellate cells (HSCs).


METHODS
The ribozyme against TGFb1 was designed with computer software. The transcripts of ribozyme, disabled ribozyme and target RNAs were prepared using the RiboMAX large scale RNA production system. The in vitro cleavage reactions were performed through incubation of 32P-labeled target RNAs with ribozyme or disabled ribozyme in different conditions. The eukaryotic expression vector encoding ribozyme and disabled ribozyme were constructed, and then transfected into HSC-T6 cells which exhibited characteristics of activated HSCs. The intracellular activity of the ribozyme was determined by detecting the ribozyme, disabled ribozyme and the TGFbeta1 expression.


RESULTS
The ribozyme cleaved target RNAs into anticipated products effectively. As expected, the disabled ribozyme possessed no cleavage activity in vitro. Further study demonstrated that the ribozyme expressed efficiently and inhibited TGFbeta1 expression in activated HSCs, while the disabled ribozyme displayed only a slight effect on TGFbeta1 expression.


CONCLUSIONS
The ribozyme with perfect cleavage activity in the cell-free system used inhibited TGFbeta1 expression effectively in activated HSCs. This ribozyme can provide a potential therapeutic approach for liver fibrosis.
Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030,China.
Yu-hu
YH
Song
Xiu-lan
XL
Xue
Qiu
Q
Zhao
De-an
DA
Tian
Nan-zhi
NZ
Liu
Huan-jun
HJ
Huang
Ju-sheng
JS
Lin
D000818:Animals; D002474:Cell-Free System; D002478:Cells, Cultured; D005822:Genetic Vectors; D022781:Hepatocytes / Q000166:cytology*; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
D016337:RNA, Catalytic; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; C427204:hammerhead ribozyme; D012313:RNA