Inhibitory effect of retroviral vector containing anti-sense Smad4 gene on Ito cell line, LI90.
BACKGROUND
Transforming growth factor-beta1 (TGF-beta1) exerts strong fibrogenic potential in culture-activated HSCs. Smad4 is a key intracellular mediator for the transforming growth factor-beta (TGF-beta) superfamily of growth factors. The aim of this study was to assess the effects of the antisense Smad4 gene on Ito cell line, LI90.


METHODS
The recombinant retroviral vector pLXSN-Smad4 was constructed by cloning the rat antisense Smad4 cDNA into the retroviral vector pLXSN. Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad4 and pLXSN vectors into PA317 cells. Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418. The expression of Smad4 was detected by Northern and Western blots. Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed.


RESULTS
mRNA and protein expressions of Smad4 in LI90 cells transfected with retrovirus containing the antisense Smad4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells. Cells hypoexpressing the Smad4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline (P < 0.01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus.


CONCLUSIONS
The antisense Smad4 gene can suppress the expression of the Smad4 gene, reduce endogenous production of Smad4 mRNA and protein, block TGF-beta1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM). Our results may provide a basis for the development of antifibrotic gene therapy.
Xu
Xin-bao
XB
Hepatobiliary Surgery Center, People's Hospital, Beijing University, Beijing 100044, China. x_xb@163.net
Leng
Xi-sheng
XS
He
Zhen-ping
ZP
Liang
Zhi-qing
ZQ
Lin
Kai
K
Wei
Yu-hua
YH
Yu
Xin
X
Peng
Ji-run
JR
D002460:Cell Line; D016373:DNA, Antisense / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008103:Liver Cirrhosis / Q000628:therapy; D012190:Retroviridae / Q000235:genetics*; D051901:Smad4 Protein; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000235:genetics*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000502:physiology; D053773:Transforming Growth Factor beta1
D016373:DNA, Antisense; D004268:DNA-Binding Proteins; C497796:SMAD4 protein, human; D051901:Smad4 Protein; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1