Comparison between the suppression of tissue inhibitor of metalloproteinase-1 gene expression by recombinant adeno-associated virus carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 in rat hepatic stellate cells / 中华肝脏病杂志
Chinese Journal of Hepatology
;
(12): 742-747, 2006.
Article
Dans Chinois
| WPRIM
| ID: wpr-260610
ABSTRACT
<p><b>OBJECTIVES</b>Elevated tissue inhibitor of metalloproteinase-1 (TIMP-1) expression contributes to excess extracellular matrix in liver fibrosis. This study was designed to construct two recombinant adeno-associated viruses (AAV) carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 (rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo), and then to compare the suppression of TIMP-1 gene expression on rat hepatic stellate cell (HSC)-T6 cells infected by these two types of viruses in vitro.</p><p><b>METHODS</b>Antisense RNA amplified by rat HSC-T6 and U6 promoter followed by the annealing siRNA were cloned into the AAV vector (pdl6-95/neo) and packed in 293 cells to construct the recombinants rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo. Rat HSC-T6 cells were infected with these recombinant AAVs and selected by using G418, and real-time PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene in these cells.</p><p><b>RESULTS</b>The results of PCR, restrictive enzyme digestion and gene sequencing confirmed that the pdl6-95/ANTI-TIMP-1/neo and pdl6-95/siRNA-TIMP-1/neo had been reconstructed successfully. After they had been packed in 293 cells to form rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo, they were used to infect HSC-T6. Thirty days after the infection, the transcription level of TIMP-1 in HSC-T6 cells infected by rAAV/siRNA-TIMP-1/neo decreased dramatically compared with the mock control and normal HSC-T6 cells (P less than 0.01), and the expression level of TIMP-1 gene in HSC-T6 cells decreased significantly (60%), while the transcription and expression level of TIMP-1 in HSC-T6 cells infected by rAAV/ANTI-TIMP-1/neo had no significant difference with mock control and normal HSC-T6 cells (P more than 0.05).</p><p><b>CONCLUSION</b>RNA interference can exert a suppression of TIMP-1 gene in rat HSC, and when this function combines with AAV infection, it can suppress the specific gene expression for a long time by chromosomal integration.</p>
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
Sujet Principal:
Cellules cultivées
/
ARN antisens
/
Dependovirus
/
Inhibiteur tissulaire de métalloprotéinase-1
/
Petit ARN interférent
/
Cellules étoilées du foie
/
Vecteurs génétiques
/
Génétique
/
Métabolisme
Limites du sujet:
Animaux
langue:
Chinois
Texte intégral:
Chinese Journal of Hepatology
Année:
2006
Type:
Article
Documents relatifs à ce sujet
MEDLINE
...
LILACS
LIS