Résumé
Objective To evaluate the influence of preconditioning with and anti-myosinmonoclonal antibody (mAb2G4)-nuclear factor-kappa B decoy oligodeoxynucleotide (ODN)-lipofectamine (lip) on hypoxia-reoxygenation (H/R) injury in H9c2 cardiomyocytes.Methods H9c2 cardiomyocytes were seeded in 6-well plate at the density of 1×105/ml (2 ml/well),and were divided into 3 groups (n=9 each) using a random number table:control group (group C),H/R group and mAb2G4-ODN-lip group (group MOL).The cells underwent 2 h of hypoxia in an air-tight bag,followed by 1 h reoxygenation.In MOL group,the cells were treated with mAb2G4-ODN-lip (2 μg ODN) for 4 h and then cultured in the common culture medium for 8 h before hypoxia.At the end of reoxygenation,proliferation of cells was measured using MTT assay,and the cells and supernatant of the culture medium were collected to determine the activity of lactate dehydrogenas (LDH),content of malondialdehyde (MDA),concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (by ELISA).The rate of proliferation inhibition was calculated.Results Compared with group C,the rate of proliferation inhibition,LDH activity,MDA content,and concentrations of TNF-α and IL-6 were significantly increased in the other two groups.Compared with group H/R,the rate of proliferation inhibition,LDH activity,MDA content,and concentrations of TNF-α and IL-6 were significantly decreased in MOL group.Conclusion mAb2G4-ODN-lip can mitigate H/R injury in H9c2 cardiomyocytes.
Résumé
Objective: As a potential gene therapeutic method, transcription factor decoy strategy has been used to block the transcription function of multi-transcription factors. This study aims to further verify the inhibitory effect of signal transducer and activator of transcription 5 (STAT 5) decoy oligodeoxynucleotides (ODN) on tumor growth in nude mice. Methods: The xenografted tumor model was established by subcutaneously injecting leukemia K 562 cells in nude mice. Liposome-ODN complex was directly injected into tumor body once daily every 3 days. The tumor growth and tumor size were measured everyday. Nude mice were sacrificed at the end of the experiment. The tumor was removed and weighed. RT-PCR and Western blotting were performed to detect mRNA and protein expression of bcl-xL, cyclin D1, and c-myc, respectively. Results: The tumorigenicity of K 562 cells was significantly inhibited by decoy ODN. The tumor weight was markedly reduced in decoy ODN group than mutant ODN group [(0.485 ± 0.178) g vs (0.928 ± 0.223) g, P < 0.05 ]. The tumor growth inhibition rate was 47.7%. RT-PCR and Western blotting showed that the mRNA and protein expression level of bcl-xL, cyclinD1, and c-myc were down-regulated. Conclusion: Transcription factor decoy strategy effectively blocks transcription of STAT 5 target genes in xenografted tumor of nude mice.
Résumé
This study is to examine the relationship between TGF-b1 expression and CTGF expression, and to evaluate the effect of Sp1 blockade on the expression of TGF-b1, CTGF and extracellular genes, clones of fibroblasts stably transfected with Sp1 decoy ODN. R-Sp1 decoy ODN was highly resistant to degradation by nucleases or serum, compared to the linear or phosphorothioated-Sp1 decoy ODN. Skin wounds were created on the back of 36 anesthetized rats. They were divided into four groups-the rats with normal skin, with wounded skin without decoy, with wounded skin injected with R-Sp1 decoy, and with wounded skin injected with mismatched R-Sp1 decoy, respectively. Skins were collected at 3rd, 5th, 7th, 14th day after wounding. Cellular RNA was extracted by RT-PCR analysis. TGF-beta1 and CTGF were deeply related with skin fibrosis during scar formation and it appeared that TGF-beta1 may cause the induction of CTGF expression. R-Sp1 decoy ODN inhibited TGF-beta1 and CTGF expression both in cultured fibroblasts and in the skin of rats. These results indicate that targeting Sp1 with R-type decoy efficiently blocks extracellular matrix gene expression, and suggest an important new therapeutic approach to control the scarring in normal wound healing and fibrotic disorders.