| Hypertrophic scar and keloid scar, results of over-repair of wounds, not only influence beauty, but also are often accompanied with picking pain, pruritus, dyskinesia or dysfunction of limbs, so as to affect the physical and mental health of the patients. At present, there are many methods to treat pathological scars, however none of the ideal ways has been developed because of the their mechanism are not clear up to now.Compared with normal scars, the main histological features of pathological scars are the sedimentation of large amount of extracellular matrix. The main composition is Type I collagen (including 2 subtypes, Type a i I and a 21). It has been proved that the main reason of sedimentation is the interaction of various cells and various cytokines. Many researches indicated that the trans-membrane signal transduction is the key step during the reaction procedure between the cells and cytokines, and then the expression of type I Collagen occurs. Nuclear transcription factor, activator protein-1 (AP-1), is in the core link of gene expression of Type I collagen. Meanwhile the activated AP-1 can increase the synthesis of tissue inhibitor of metalloproteinase (TIMP), so as to specifically inhibit the activity of matrix metalloproteinase (MMP), which can degrade collagen in tissues. Therefore, inhibiting the activity of AP-1 can not only inhibit the synthesis of Type I collagen but also enhance the degradation of collagen to treat pathological scars.Transcription factor decoy strategy (TFDS) is one of the gene therapies inIVrecent years. Double-chain oligodeoxynucleotides (ODNs) fragments were artificially synthetic, which includes the sequence similar to the key sequence of the cis-elements acting with nuclear transcription factors. After ODNs were conducted into cells, ODNs and DNA could competitively combine the nuclear transcription factors so as to competitively inhibit the interation between nuclear transcription factors and its cis-elements and finally reach the aim of gene therapy.Decoy-ODNs and ODNs with unrelated sequence targeting AP-1 were synthesized in this study. The synthetic Decoy-ODNs were analyzed and tested with electrophoretic mobility shift assay (EMSA) to estimate their inhibition. Then decoy-ODNs, labeled with fluorescein and wrapped up by cationic Hposomes, were transfected into the NIH3T3 cells from fibroblast cell lines in rats, and the distribution and changing rule of Decoy-ODNs were observed by cofocal microscope. Finally, reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsorbent assay (ELISA) were used to analyze the effect of decoy-ODNs on gene expression of Type a 2 I collagen in NIH3T3 cells.The result showed that decoy-ODNs could competitively inhibit the activity of AP-1 in extracellular environment and the inhibiting effect was better than that of cold probes with same concentration. Decoy-ODNs with unrelated sequence had no inhibiting effect at all. Dim fluorescent light could be observed in NIH3T3 cells after Decoy-ODNs was transfected for 30 minutes. And 1 hour later, fluorescent signals became bright. With time passing by, the fluorescent light became brighter gradually and the fluorescent signals were distributed from the plasma into nucleus gradually, at last concentrated in nucleus. RT-PCR and ELISA proved respectively in gene expression and protein levels that our synthetic Decoy-ODNs could inhibit the synthesis of Type a 21 collagen.It includes as follow:1. Decoy-ODNs can inhibit the activity of AP-1 in vitro and this inhibiting effect is dose-dependent.2. Decoy-ODNs can be transfected effectively into plasma and nucleus through cationic liposomes.3. Decoy-ODNs can obviously inhibit the synthesis of Type a 2 I collagen.4. ODNs with unrelated sequence have no obvious inhibiting effect.The above-mentioned results provide technological methods and theoretical basis for gene therapies for pathological scars. |
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