Preliminary Study On The Treatment Of Scar With Decorin Gene

Decorin(DCN)is widely used as a natural antagonist of TGF-β1 in the study of hypertrophic scars.However,exogenous DCN protein has problems such as easy degradation and short half-life,so repeated administration is required.Gene therapy,as a novel treatment,achieves therapeutic purposes through the expression of target gene in the host.In this study,a recombinant plasmid pDCN carrying the DCN gene was constructed,and a large amount of plasmid DNA was obtained by fermentation,isolation,purification,etc.The therapeutic effect of recombinant plasmid pDCN on rabbit ear scar was studied.The effect of DCN expression on TGF-β1 was also explored.In this study,the plasmid pUDK constructed in the laboratory was selected as the vector,and the recombinant plasmid pDCN carrying the decorin gene was constructed.It was verified that the target gene had inserted into the vector by double enzyme digestion and sequencing comparison.The recombinant plasmid was transformed into the competent state of E.coli,and a large number of bacteria were obtained by fermentation of the engineering bacteria.Then the lysate was obtained by lysis and concentration of bacteria,and the impurity such as RNA,open circular DNA(OcDNA)was removed by chromatography column to obtain high purity recombinant plasmid pDCN.Then,pDCN was transfected into 293 T cells.The expression of DCN and TGF-β1 were detected by RT-PCR and Western blot methods and the cells of empty plasmid group and normal group were used as control.Based on the cell experiments,rabbit ear scar model was constructed,pDCN was administered by drop,high dose group,middle dose group and low dose group.The wound healing and formation of scars were observed regularly when PBS was used as negative control and DCN protein was used as positive control.The hypertrophy index was as an indicator of the treatment effect of scars to observe the scar treatment effect of each group.The scars of each groups were taken for pathological examination,and the expression of DCN in scar tissue was detected by immunohistochemical staining.In the study,the recombinant plasmid pDCN carrying the decorin gene was successfully constructed by double enzyme digestion and sequencing.The plasmid with high purity was obtained by fermentation,concentration and cleavage.The results of RT-PCR and Western blot showed RNA and protein expression of DCN by transfecting 293 T cells and the amount of intracellular TGF-β1 expression is decreased.In the animal experiment,the rabbit ear scar model was established to observe the therapeutic effect of recombinant plasmid on scar.After 30 days of injury,the scar in the central was obviously higher than that of the surrounding normal skin,and the boundary between the central scar and the surrounding normal skin is obvious.The wound healing scar was pale pink,and the texture is slightly tough.The results showed that the hypertrophy index of pDCN group and rhDCN group were lower than that of PBS group,and the hypertrophy index of pDCN-M group was significantly lower than that of PBS group(P < 0.05).The pathological examination showed that the inflammatory cell infiltrated,a large amount of collagen was deposed and a large number of fibroblasts existed in PBS group.In pDCN-M group and pDCN-H group,the infiltration of inflammatory cell was not obvious and fibroblasts were less.It was indicated that the recombinant plasmid could inhibit the scar.The results of Immunohistochemical section showed that the positive expression in PBS group and pUDK group were brownish yellow,granular and filamentous,with small distribution area and sparse.The positive expression in DCN-H group、DCN-L group、DCN-M group and rhDCN group were more widely distributed and denser than those in PBS group and pUDK group.The positive expression in DCN-M group were more widely distributed and denser than those in other groups,which was consistent with the results of hypertrophy index statistics and HE section observation.This study initially showed that the recombinant plasmid pDCN has an inhibitory effect on scar formation.