| Objective: To investigate the dynamic expression of cytokeratin-15(CK15)and cytokeratin-19(CK19)in chronic refractory wound tissues of rats under different intervention schemes,and to improve the partial molecular mechanism of moist exposed burn therapy/moist exposed burn ointment(MEBT / MEBO)in promoting wound healing,so as to promote its clinical treatment To provide theoretical support.Methods: 90 healthy adult male Wistar rats(SPF grade)were randomly divided into three groups: blank group(18 rats),control group(18 rats),model group(18 rats),MEBO group(18 rats)and Rb-b FGF group(18 rats).Among them,the blank group did not do damage treatment,the acute full-thickness skin defect model was done in the control group,and hydrocortisone was injected in the model group,MEBO group and Rb-b FGF group on the basis of the control group to prepare the chronic refractory wound model.After the model was established,the wound was changed twice a day.control group and model group were treated with Iodophor.MEBO group was treated with MEBO after iodophor disinfection.Group Rb-b FGF was treated with Iodophor and smeared with Recombinant Bovine Basic Fibroblast Growth Factor Gel(Recombinant bovine basic fibroblast growth factor gel(Rb-b FGF).At last,six rats were randomly selected on the 3rd,7th and 14 th day after treatment.After abdominal anesthesia,the granulation tissue of the wound was cut completely.Part of the tissues were fixed with formaldehyde and then stained with Hematoxylin-Eosin staining.Other tissues were put into-80 ℃ refrigerator.After all time points were collected,Western blotting and real time PCR were used to detect and compare the expression of CK15 and CK19 protein and the transcription level of CK15 and CK19 m RNA.Results:(1)after Hematoxylin-Eosin staining,no change was found in the blank group.On the third day after modeling,tissue edema and neutrophil infiltration were observed in the control group.In the slow injury model group,MEBO group and Rb b FGF group,there was edema and a small amount of lymphocyte infiltration.On the7 th day after modeling,neutropenia,edema and capillary regeneration were observed in the control group.In MEBO group and Rb-b FGF group,the infiltration of lymph was also reduced,and the edema between tissues was improved.In addition to neovascularization,a small amount of hair follicles were also seen.However,in the model group,the infiltration of lymphocytes was not reduced,tissue edema was still obvious,and new capillaries were absent.On the 14 th day after modeling,there was no inflammatory cell infiltration in the tissues of the control group,MEBO group and Rb-b FGF group,and the edema was not obvious.The new stratified epidermis structure was seen,and the skin appendages such as hair follicles and sebaceous glands were also seen.However,in the model group,there were still lymphocytic infiltration,edema and no obvious epidermis and hair follicle.(2)Western blotting:CK15 protein expression results: on the 3rd,7th and 14 th day after the treatment of the wound,there was no significant change in CK15 protein expression in the blank group(P> 0.05),but in the other groups,the expression of CK15 protein in the wound tissue was significantly changed(P< 0.05),and showed a trend of first decreasing and then increasing,the expression of CK15 protein was 14 days > 3 days > 7 days.There was no significant difference in CK15 protein expression between the control group,MEBO group and Rb-b FGF group(P > 0.05),and the expression of CK15 protein in the three groups was lower than that in the model group(P < 0.05).On the 14 th day,there was no significant difference in CK15 protein expression between the control group,MEBO group and Rb-b FGF group(P > 0.05),and CK15 protein expression in the three groups was higher than that in the model group(P < 0.05).CK19 protein expression results: on the 3rd,7th and 14 th day after wound treatment,there was no significant change in CK19 protein expression in skin tissue of blank group(P > 0.05),and CK19 protein expression in wound tissue of other groups was more significant(P< 0.05).There was no significant difference in CK19 protein expression between the control group,MEBO group and Rb-b FGF group(P > 0.05),and the expression of CK19 protein in the three groups was higher than that in the model group(P < 0.05).On the 14 th day,there was no significant difference in CK19 protein expression between the control group,MEBO group and Rb-b FGF group(P> 0.05),and CK19 protein expression in the three groups was lower than that in the model group(P <0.05).(3)Real time PCR result: it was consistent with Western blotting result.Results:there was no significant change of CK15 m RNA transcription level in the skin of the blank group on the 3rd,7th and 14 th day after wound treatment(P > 0.05).The expression of CK15 m RNA in the wound tissue of other groups was decreased first and then increased(P < 0.05),the transcription level of CK15 m RNA was 14 days > 3days > 7 days.There was no significant difference in CK15 m RNA transcription between the control group,MEBO group and Rb-b FGF group(P > 0.05),and the three groups were lower than the model group(P< 0.05).On the 14 th day,there was no significant difference in CK15 m RNA transcription level between the control group,MEBO group and Rb-b FGF group(P > 0.05),and CK15 m RNA transcription level in the three groups was higher than that in the slow injury model group(P <0.05).Results: on the 3rd,7th and 14 th day after the wound was treated,there was no significant change in CK19 m RNA transcription in the skin of the blank group(P >0.05).The expression of CK19 m RNA in the wound tissue of other groups was higherthan that in the other groups(P>0.05).There was no significant difference in CK19 m RNA transcription between the control group,MEBO group and Rb-b FGF group(P> 0.05),and the three groups were higher than the model group(P< 0.05).At the14 th day,there was no significant difference in CK19 m RNA transcription level between the control group,MEBO group and Rb-b FGF group(P > 0.05),and CK19 m RNA transcription level in the three groups was lower than that in the model group(P <0.05).Conclusion:(1)MEBT / MEBO can improve inflammatory response,reduce lymphocyte infiltration and promote the regeneration of skin and its accessory structures.(2)MEBT / MEBO can change the extracellular environment,regulate the expression of CK15 and CK19 protein and the transcription level of m RNA in granulation tissue of wound,so as to promote the proliferation and differentiation of epidermal stem cells,and accelerate the repair of chronic wound.(3)MEBT / MEBO can promote the healing of chronic wounds as well as Rb-b FGF,which can be further promoted in clinical application. |
