| Hyperplastic scar was the most common pathological changes skin occurring in the process of healing skin after burns.The incidence of it is 70 % and morbidity is about 48 %.In recent years,the life quality and survival rate of burn patients greatly improved along with the rapid development of medical technology.However,severely burned patients usually accompanied with a large number of hyperplastic scar formation,which not only affects beautiful,but also along with pain and itching,even affecting the movement.Causing psychological trauma and seriously affects the life quality of patients.Therefore,the formation mechanism and treatment of hyperplastic scar increasingly brought to attention of people.So,to explore effective measures to eliminate the scar has important clinical significance.Micrornas(miRNA)is a kind of endogenous non-coding RNA,which general length is 21-25 nt,usually plays an important role in transcription regulation,participates in the occurrence and development of skin diseases,and the skin repair process after trauma.Previous studies have shown that abnormal expression of miR-29 b are closely related with a variety of organs such as heart,kidney,lung fibrosis lesions.And it was shown to targeting inhibition of collagen,elastin and fibrillar protein 1 expression.In addition,miR-29 b affected tendon healing process through regulating the expression of TGF-?1.More recent researchs finding that the expression of miR-29 b is significant reduction in scar tissue comparision with normal skin tissue,which overexpression could effectively inhibit Collagen ? expression in fibroblasts.Hinting that miR-29 b is likely to be closely related with hyperplastic scar formation,but the specific molecular regulatory mechanism is not yet clear,whichstill needs to explore.Bone morphogenetic proteins-7 is a member of TGF-? superfamily,regulation cell proliferation,differentiation and apoptosis life process,participation in tissue regeneration after injury repair process and closely related with the development of liver,kidney and corneal fibrosis.Existing studies showed that the expression of BMP-7 significantly decreased in renal fibrosis mice.Vitamin E relieved renal fibrosis through regulating BMP-7 expression.Which was confirmed that could inhibit the rat hair papilla cells(DPCs)to fibroblasts.Furthermore,BMP-7 could reduce liver fibrosis through lowering the expression of ?-SMA and p-Smad2/3.Hinting that BMP 7 is likely to be involved in wound healing and inhibition hyperplastic scar formation.This study established scald mice model and subcutaneously injected miR-29 b mimics or rh BMP-7 to observe the effect of them on wound healing,scar tissue pathological change,collagen protein,fibrosis protein and cell apoptosis.Further explored the regulatory effect of miR-29 b on TGF-?1/Smad/CTGF signaling,and regulatory effect of BMP-7 on BMP-7/Smad1/5/8 signaling.Investigated the molecular mechanisms and effect of miR-29 b and BMP-7 on hyperplastic scar formation.Aimmed to elucidate the pathogenesis of hyperplastic scar and provide new molecular targets and more theoretical basis for the clinical treatment.1.The effect of miR-29 b overexpression on wound healing and scar pathological changesObjective: Analysis of whether miR-29 b mimics has influnce on burn wound healing and scar tissue pathology change.Making clear that miR-29 b participates in the hyperplastic scar formation.Method: Mouse scald model was established,subcutaneously injected miR-29 b mimics and randomly divided into four groups: Control group,thermal injury group,thermal injury+negative control group and thermal injury+miR-29 b group.Observedwound healing and made photographic record in 0 d,3 d,7 d,10 d,14 d after scald.Observed scar tissue pathological changes,collagen fiber formation by HE staining and masson staining respectively.Detected the expression level of miR-29 b by Real-time PCR.Result: Real-time PCR detection results showed that miR-29 b expression declined significantly after scald and the difference is statistically significant(P<0.001).The wound healing observations showed that obvious wound area was formed after mice scald,wound area in thermal injury+miR-29 b group was significantly reduced comparison with thermal injury group in 10 d and 14 d(P<0.01 or P<0.001).HE and Masson staining results showed that scar tissue epidermal stratum spinosum obvious thickening,perivascular inflammatory cell infiltration,fibroblasts hyperplasia in great quantities,number of collagen fibers significantly increased,bulky density and disordered arrangement in thermal injury group.Mice skin epidermal stratum spinosum obviously thinning,visible complete hair follicle structure,inflammatory cells basic disappear,collagen fiber content reduction and thinner,orientation is more neat and fiber bundle gap is bigger after intervention with miR-29 b mimics.Conclusion: MiR-29 b can effectively improve scar pathological changes,participate and regulate the development process of hyperplastic scar.Which laids a solid foundation for subsequent exploration for molecular mechanism.2.The effect of miR-29 b overexpression on TGF-?1/Smad/CTGF signaling and scarring formation associated proteinsObjective: Analysis of the effect of miR-29 b mimics on TGF-?1/Smad/CTGF signaling and Collagen ??Collagen ???-SMA expression.Making clear the specific molecular mechanisms affected hyperplastic scar formation.Method: The gene expression of Collagen ??Collagen ???-SMA?TGF-?1 and CTGF were detected with Real-time PCR.The protein expression of Collagen ??Collagen ???-SMA?TGF-?1 and CTGF were detected with Western blot.Result: Real-time PCR detection results showed that the expression of Collagen??Collagen ???-SMA?TGF-?1?CTGF m RNA was significantly increased in thermal injury group comparison with Control group,and the difference is statistically significant(P<0.001),which were significantly reduced comparison with thermal injury after subcutaneous injection miR-29 b mimics(P<0.05 or P<0.01 or P<0.001).Western bot detection results showed that the protein expression of Collagen ??Collagen ???-SMA?TGF-?1?CTGF and p-Smad2/3 was significantly increased in thermal injury group comparison with Control group,and the difference is statistically significant(P<0.05 or P<0.01),which were significantly reduced after subcutaneous injection miR-29 b mimics(P<0.05),the trend is consistent with the molecular level.Conclusion: MiR-29 b could regulate activity of TGF-?1/Smad/CTGF signaling,inhibit expression of scar formation related protein.Which eventually improves collagen deposition and reduces pathological scar formation.3.The effect of BMP-7 on wound healing,scar tissue pathology change and expression of related proteinObjective: Analysis of the effect of rh BMP-7 on burn wound healing,scar tissue pathology change and Collagen ?,Collagen ?,?-SMA,TGF-?1,CTGF protein expression.Method: Mouse scald model was established,subcutaneously injected different dose(25,50,100 ?g)rh BMP-7 and randomly divided into Control group,thermal injury group and thermal injury+BMP-7 group.Observed wound healing and made photographic record in 0 d,3 d,7 d,10 d,14 d after scald.Observed scar tissue pathological changes,collagen fiber formation by HE staining and Masson staining respectively,.Based on the above experimental results,rh BMP-7 best dosage was determined and the protein expression of Collagen ??Collagen ???-SMA?TGF-?1?CTGF was detected by Western blot.Result: The wound healing observations showed that wound area of thermal injury++BMP-7 group was reduced obviously in 7 d?10 d?14 d comparison with thermal injury group,and the difference is statistically significant(P<0.05 or P<0.01 or P<0.001).HE and Masson staining results showed that scar tissue epidermal stratum spinosum was obvious thickening,inflammatory cell infiltration,fibroblasts and collagen fiber number increased significantly,collagen fiber was bulky and chaotic in thermal injury group.Above scar tissue pathology changes dramatically improved after subcutaneous injection of rh BMP-7 and a dose dependent.Western bot detection results showed that the protein expression of Collagen ??Collagen ???-SMA?TGF-?1?CTGF markely increased in the scar tissue after scald(P<0.05 or P<0.01),which could be reduced obviously after subcutaneous injection 100 ?g rh BMP-7(P<0.05).Conclusion: BMP 7 could effectively reduce Collagen ? and Collagen ?content,inhibit collagen deposition,reduce ?-SMA,TGF?1 and CTGF expression level,prevent collagen fiber hyperplasia in great quantities,improve scar tissue pathology change and ultimately influence scar formation.4.The effect of BMP-7 on cell apoptosis and Smad pathway in the scar tissueObjective: Analysis of the effect of rh BMP-7 on cell apoptosis and expression of apoptosis related proteins,BMP-7,Smad1/5/8 and p-Smad1/5/8 in scar tissue.Method: Detected cell apoptosis of thermal injury group and thermal injury+BMP-7 100 ?g group with tunel method.Apoptosis related proteins Bax,Bcl-2,cleaved capase-3 and signaling pathway related protein BMP-7,Smad1/5/8,p-Smad1/5/8 were determined with Western blot.Result: TUNEL detection results showed that cell apoptosis number significantly increased in thermal injury+BMP-7 100 ?g group comparision with thermal injury group.Western blot detection results showed that subcutaneous injection of 100?g rh BMP-7 could significantly increase Bax/Bcl-2 proportion,and cleaved capase-3,BMP-7,p-Smad1/5/8 expression,difference was statistically significant.Conclusion: BMP-7 could activate BMP-7/Smad1/5/8 signaling pathway,increase Bax/Bcl-2 ratio and the cleaved capase-3 expression,promote fibroblast apoptosis,ultimately influence the formation of pathological scar. |
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