| Background:Hypertrophic scar(HS)is the product of over-healing of wounds,and it is common in deep skin injuries caused by severe burns,surgical operations and other reasons.The clinical manifestation is that the scar protrudes from the surrounding normal skin tissue,and the texture is hard,often accompanied by symptoms such as pain and itching.It not only affects the appearance,but even scars contracture deformity,which greatly affects the patient's physical and mental health.In recent years,domestic and foreign scholars have conducted a lot of research on HS,but its pathogenesis has not been fully elucidated,and there is no ideal prevention method.Therefore,elucidating the pathogenesis of hypertrophic scars and finding new intervention targets will provide new ideas for the treatment of hypertrophic scars.It has been confirmed that the activity of TGF-?/ Smad signaling is abnormally increased in HS.The hyperactive TGF-?/Smad signaling leads to fibroblast proliferation,induces transformation of fibroblasts to myofibroblasts,increases collagen synthesis,inhibits matrix degradation and eventually leads to HS formation.Therefore,the regulation of the transduction activity of the TGF-?/Smads signaling pathway is of great significance for the treatment of hypertrophic scars.As an important protein post-translational modification,ubiquitination participates in the regulation of cell cycle,apoptosis,DNA repair,antigen presentation,receptor endocytosis,intracellular signal transduction and other biological processes,and is of great significance for maintaining cell homeostasis.Recent studies have found that deubiquitination mediated by deubiquitinating enzymes(DUBs)as the reverse process of ubiquitination also plays an important role in regulating intracellular signal transduction.USP15 is an important member of the ubiquitin-specific protease family.It is located in the 12q14 zone of chromosome and consists of 952 amino acids.Its relative molecular mass is 109.2k Da.USP15 has DUSP and UBL double domain units.Under normal circumstances,USP15 can be used as a "thermostat" in the TGF-?/Smad signaling pathway to regulate its transduction activity.In the case of weak TGF-?/Smad signal activity,USP15 binds tightly to the Smad7-Smurf2-T?RI complex to enhance TGF-? transduction activity,while in the case of strong TGF-?/Smad signal activity,USP15 dissociate from Smad7-Smurf2-T?RI complex,thereby promoting the degradation of the complex to weaken the TGF-? signal transduction activity.Therefore,USP15 plays an important role in regulating TGF-?/Smad signal transduction activity.However,in some diseases such as glioblastoma,breast cancer,and ovarian cancer,USP15 gene over-amplification leads to abnormally high expression of USP15.The USP15 thermostat is destroyed and only the "cold signal" can be detected,leading excessive activation of TGF-?/Smad signaling pathway.However,whether USP15 has abnormal activation in hypertrophic scars and whether it can play a deubiquitination effect to regulate TGF-?/Smad signal transduction and promote the occurrence and development of hypertrophic scars is still unclear.Based on the above considerations,this study first clarified the expression of USP15 in hypertrophic scars,and further planned to use USP15's regulation of TGF-?/Smad signal transduction as the main line.Integrated application of scar fibroblast culture in vitro,gene overexpression,RNAi interference technology,CCK-8cell proliferation,cell scratches,Cell invasion assay,quantitative real-time PCR(q RT-PCR),Western blot,co-immunoprecipitation(Co-IP),proteasome inhibition assay,ubiquitination assay,construction of hypertrophic scar animal model in nude mice were used to observe the effects of USP15 on the biological behavior of fibroblasts derived from hypertrophic scars,the interaction and changes between USP15 and related molecules in TGF-?/Smad signaling pathway,and the effects of interfering USP15 on the outcome of hypertrophic scars.To explore the role and molecular mechanism of USP15 in regulating TGF-?/Smad signaling pathway in hypertrophic scar at the tissue,cellular and animal levels,in order to provide new ideas and basis for the formation and prevention of hypertrophic scar.Part? The expression of USP15 and the TGF-?/Smad signaling pathway related molecules in hypertrophic scar tissues Objective:To detect the expression of USP15 and the TGF-?/Smad signaling pathway related molecules in hypertrophic scar tissues Method:The hypertrophic scar tissue specimens of patients undergoing scar resection were collected,and the remaining normal skin tissue of autologous skin grafting was used as a control.HE and Masson were used to detect the histological difference between hypertrophic scar and normal skin.Real-time fluorescent quantitative PCR and Western blot were used to detect USP15,TGF-?/Smad signaling pathway related molecules T?RI,Smad2,Smad3,and fibrosis markers ?-SMA,COL1,COL3 in hypertrophic scar and normal skin tissue.Result:HE staining and Masson staining showed that the disordered collagen fibers in the dermis layer and the number of fibroblasts were increased in HS tissues compared with the matched normal skin tissues.q RT-PCR showed that the m RNA expression level of USP15 and TGF-?/Smad signaling-related molecules T?RI,Smad2,Smad3 and fibrosis markers ?-SMA,COL1,and COL3 was significantly upregulated in HS compared with the matched normal skin tissues.Western blot results showed that the protein expression level of USP15 and TGF-?/Smad signaling-related molecules T?RI,Smad2,Smad3 and fibrosis markers ?-SMA,COL1,and COL3 was significantly upregulated in HS compared with the matched normal skin tissues Conclusion:Compared with the matched normal skin tissues,USP15 is highly expressed in hypertrophic scar tissue;the abnormal activation of TGF-?/Smad signaling pathway in hypertrophic scar can lead to scar fibrosis.Part? The effect of USP15 on the biological behavior of hypertrophic scar fibroblasts Objective:To detect the effect of USP15 on the biological behavior of hypertrophic scar fibroblasts Method:The tissue block adherence method was used to isolate and culture hypertrophic scar fibroblasts in vitro;Immunofluorescence was used to identify the fibroblast specific marker;a lentivirus carrying USP15 interference sequence and overexpression sequence was constructed,and lentiviral transfection and puromycin selection were used.The down-regulated and over-expressed stable cell lines of USP15 were used to verify the down-regulation and over-expression efficiency of USP15 in hypertrophic scar-derived fibroblasts by real-time fluorescent quantitative PCR and Western blot.CCK-8 assays was used to detect the effect of USP15 on the proliferation of hypertrophic scar fibroblasts.The cell scratch and Matrigel assays were used to detect the effect of USP15 on the migration and invasion of hypertrophic scar fibroblasts.Result:The tissue block adherence method successfully isolates and cultures hypertrophic scar fibroblasts.Immunofluorescence staining showed that the nuclear of fibroblast was stained blue and the cytoplasm of fibroblast was stained green due to vimentin-specific marker was regarded as positive stained cell.The percentage of positive stained cells was nearly 100%.It was confirmed that the cells cultured in vitro were fibroblasts.Lentiviral transfection and puromycin selection were used to successfully construct a stable transfected cell line with USP15 down-regulation and over-expression.The results of the CCK-8 assays showed that compared with the blank control group and the negative control group,the proliferation ability of hypertrophic scar fibroblasts in the USP15 interference group was significantly inhibited,and the difference was statistically significant(P<0.05);Compared with the blank control group and the empty vector group,the proliferation ability of hypertrophic scar fibroblasts in the USP15 overexpression group was significantly enhanced,and the difference was statistically significant(P<0.05).The results of the cell scratch and Matrigel assays showed that compared with the blank control group and the negative control group,the migration and invasion ability of hypertrophic scar fibroblasts in the USP15 interference group was significantly inhibited,and the difference was statistically significant(P<0.05);Compared with the blank control group and the empty vector group,the migration and invasion ability of hypertrophic scar fibroblasts in the USP15 overexpression group was significantly enhanced,and the difference was statistically significant(P<0.05).Conclusion:USP15 promotes the proliferation,migration and invasion of HSFs.Part? The molecular mechanism of USP15 regulates the TGF-?/Smad signaling pathway in hypertrophic scar fibroblasts Objective:To explore the effect of USP15 on TGF-?/Smad signal transduction in hypertrophic scar fibroblasts and its specific molecular targets Method:Construct a lentivirus carrying USP15 interference sequence and overexpression sequence,use lentiviral transfection and puromycin to select down-regulated and overexpressed stable cell lines of USP15,the interference and overexpression efficiency in hypertrophic scar fibroblasts were detected by real-time fluorescent quantitative PCR and Western blot.Real-time fluorescent quantitative PCR and Western blot were used to detect the m RNA and protein expression of USP15 on TGF?/Smad signaling pathway related molecules T?RI,Smad2,Smad3 and fibrosis marker ?-SMA,COL1,COL3.In addition,proteasome inhibition experiments are used to detect the degradation of T?RI protein;proteomics techniques such as co-immunoprecipitation(Co-IP)and ubiquitination experiments are used to detect the interaction between USP15 and T?RI and the level of ubiquitination modification of the molecular target.Result:The results of qPCR showed that compared with the blank control group and the negative control group,the m RNA level of T?RI,Smad2,Smad3,?-SMA,COL1 and COL3 were significantly down-regulated in USP15 interference group,and the difference was statistically significant(P<0.05);USP15 overexpression showed the opposite trends(P<0.05).Western blot results showed that compared with the blank control group and the negative control group,the protein level of T?RI,Smad2,Smad3,?-SMA,COL1 and COL3 were significantly down-regulated in USP15 interference group,and the difference was statistically significant(P<0.05);USP15 overexpression showed the opposite trends(P<0.05).The results of proteasome inhibition experiments showed that T?RI protein was degraded by ubiquitin-proteasome in HSFs,and the results of Co-IP and ubiquitination experiments showed that USP15 interacted with T?RI,and confirmed that USP15 can regulate the ubiquitination of T?RI.Conclusion:USP15 deubiquitinates T?RI and inhibits its degradation,thereby stabilizing T?RI protein levels and enhancing TGF-?/Smad signal transduction in hypertrophic scar fibroblasts.Part IV: In vivo intervention study of USP15 shRNA recombinant plasmid on nude mouse model of human hypertrophic scar Objective:RNA interference(RNAi)technology was used to intervene in a nude mouse model of human hypertrophic scar by USP15 sh RNA recombinant plasmid.Explore the possibility and significance of USP15 as a therapeutic target for anti-hypertrophic scar fibrosis.Method:First,transplant human hypertrophic scar tissue under the skin of the shoulder area of the back of nude mice to construct a total of 3 nude mouse models of human hypertrophic scar.The survival rate,biological behavior,diet,weight change,local redness,infection,ulcer,necrosis,etc.of the nude mice were observed.After 16 days,collect the scar graft,HE and Masson staining were used to observe its cells composition and collagen arrangement compare the hypertrophic scar tissue of the same patient before transplantation.A total of 9 human hypertrophic scar nude mice animal models were constructed again,and the experiment was divided into three groups: USP15 interference group(16 days after modeling,the scar tissue was injected with 250?l of USP15 sh RNA plasmid,liposome and PBS mixture at multiple points,n=3);USP15 negative control group(injection of USP15 negative interference plasmid,liposome and PBS mixture 250?l,n=3);blank control group(injection of PBS 250?l,n=3).All animals were raised in a single cage.After injection,the survival rate,biological behavior,diet,weight change,local redness,infection,ulcer,necrosis,etc.of the nude mice were observed.After 7 days,the specimens were collected,the volume and weight of the specimens were measured,and then the m RNA and protein expression of USP15,COL1,COL3 were detected by q RT-PCR and Western blot.Result:All the modeled nude mice were alive,eating normally,no weight loss,sluggishness,sluggishness,etc.,and no local redness,swelling,infection,ulcers,necrosis,etc.,scar grafts can be palpable on the back shoulder area of the nude mice,and they can be pushed along.Visually,the surface of the graft is wrapped with a fiber envelope,and a rich capillary network can be seen on the envelope.The results of HE and Masson staining showed that compared with the scar tissue before transplantation,the original histological structure was maintained after 16 days,the cell components were consistent,the collagen fibers were dense and the arrangement was disordered.The above results indicated that this experiment constructed a nude mouse model of human hypertrophic scar is safe,stable and reliable.In addition,by measuring the volume and weight of scar tissue before and after transplantation,it was found that compared with the blank control group and the negative control group,the graft volume and weight of the USP15 interference group decreased,and the difference was statistically significant(P<0.05).The results of q PCR showed that after injecting USP15 sh RNA plasmid at the m RNA level,USP15,COL1 and COL3 were significantly down-regulated compared with the blank control group and the negative control group,and the difference was statistically significant(P<0.05).Western blot results showed that after injection of USP15 sh RNA plasmid at the protein level,USP15,COL1 and COL3 were significantly down-regulated compared with the blank control group and the negative control group,and the difference was statistically significant(P<0.05).Conclusion:In this experiment,a nude mouse model of hypertrophic scar was successfully constructed.At the same time,interference with the expression of USP15 can prevents the progression of scar fibrosis,indicating that USP15 can be used as a new target for hypertrophic scar treatment. |
PDF Full Text Request