The Role And Mechanism Of 5-hmC On The Development Of Pathological Scar Formation

Part 1Comparing the Expression of 5-hmC on Fibroblast in Different Human ScarBackground:Injury or operation can result in pathological scar,which brings extremely burden to patients.The way of how to inhibit the scar formation,become the most difficult issue in the whole world.Currently,the treatment of scar including:scar resection,intrascar injection,laser therapy as well as radiotherapy.Research regarding to the mechanism of molecular and gene level would help clinicians to solve the issue.Many researchers have been focusing on the modulation of scar inculding p53,MiRNA-21 as well as DNA demethylation.5-hmC(5-hydroxymethylcytosine)is a very important epigenetic marker,which is converted from 5-mc(5-methylcytosine)under the catalyze function of TETs(ten-eleven translocation family).Recent studies have found that the loss of 5-hmC related to tumor formation and some neural disease.However,there is little study relating to pathological scar.In this study,we hypothesis that the level of 5-hmC has some unknown relationship with pathological scar formation.We tried to compare 5-hmC expression between normal skin and scar tissue,which may provide the idea for treating pathological scar formation from gene level.Objective:The aim of our study is to compare the 5-hmC expression on pathological scar and normal skin fibroblasts in dermal level on humans,in order to judge if there is 5-hmC loss in pathological scar.Methods:Seven pathological scar specimens and seven normal skin control specimens were retrieved from pathology archive of the Department of Pathology at Brigham and Women’s Hospital.Samples were randomly selected for the study and the tissue from the procedures where they were harvested.All the patients have signed the patient consent form.The excised tissue was embedding in parffarin and cut into slices for HE staining and Trichrome staining as well as the lmmunohistochemistry(IHC)staining for 5-hmC.The analysis and the counting of 5-hmC have been completed under microscope.The statistical analysis of 5-hmC was calculated by Intensity multiply by percentage of 5-hmC positive fibroblast cells.All the data was analyzed by Graphpad Prism 7.0.Results:H&E staining and Trichrome staining showed the difference between normal skin and pathological scar tissue.The IHC staining of 5-hmC showed positive expression in normal skin,but loss in pathological scar.As we evaluated the percentage and intensity of 5-hmC expression on dermal fibroblast.5-hmC expression on normal tissue fibroblasts are 2.4 times more strong than that of pathological scar tissue(2.857 ± 0.356 VS 1.143 ± 0.971,p<0.001)Conclusions:The expression of 5-hmC on dermal fibroblasts in pathological scar tissue express less than that of normal tissue.Part 2The Effect of Hypoxia Condition in vitro on Human Fibroblast 5-hmC ExpressionBackground:Previous studies have found the loss of 5-hmC in various kinds of tumors and cancers.Our previous study has shown that the expression of 5-hmC on dermal fibroblast on pathological scar tissue,which was less than that of normal tissue.Tumor and scar has some similarity such as both are based on cell proliferation.Also,some tumor related genes have been found expressed in scar Hypoxia exists in scar tissue just like tumor dose.To imitate the environment of scar,we use human fibroblast under the treatment of hypoxia to process our experimentObjective:The purpose of our study is to observe the change of 5-hmC and its related genes to evaluate the effect of hypoxia on human dermal fibroblastMethods:The human fibroblast was cultured in vitro.We used CoCl2 to treat cells until they were spread to 100%.The concentration of CoCl2 we used were 0,100 μM,200μM respectively for 24 hours.After that,we used western blot to detect the protein expression of Hif-1α,FAK,TET3,and the evaluation of 5-hmC on fibroblasts under hypoxia was completed by dot-blot.RT-PCR was used to evaluate the mRAN expression of FAK and TET3.Immunofluorescent staining was used to evaluate the positive expression of 5-hmC and FAK.All the tests were repeated for 3 times and the data has been analyzed by Graphpad 7.0.Results:Western blot showed that in hypoxia status,Comparing with blank control group(FBO),HIF-1α of CoCl2 100μM group(FB100)increased(0.825±0.114 vs4.60±0.911;p<0.001),CoCl2 200μM group(FB200)HIF-1α increased(0.825 ±0.114 vs 7.29 ± 1.119;p<0.001).Comparing with FB100,HIF-1α expression in FB200 group elevated(4.60±0.911 vs 7.29 ±1.119;p<0.001).TET3 decreased after hypoxia treated(FBOvs FB100:3.367±0.208 vs 1.433±0.058;p<0.001,FBOvs FB200:3.367±0.208 vs 1.60±0.1;p<0.001).p-FAK expression increased with hypoxia(FBOvs FB100:0.703±0.024 vs 1.074±0.009;p<0.001,FBOvs FB200:0.703±0.024 vs 1.60±0.017;p<0.001).RT-PCR showed FAK mRNA increased with oxygen decreased(FBOvs FB100:1.000±0.0 vs 1.9±0.1;p<0.05,FBOvs FB200:1.000±0.0vs 2.267±0.153;p<0.05).TET3 expression decreased under hypoxia status.(FBOvs FB100:1.000±0.0 vsO.633±0.058;p<0.05,FBOvs FB200:1.000±0.0vs0.433±0.058;p<0.05).Dot-blot showed that 5-hmC DNA decreased with oxygen decreased(FBOvs FB100:20.56±1.25 vs 15.60±1.039;p<0.05,FBOvs FB200:20.56 ± 1.25vs 12.33 ±0.058;p<0.05).5-hmC immunofluorescent staining and F-actin&FAK double immunofluorescent staining showed the same tendency.Conclusions:5-hmC loss exist in pathological scar tissue fibroblasts,as well as the reduction of TET3.Additionally,the increase of FAK in hypoxia condition was consistent with former report regarding to FAK loss in scar tissuePart 3Explore the Effect of Vitamin C on Human Fibroblast under Hypoxia ConditionBackground:Our previous study has found that 5-hmC loss not only in human pathological scar tissue,but also in hypoxia condition in fibroblasts.Prior studies have demonstrated that the adjunvant function of Vitamin-C on wound healing,such as immune function,collagen deposition,antioxidant activity,neutrophil function as well as angiogenesis.However,the research regarding to the scar formation is very rare.We tried to examine whether Vitamin-C has modulation function on 5-hmC signal pathway in pathological scar formationObjective:The aim of our study is to evaluate the change of 5-hmC,TET3,FAK after treated with Vitamin C after hypoxia treatment,as well as to explore the Vitamin C on fibroblast demethelaytionMethods:We use ascorbate acid 100μM to treat fibroblasts,which have been treated with CoCl2,for 24 hours.Western blot of Hif-1α was used to evaluate the hypoxia condition of fibroblasts.dot-blot was used to confirm the change of 5-hmC.Western-blot and RT-PCR were used to explore the expression of FAK and TET3.All the tests were repeated for 3 times and data analysis was completed by Graphpad Prism 7.0Results:Western blot showed that HIF-1α expression decreased after vitamin C treated(FB100vs FB100+VC:4.604 ±0.911 vs 0.726 ±0.077;p<0.001;B200vs FB200+VC:7.289±1.119vs3.311±0.468;p<0.001).TET3 increased after vitamin C treated.(FBOvs FBO+VC:3.367±0.208 vs 8.15±0.30;p<0.001,FB100vs FB100+VC:1.433±0.057vs 4.4±0.1;p<0.001;FB200vs FB200+VC:1.6±0.1vs3.12±0.34;p<0.001).p-FAK decreased after vitamin C treated(FBOvs FBO+VC:0.703±0.024vs 1.264±0.076;p<0.05,FB100vs FB100+VC:1.074±0.009vs0.301±0.025;p<0.001;FB200vs FB200+VC:1.599±0.018vs0.316±0.031;p<0.001).RT-PCR showed that TET3 mRNA increased after vitamin C treated(FBO vs FBO+VC:1.000±0.0 vs 1.233±0.231;p<0.001,FB100vs FB100+VC:0.633±0.057vs 0.733±0.116;p<0.05;FB200vs FB200+VC:0.433 ±0.058 vs0.467±0.058;p<0.001.FAK decreased after vitamin C treated(FBO vs FBO+VC:1.000±0.0 vs 1.62±0.105;p<0.001,FB100vs FB100+VC:1.9±0.1vs1.053±0.186;p<0.001).Dot-blot showed that,5-hmC DNA increased after vitamin C treated.(FBO vs FBO+VC:20.56±1.25vs 36.33±3.215;p<0.001,FB100vs FB100+VC:15.6±1.039vs 27.67±2.082;p<0.001).Except for FB200 group,which has no significant change after vitamin C treated(FB200vs FB200+VC:12.33±0.578 vs13.42±0.947;P>0.05).5-hmC immunofluorescent staining and F-actin&FAK double immunofluorescent staining showed the same tendencyConclusions:The expression of 5-hmC and TET3 decreased,FAK and Hif-Icc increased under hypoxia conditions.Vitamin-C can partially reverse the expression of 5-hmC and TET3.Additionally,Vitamin C can partially reverse the hypoxia status of fibroblast,as well as decrease the expression of FAK under hypoxia.In a word,Vitamin C may provide us new horizon on how to reduce scar formation from epigenetic point of view.