The Study Of 2-Methoxyestradiol Effects Towards Apoptosis And Proliferation Level In Keloid Fibroblasts And Its Mechanisms

Objective:1.To study the cell apoptosis and proliferation level in keloid tissue,and compare these levels with physiological scar tissue and normal skin tissue.2.To study the concentration-time-effect curve of 2ME2 towards keloid fibroblasts,then obtain the proper half maximal inhibitory concentration and working time.3.To study the effect of 6.975μM 2ME2 towards normal skin fibroblasts activity.4.To study the effect of 2ME2 in keloid fibroblasts mitochondrial apoptotic pathway key factors expression and PCNA expression,then explore and test its mechanisms.Methods:1.Thirty samples from thirty patients:10 keloid tissue(marked group K),10 physiological scar tissue(marked group C)and 10 normal skin tissue(marked group S).Morphological differences were observed by H&E staining and Masson staining.The qualitative and quantitative expression of key factors in mitochondrial apoptotic pathway and PCNA were analyzed by immunohistochemical staining and Western Blot.2.Primary culture of keloid fibroblasts from three different patients.In this part,all keloid fibroblasts were divided into three groups:①2ME2 group(with 7 different medicine concentrations and 4 different working times);②triamcinolone acetonide group(with 9 different medicine concentrations and 3 different working times);③5-FU group(with 10 different medicine concentrations and 3 different working times).Cell activity were measured by CCK-8 kit to draw the curve of concentration-time-effect and obtain the IC50 of different groups.3.Primary culture of keloid fibroblasts from three different patients and normal skin fibroblasts from three different patients.In this part,4 different working times and 6 different groups were established:①0μM 2ME2+normal skin fibroblasts(marked group OS);②0μM 2ME2+keloid fibroblasts(marked group OK);③6.975μM 2ME2+normal skin fibroblasts(marked group S);④6.975μM 2ME2+keloid fibroblasts(marked group K);⑤0.07%DMSO+normal skin fibroblasts(marked group DMSO-S);⑤0.07%DMSO+keloid fibroblasts(marked group DMSO-K).2ME2 activity towards normal skin fibroblasts were measured and analyzed by CCK-8 kit.4.Primary culture of keloid fibroblasts from six different patients and normal skin fibroblasts from six different patients.In this part,6 groups were established:①normal skin fibroblasts group(marked group S);②keloid fibroblasts(marked group K);③6.975μM 2ME2+keloid fibroblasts(marked group 2ME2);④0.07%DMSO+keloid fibroblasts(marked group DMSO);⑤8μM Ac-DEVD-CHO+keloid fibroblasts(marked group IN);⑥8pM Ac-DEVD-CHO+6.975μM 2ME2+keloid fibroblasts(marked group IN+2ME2).After 48h,the fibroblasts activity was measured.After 24h,TUNEL staining was used to observe morphological changes,immunofluorescence staining and Western Blot were used to assess the key factors expression in mitochondrial apoptotic pathway and PCNA expression qualitatively and quantitatively.Results:1.There were inflammatory cells infiltration in keloid tissue which has thicker epidermis and dermis arrange disorderly.The dermis of physiological scar arranges orderly.Normal skin tissue has loose dermis and thin epidermis.Compared with normal skin tissue,keloid tissue presents higher expression of caspase-3.Compared with other two groups,caspase-8 expression was also increased significantly.But,the expression of other key factors and Bcl-2/Bax ratio showed no significant differences among these three groups.The PCNA expression was remarkably increased in keloid tissue.2.The best IC50 of 2ME2,5-FU and triamcinolone acetonide is 6.975μM,5520μM and 152.4μM respectively.The best working time of all medicine is 48h.3.The activity of normal skin fibroblasts was slightly decreased during 6h to 12h after 6.975μM 2ME2 intervention.There is no significant difference and decreasing trend were found among 12h,24h and 48h.The activity of keloid fibroblasts was decreased obviously within 6h.During 12h to 24h,keloid fibroblasts activity decreased relatively slow,but still lower than normal skin fibroblasts.At 48h,normal skin fibroblasts activity showed no decreasing trend,on the contrary,keloid fibroblasts activity decreased significantly.4.2ME2 significantly decreased keloid fibroblasts activity and lead to typical cell apoptotic appearance.Besides Bcl-2,2ME2 group showed highest expression of other key factors in mitochondrial apoptotic pathway.Most key factors showed similar expression level among K group,DMSO group and IN group.Compared with 2ME2 group,the protein expression of caspase-3,caspase-8 and caspase-9 were decreased obviously.2ME2 also remarkably decreased the expression of PCNA.Conclusions:1.The overall level of cell apoptosis in keloid tissue is quite similar with physiological scar tissue and normal skin tissue.But the proliferation level is significantly higher in keloid tissue.These phenomenon means that keloid tissue is in an imbalance condition of extremely high cell proliferation and relatively low cell apoptosis.2.The best working time of 2ME2,5-FU and triamcinolone acetonide were all 48h.But 2ME2’s IC50 is much lower than any of other two medicine,which implies 2ME2 dosage is not as much as 5-FU and triamcinolone acetonide to obtain clinical effect and might company less complications as well when treating keloid patients.3.6.975μM 2ME2 could slightly inhibit normal skin fibroblasts activity.The inhibitory effect of 2ME2 does not last long.In this case,the side effects of 2ME2 toward normal skin may not obvious when clinical use.4.2ME2 could remarkably decrease keloid fibroblasts activity through improving cell apoptosis and inhibiting cell proliferation.2ME2 has significant influence on the expression of key factors in mitochondrial apoptotic pathway.This effect is partially achieved by caspase-dependent way.5.DMSO in 6.975 μM 2ME2 has no influence on cell activity of keloid fibroblasts and normal skin fibroblasts,does not change keloid fibroblasts morphological appearance,does not affect the expression of key factors in mitochondrial apoptotic pathway and PCNA.