| Scleroderma is a chronic localized or diffuse progressive connective tissue disease characterized by skin thickening and visceral fibrosis,which may involve multiple organs such as the lungs,gastrointestinal tract and heart.The mechanisms and pathogenesis of this complex disorder are not well understood,leeking in effective treatment for the disease to date.The purpose of treatment is to prevent new skin and organs developed in the early stage and to improve the present symptoms in the advanced stage.UVA1(340 400nm) phototherapy is a new physical therapy in recent years.It is effective in the treatment of atopic dermatitis,scleroderma,mycosis fungoides,and other skin diseases, but its mechanism is unknown yet,maybe it is related to regulate immune function and induce T-cell apoptosis.There are arguments on the irradiation dose which have been reported;however the effectiveness of therapy is confirmed,especially in the improvement of skin symptoms.In order to look for the suitable irradiation dose in the clinical treatment for scleroderma,the changes of skin thickness,vascular endothelial cells,hydroxyproline and collagen content of mice models of scleroderma with different doses UVA1 treatment will be compared,which can provide a theoretical basis for clinical therapy.ChapterⅠExperimental study of establishing mouse model for sclerodermaObjective To establish a stable mouse model of scleroderma by Bleomycin(BLM), which to prepare for the next step of experiment.Methods Twenty BALB/c mice characterized as specefic pathogen free,female,aged 6 weeks,in weight 18～22g,were averagely divided into models and controls at random.Daily 100 UL BLM at a concentration of 400μg / ml was injected hypodermically into the backs of the models for 28 days.The pathological changes of skin tissue,thickness of skin and content of collagen were observed in the end of experiment.Results In the skin of the model group,cutaneous changes were generally localized to the area surrounding the injected site,where the sclerotic skin with reduced elasticity and hairless exhibited was observed. The control group's skin was reddish with elasticity and burnish hair.Histopathological examination revealed dermal sclerosis characterized by thickened derma and homogenous collagen bundles in model group,thinness skin and less collagen fibers in control group.There was a statistical significance of the differences between model and control groups in the skin thickness of skin(10.68±2.29μm and 6.96±0.64μm,t =4.945,P＜0.001),hydroxyproline(5041.10±1720.92μg / mg wet weight and 2094.40±586.01μg / mg wet weight,t =3.712,P＜0.01),content of collagen(37606.61±12838.04μg / mg protein and 15624.22±4371.63μg / mg protein,t =3.712,P＜0.01).Conclusions The mouse model of scleroderma could be successfully established by injection of BLM.ChapterⅡExperimental study on the effect of different dose UVA1 in the treatment of mouse model of sclerodermaObjective To compare the changes of skin thickness and collagen content of mice models of scleroderma with different dose UVA1 treatment,to look for the suitable irradiation dose in the clinical treatment for scleroderma.Methods The experimental mice models of scleroderma were established by hypodermical injection 100UL of the BLM 400μg/ml into the back of BALB/c mouse for 28 days,later the mice were randomly separated into 6 groups:normal control group,model group,high-dose(HD) UVA1 irradiation group,medium-dose(MD) UVA1 irradiation group,low-dose (LD)UVA 1 irradiation group and negative control group.Phototherapy was performed 3 times weekly for 10 weeks.The pathological changes of skin tissue,thickness of skin and content of collagen were observed after phototherapy.Results There was a statistical significance of the differences between normal control and model group in the skin thickness and collagen content(P＜0.05).UVA1 phothotherapy had significant therapeutic effect on mice model,which could improve the skin sclerosis and soften the skin,there were significant differences between HD-UVA1 and model group,negative group(P＜0.05).In 3 groups of phototherapy,HD-UVA1 group was significantly more effective than MD-UVA1 group and LD-UVA1 group(P＜0.01).Conclusions The HDUVA1 phototherapy has obvious therapeutic effect on mice model,which could improve the skin sclerosis of mice and collagen fibrous proliferation,its mechanism is related with its inhibition effect on collagen fibrous proliferation and reduction of content of collagen.ChapterⅢThe effect of different dose UVA1 on vascular endothelial cells in mouse model with sclerodermaObjective To compare the changes of CD34 and M30 from skin homogenate of mice models of scleroderma with different dose UVA1 treatment,to investigate the effect of UVA1 phototherapy on vascular endothelial cell(VEC)'s function.Methods The experimental mice models of scleroderma were established by the above mentioned method,later the mice were randomly separated into 5 groups:model group,HD-UVA1 irradiation group,MD-UVA1 irradiation group,LD-UVA1 irradiation group and negative control group(models wiyhout irradition).Phototherapy was performed 3 times weekly for 10 weeks.The changes of CD34 and M30 from skin homogenate were detected after phototherapy.Results①There was a statistical significance of the differences between HD-UVA1 group and model group in CD34(t =6.753,P＜0.001), UVA1 group and model group in M30(P＜0.005).②Therewas a statistical signifcance of the differences between MD-UVA1 group,HD-UVA1 group and negative control group in.CD34(P＜0.005),HD-UVA1 group and negative control group in M30(t =6.413,P＜0.001).③There was a statistical significance of the differences among 3 UVA1 groups by single factor analysis of variance(F=15.315,P＜0.001).Conclusions The functions of VEC are adjusted by UVA1 phototherapy through up-regulation of CD34 and inhibition of apoptosis of VEC,and the efficacy is related to radiation intensity and cumulative dose,the HD-UVA1 is more effective than MD-UVA1 and LD-UVA1.ChapterⅣPrimary study on effect of UVA1 on human fibroblasts' biosynthesis and metabolism in vitroObjective To compare the effect of different doses UVA1 on human fibroblasts' proliferation and metabolism of collagen.Methods Monolayers from human fibroblasts cultured in vitro were divided into UVA1-exposed cells and UVA1-unexposed cells. UVA1-exposed cells were irradiated by different dose UVA1(100J/cm~2,60 J/cm~2,20 J/cm~2).At 0 h,24h,48h after phototherapy,the changes of fibroblasts' morphology were observed with inverted microscope,the fibroblasts' proliferation was measured by MTT method,and the content of hydroxyproline and collagen of the cells were detected by digestion method.Results UVA1-exposed cells manifested edema,breek and rounded cells,and the destruction of HD-UVA1 was the biggest.The proliferation of cells,the content of hydroxyproline and collagen were inhibited after phototherapy,and there was statistical difference between the UVA-exposed cells and the UVA1-unexposed cells (P＜0.001).At the same time,phototherapy could decrease the proliferation of cells,the content of hydroxyproline and collagen in dose-dependent manner,there was a statistical difference in various doses of UVA1.Conclusion UVA1 radiation could reduce the proliferation of cells,the content of hydroxyproline and collagen in dose-dependent manner.The decrease of collagen content maybe related to the reduction of the number of cells and activity,as well as the inhibition of the synthesis of collagen.
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