Research Of Apoptosis And Mechanism By ALA-PDT Therapy On Fibroblast Cells From Hypertrophic Scar

Hypertrophic scars(HS)belongs to pathological scar.Treatment methods have been adopted,such as the efficacy of surgery,physics,lasers,and other drugs is not ideal,there is some degree of disadvantages and side effects,so that the lack of real control methods over clinical.5-aminolevulinic acid photodynamic therapy(ALA-PDT)is a new technology that treatment cancer significantly and rise in the 80-90s,local treatment is the use of physical principles,promising.Studies have shown that ALA-PDT effect is based on the optimum concentration of ALA combined with optimum laser energy density,people vitro scar fibroblast(Fb)is proliferation significantly inhibited.The mechanism may be associated with decreased activity and increased fibroblasts autophagic capacity.Fb proliferation of strong,active function,as more uptake,retention photosensitizers provide the conditions,but also for the prevention of scar formation provides the best method of treatment.Accordingly,we hypothesized that ALA-PDT may be a new treatment for hypertrophic scars,but the effectiveness and safety of its mechanism of action and clinical use pending further study.Objective:We cultured fibroblasts of people(scar)as experimental subjects,the use of ALA-PDT on fibroblast intervention,observation ALA group and laser group,ALA-PDT group and the control group(normal skin fibroblasts,scar tissue fibroblasts)on proliferation and apoptosis of Fb.In addition,by molecular biology methods to monitor Fb extracellular matrix proteins(α-SMA,VEGF,TGF-β，FGF2)secretion and expression,according to the results of ALA-PDT inhibit fibroblast mechanism of action related to elaborate while providing a theoretical basis for the molecular mechanism of hypertrophic scarring,but also for the prevention and treatment of hypertrophic scar formation of new ideas.Method:(1)collected from patients diagnosed with hypertrophic scar tissue,using cultured fibroblasts into separate culture,with 5-8 generations subcultured fibroblasts study.(2)were used to detect different concentrations(0,0.015,0.15,1.5,15,150,1500mmol/1)5-aminolevulinic acid for 2h and different energy(5,10,50,100,150J/cm2)of laser action in OD490 value Fb 10min after,observe the effect of Fb proliferation rate:(3)by MTT assay(ALA concentration:0,0.01,0.05,0.1,1.0,2.0J/cm2.incubated 2h;laser density:0,10,30,50J/cm2,irradiation 10min)for 48h of Fb proliferation inhibition,to find the best drug concentration and the optimum laser irradiation density;(4)the use of optimal drag concentration,and the laser irradiation dose cultured cells 48h,flow cytometry cell cycle and apoptosis;(5)cell supernatants were collected,ELISA assay α-SMA,TGF-β,VEGF and FGF2 collagen content;(6)cultured 48h,Western blotting(Western-Blot)to detect the expression of cell a-SMA,VEGF A,TGF-β and FGF2 protein.Results:1,Fb culture Results:Fb morphology see mature fibroblasts were mutually cross between spindle or irregular-shaped,long projections associated cell-cell growth,even abundant cytoplasm and nucleus were round or round.2,the test results MTT assay showed that ALA group and laser group irradiation on proliferation of fibroblasts into the increase drug concentration and laser energy is inhibited,but the inhibition was not obvious;ALA-PDT role of fibroblasts,the laser energy density when 30 J/cm2 or less,combined with the photosensitizer concentration was statistically significant at 1.0 mmol/L or less influence on the proliferation of fibroblasts had no(P>0.05).3,when the laser energy density(>50 J/cm2)combined with drug concentration(>2.0mmol/L),the concentration of the drug in the same group or the same energy density of the laser group compared to the inhibition rate increased significantly(P<0.05).In this experiment,the concentration of ALA in 2.0mmol/L,the laser irradiation dose 50J/cm2 for the best combination drug concentration and laser energy density.4,flow cytometry apoptosis and cell cycle results show that ALA-PDT Fb group and the control group scar,early apoptosis and late apoptosis was significantly increased.Inhibiting effect of S phase was significantly higher than the control group and the scar alone ALA group or laser group,significant inhibition of cell cycle progression.It showed that ALA-PDT laser can promote scar Fb cycle progression and inhibition of apoptosis Fb.5,ELISA assay ALA-PDT group influence the outcome of protein secretion display:secretion Fb inα-SMA,TGF-β1,VEGFA and FGF2 and other protein significantly decreased,and the scar in the control group,alone ALA group,individual laser group the difference was statistically significant.It showed that ALA-PDT can significantly reduce the associated protein to promote secretion of Fb proliferation.6,Western Blot assay ALA-PDT on HSFb protein expression showed:Fb in α-SMA,VEGF,TGF-β and the like FGF2 protein and scar control group,ALA group alone,alone laser group was significantly inhibited,suggesting that ALA-PDT can significantly reduce the expression HSFb of α-SMA,VEGF,TGF-β and FGF2 protein.Conclusion:1,derived ALA-PDT therapy on proliferation of fibroblasts into the optimal concentration and laser energy;2,ALA-PDT therapy in vitro culture system can inhibit α-SMA,VEGF,TGF-β and FGF2 secretion and expression;3,ALA-PDT therapy HSFb significantly inhibit proliferation and induce apoptosis.