Effect Of Aminolevulnilic Acid Photodynamic Therapy On Keloid Fibroblast Cycle And The Research Of Regulatory Mechanism

objective:To investigate the effect of 5-aminolevulinic acid photodynamic therapy(ALA-PDT)on keloid fibroblasts(KFB)and the research on its regulatory mechanism.Methods:1.Tissue piece method was used to cultivate keloid fibroblasts and human skin fibroblasts(HSFs)in vitro and immunocytochemistry method was performed to identify fibroblasts(FB).2.After passaged,the 3rd ~ 5th generation of the logarit-hmic growth phase fibroblasts were choosed for further research.Added different concentrations of ALA,which was 0.25 mmol/L?0.5 mmol/L?1.0mmol/L?2.0mmol/L?4.0mmol/L ? 8.0mmol/L respectively,in high glucose with serum-free DMEM,avoid light 4h,optical power meter,light dose of 10J/cm~2?20J/cm~2 ?40J/cm~2?80J/cm~2?120J/cm~2.After incubation for 24 hours,CCK8 method was used to detect fibroblasts activity after the intervention,calculated the proliferation level,found the optimal combination of photosensitizer + energy(ALA concentration was 4.0mmol/L,PDT energy was 80J/cm~2).3.The growth of subculture in good condition of KFB were divided into four groups,blank control group,ALA+PDT group(AP group),p79350(the specific agonist of p38 MAPK signaling pathway)+ALA+PDTgroup(PAP group),SB203580(a specific repressor of p38 MAPK signaling pathway)+ALA+PDT group(SB group);human skin fibroblasts was marked as HSFs group.With concentration of ALA four mmol/L,PDT energy of eighty J/cm~2(the optimal concentration of photosensitizer + light energy combination)to intervene KFB each groups.Flow cytometry instrumentwas used to detect the distribution proportion in cell cycle of all fibroblasts groups.Western blot was used to detect the expression of CyclinD1 protein and phospho-p38 mitogen-activated protein kinase(p-p38 MAPK)protein.Results: 1.Cells climbed out of the tissue blocks after about 5 ~ 7 days.about 30~40 days,cell crawled about 80% of the culture bottle bottom,some prominences stretched out from the edge,vimentin immunocytochemistry staining result was positive,confirmed as fibroblasts.2.The results from the detection of CCK8 showed that the proliferation activity of KFB was restrained after ALA-PDT intervention,,and within a certain range,the proliferation activity reduced with the increase of concentration of photosensitizer and light dose.3.The results of the cell cycle from flow cytometry instrument showed that: the G0/G1 phase cells were the main peak in five groups,the S phase cells in blank control group were higher than HSFs group,S phase proportion cells were significantly higher(49.32 + 2.8%),PI was statistically difference(P<0.05);the S phase cells in AP group?PAP group and SB group were lower than the blank control group,S phase proportion cells were significantly reduction,were(20.26+2.5%?24.45+2.3%?16.97+2.6%),PI was statistically difference(P<0.05);the S phase cells in AP group were higher than SB group(16.97+2.6%),and lower than PAP group(24.45+2.3%),PI wasstatistically difference(P<0.05).4.Western blot test showed that: the expression of cyclin D1 and p-p38 MAPK rised in blank control group compared with HSFs group(P <0.05);the expression of cyclin D1 and p-p38 MAPK decreased significantly in AP group?PAP group and SB group,compared with blank control group(P < 0.05);while compared with AP group,the expression of cyclin D1 and p-p38 MAPK(P < 0.05)in PAP group rised,in SB group decreased(P<0.05).Conclusion: 1.The abnormal expression of p-p38MAPK/Cyclin D1 in KFB can lead to abnormal proliferation of fibroblasts;2.The proliferation cycle of KFB can be inhibited by ALA-PDT,its mechanism may be related to inhibition of p-p38 MAPK signaling pathways and reduce Cyclin D1 expression to induce G1 phase retardation.