Quercetin In Keloid Fibroblasts Radiotherapy Sensitization Effect And The Lack Of Research On The Effects Of Factor Expression

Background:Keloid is regarded as benign tumor since its growth characteristic that infiltrating into the surrounding normal tissues. The symptom of keloid accompany by unbearable itching and pricking. Long-term keloid may lead to tissue necrosis and repeated-infection, and eventually cause scar cancer. These have serious negative impacts on the patient’s life quality. So far, keloid is still lack of effective treatments. The topic of this study is based on the similar biological characteristics of malignant tumor and keloid that they both highly express the hypoxia inducible factor-α (HIF-la). In light of our previous achievements and the existing oncology research work of HIF-1α, we attach importance to the molecular level researches be performed on the keloid fibroblast cells after radiotherapy and quercetin intervention to clarify the impact of quercetin and radiotherapy on HIF-la protein expression.Objective:1. To investigate the growth situations of the keloid fibroblast cells under normoxic and hypoxic condition.2. To compare the radiation sensitivity of the fibroblasts derived from normal skin and keloid, then to determine the ideal radiation dose that cause the biggest difference between the two groups.3. To investigate the HIF-1α expression of the cultured keloid fibroblast cells and the fresh keloid tissue slices.4. To investigate the apoptosis-promote effects of quercetin on keloid fibroblast cells and the dose-response relationship.5. To investigate the relationships among quercetin, radiation and HIF-la expression.6. To determine whether quercetin could act as a radiation-promote reagent. Methods:1. Part Ⅰ:fibroblast cells were recovered and cultured for3～6generations before using in this experiment. We replaced the gas supply with1%O2,5%CO2,94%N2mixed gas to imitate the hypoxia condition. Two groups were included:the normoxia group and the hypoxia group. Cells were cultured in96-well plates with three different densities (1000cells,3000cells and5000cells per100μL),5wells for each density. CCK-8kits were used to determine the cell proliferation condition.2. Part Ⅱ:fibroblast cells that derived from the normal skin (the Norm group) and the keloid (the K1d group) were cultured regularly. Cell apoptosis rates were tested with flow cytometry24hours or48hours after radiation. Three radiation doses were used.3. Part Ⅲ:fibroblast cells from the normal skin (the Norm group) and the keloid (the Kid group) were treated with or without20gray radiation. HIF-1α expression of the fibroblast cells from the two groups were tested with western blot, real time-PCR and immunofluorescent staining technologies.4. Part Ⅳ:all the fibroblast cells were cultured under hypoxia condition in this experiment.0μmol/L,20μmol/L,40μmol/L and80μmol/L quercetin was used as intervene agents to the keloid fibroblast cells that with or without radiation treatment. Cells apoptosis rates were tested with flow cytometry and the ideal quercetin dose was determined based on the apoptosis results. Fibroblast cells from the normal skin (the Norm group) and the keloid (the K1d group) were treated with or without20gray radiation.24hours after radiation, HIF-1α protein expression were tested.40μmol/L quercetin was added to cells from the Kld group or not. HIF-1α expression was tested.Results:1. Part Ⅰ:the keloid fibroblast cells from the hypoxia group grew faster than the normoxia group under1000,3000,5000per100μl culture densities. On the5th day of culturation, the OD value of the hypoxia group was higher than the normoxia group significantly (P<0.05).2. Part Ⅱ:24hours after radiation, the apoptosis rate of the K1d group were lower than the Norm group after radiation with all the doses. After20gray radiation, the apoptosis rate of the Nor group (12.5±0.4%) increased significantly (P=0.000), compared with the K1d group (6.3±0.1%).48hours after radiation, the apoptosis rate of the K1d group decreased compared with the Norm group, and the rates of the two groups decreased compared with that tested24hours after radiation.3. Part Ⅲ:compared with the Norm group, the HIF-1α protein expression in the K1d group increased siginficantly. After20gray radiation, HIF-la protein expression increased by20%compared to the non-radiation cells. There was no HIF-1α expression could be determined in cells from the Norm group with or without radiation, while the cells from the K1d group positively expressed HIF-la. After radiation, HIF-1α expression in the K1d group strengthened compared with the cells treated with no radiation. The immunohistochemical staining results showed that HIF-1α existed mainly in the basal layer epidermis cell cytoplasm.4. Part Ⅳ:under radiation or non-radiation condition, the apoptosis rate of the keloid fibroblast cells increased according to the augmentation of the quercetin concentration. After40μmol/L quercetin intervention, the difference between the radiation and the non-radiation groups was maximum. The HIF-1α protein expression of the radiation group was higher than the non-radiation group, and the expression decreased significanly after treating with40μmol/L quercetin. The HIF-la gene expression of the keloid cells that treated with20gray radiation and40μmol/L quercetin decreased significantly compared with the cells with no treatement. Cells treated with40μmol/L quercetin expressed less HIF-1α compared with the cells without quercetin treatment according to the immunoflurescent test. Conclusion:1. During in vitro culture process, the fibroblast cells from the keloid grow faster under hypoxia condition than nomoxia condition.2. The keloid fibroblast cells are radiation resistant and this resistant effect is mainly related to the apoptosis level decline.3. The keloid fibroblast cells expressed more HIF-1α than the normal skin fibroblast cells. Radiation promotes HIF-1α expression.4. Quercetin can increase the apoptosis level of the keloid fibroblast cells. This effect strengthen when the dose of quercetin increased.5. Under hypoxia condition, quercetin can enhance the HIF-1α expression of the keloid fibroblast cells.6. Quercetin can improve the radiation sensitivity of the keloid fibroblast cells.