| Background and objectsKeloids represent a form of abnormal wound healing in genetically susceptible individuals. This disease is unique to human. They are more common in Asian people and no appropriate animal models to research potential mechnism. Currently, it is one of the difficulties of dermatology and plastic surgery treatment. They are often resistant to treatment and have a high recurrence. After the complete excision of keloids, radiation therapy has been demonstrated as one of the most effective methods to prevent recurrence. The recurrence rate was correlated with total dose of ionizing radiation. Keliod fibroblasts (KFb) are the main function cells, which have epigenetic modifications like tumor cells. The characteristic of biological behavior is abnormal fibroblasts proliferation and secretion of extracellular matrix. Comparing, two kinds of primary fibroblasts from keloids and normal skin, we try to illustrate whether multiple genes and their relevant gene pathways were involved in this progress? Whether these genes have epigenetic changes influencing the transcription, then affecte proliferation, cell cycle, cell metabolism function? Whether different doses of ionizing radiation contribute to these links? Radiation induced keloid fibroblast apoptosis, senescence or other phenotypes at different stages after ionizing, which produce therapeutic effect. Our study aimed to explore possible epigenetic mechanisms in the therapeutic effect by the primary culture fibroblasts from keloids and normal skin. We hope these research will provide new ideas, diagnosis and therapeutic targets for Keloids treatment.Methods and material1. First, different clinical stage keloid patients were included, signed the informed consent, judge the severity of keloid, and we obtained the composite grade points. In this study, isolated primary cells of keloid fibroblasts (KFb) and normal human dermal fibroblasts (NFb) were identified, the cell viability and proliferate potential were determined by MTT/CCK-8, the cell cycle was detected by flow cytometry instrument. At same time, the difference on the collagen synthesis between KFb and NFb were tested.2. Epigenetic research of p16gene promoter region function disorder was conducted on keloids tissue and KFbs by bisulfite sequencing (BSP).The correlation between severity of keloid and clinical staging was analyzed. This study would detect of p16gene methylation status and judge DNMTs gene expression difference between KFb and NFb. Difference in p16, p21and p27gene expression was dectected by Real-time fluorescent quantitative PCR. Immunohistochemical method was used to detect p16, p21, p27, and p53expression in keloid tissue. Comparison the pathological difference of keloid lesion tissue, normal skin and fibroblasts source tumour-fibrosarcoma, and proliferation differences in keloids and fibrosarcoma through Ki-67immunohistochemical staining.3. Ionizing radiation induced keloid fibroblasts premature senescence and its related gene expression changes. After exposed to2,4,6, or8Gy of X-Ray radiations for24hr,48hr,72hr, or96hr, respectively the cell viability and proliferative potential of KFb and NFb were detected at different time using the trypan blue exclusion and CCK-8assay. The analysis of cell cycle was performed by flow cytometry. The percentage of cells positive for Senescence-associated β-galactosidase (SA (3-gal) activity was assessed. Moreover, the expression of cell cycle-associated genes such as p21, p16and p27, was assessed at mRNA and protein levels with Real-time PCR and western-blot. According to the situation of proliferation inhibition, expression of p16, p21, p27and former type I collagen gene, and the correlation with the cell cycle distribution, we can determine the most significant inhibition effect of ionizing radiation dose and time.Results1. Twelve patients with keloid were included. The gender and age composition from patients and control group was no significant difference. The primary cultivation of keloid fibroblasts (KFb) is8cases and NFb is6cases. The phase contrast optical microscopy imaging showed that both KFb isolated from keloid tissues and NFb from normal skin tissues possessed classic and similar fibroblast cell morphology. But there was a significant difference between cell proliferation and metabolism ability, compared with NFb, KFb exhibited more percentage of G2/M phase cells and less percentage of G0/G1phase cells.2. Ionizing radiation influences KFb cells p16genes’ methylation state and then influences transcription and expression. The results showed that low expression of cell cycle related genes p16, p21and p27in KFb cells and keloid tissues, high expression of P53protein in keloid. Three kinds of DNA methyltransferases (DNMTs) were high expression at mRNA levels. Ki-67expression positive of Keloid lesion was higher than that of normal skin, but far below the fibroblasts source tumour-fibrosarcoma. The cells were absent atypia and mitotic figure, collagen content and nucleoplasm ratio have difference.3. X-ray radiation inhibited cell proliferation and induced cell senescence in KFb in a dose-dependent manner. Compared to the NFb cells, KFb cell proliferation inhibition rate is higher. Inhibition of cell proliferation and induction of cellular senescence were mediated by interruption of the cell cycle with an extended G1/G1phase. After exposure to4Gy irradiation for72hours, KFbs presented senescence phenotype with S A β-gal positive. Its proliferation potential and collagen production were reduced. The expression of p21, p16and p27genes in KFb was up-regulated after irradiation both at mRNA and protein levels. The mRNA level of DNMTs was decrease. Using PI staining assay, we found that the treatment with4Gy X-ray irradiation resulted in an accumulation of G0/G1phase in the keloid fibroblast cellsConclusion:The proliferation and collagen synthesis of keloid fibroblast might be affected by regulating the expressionsof cell cycle-related genes which was mediated by its epigenetic changes. The formation of keloid likely involves aberrant interactions of some genes which affected its development at different extents. Overall, our data indicate that ionizing radiation prevents recurrence of keloid probably through regulating the keloids fibroblast proliferation, inhibiting cell cycle process and inducing cell premature senescence. The processes of keloid development are between normal cells and tumor cells. Study about epigenetic changes of keloid fibroblasts in a variety of micro environment will be helpful to understand the mechanism of keloids and tumor development, and provide help for the final cure these diseases. |
PDF Full Text Request