| CHAPTER 1 EXPRESSION AND SIGNIFICANCE OF CYLD IN KELOID AND NORMOL SKIN TISSUESObjective:To investigate the expression and significance of cylindromatosis (CYLD) in Keloid tissues and fibroblasts.Methods:72 skin specimens were collected from clinical patients and were divided into normal skin group (group A), normal scar group (groupB), Keloids group (groupC) and hypertrophic scars group (groupD). Immunohistochemistry and Western-blot were used to detect the expression of CYLD in each group and the results were compared. Collect keloids, hypertrophic scar and surrounding normal skin tissues for primary fibroblasts culture with tissue adherent method. Expressions of CYLD protein in the three kinds of fibroblasts were detected by Western-blot.Results:The results of Immunohistochemistry showed that the positive expression of CYLD protein in group A (0.776±0.150) and groupB (0.706±0.065) were significantly higher than that in groupC (0.173±0.070) and groupD (0.213±0.107). Group A compared with groupC and groupD, respectively (P=0.00<0.05), groupB compared with groupC and groupD(P=0.00<0.05), the differences were statistically significant。 ②Measuring by Western-blot showed that the expression of CYLD protein in normal skin tissues (0.550±0.024) and normal scars tissues (0.442±0.075) were higher than keloids (0.173±0.070) and hypertrophic scars (0.178±0.026). ③Expressions of CYLD protein in nomral skin fibroblasts (0.564±0.037) is also significantly higher than keloid fibroblasts(0.240±0.022) and hypertrophic scars fibroblasts (0.256±.038), respectively.Conclusion:Expression of CYLD in Keloid tissues and fibroblasts were lower than that of normal skin. This indicated that the low expression of CYLD may play an important role in the formation of Keloid.CHAPTER 2 EFFECT OF TRICHOSTAIN A AND GENISTEIN ON PROLIFERATION AND EXPRESSION OF CYLD IN KELOID FIBROBLASTSObjective:To study the effect of histone deacetylase inhibitor trichostatin A (TSA) and three hydroxy isoflavone (Genistein) on proliferation and apoptosis of Keloid fibroblasts (KFb) and the expression of cylindromatosis(CYLD).Methods:Keloids tissues were collected for primary fibroblasts culture with tissue adherent method. KFb were stimulated with TSA and Genistein in various concentrations for different time, the optimal intervention time and median inhibitory concentration (IC50) were determined with the MTT colorimetric assay,which served as the intervention concentration of the subsequent experiments.The experiment was divided into four groups, TSA group (T,treated with TSA in IC50)> Genistein group (G,treated with Genistein in IC50)、TSA+Genistein group(T+G,treated with TSA,Genistein in IC50) and control group(treated with medium solution). Trypan blue staining was used to observed the influence of two drugs on the growth of KFb; The cycle and apoptosis of KFb in each growp were detected with flow cytometric assay, and the nucleic acid and protein expressions of CYLD in each group were determined with fluorescence quantitative PCR and Western-blot.Results: ① According to the results of MTT,600nmol/L TSA and 100 μ mol/L Genistein were selected as the intervention concentration; The results of flow cytometric assay showed that, The percentage of cells in G2/M phase of group G、group T and group T+G were (10.82±0.31)%, (11.11 ± 0.58)%, (17.60 ± 0.62)%, respectively. higher than the control group(7.11 ± 0.58)%(F=198.714, P=0.000); The percentage of cells in G0/G1 phase were (59.25 ± 1.23)%, (60.26 ± 0.74)% and (48.69 ± 1.55)%, lower than the control group (74.21 ± 2.40)%(F=128.458, P=0.000). The apoptotic rates of group G、group T and group T+G were (31.40 ± 4.07)%, (32.90 ± 3.87)%, (61.09 ± 0.55)%, which were significantly higher than that of control group (4.31 ± 2.12)%(F=177.439, P=0.000). ③ The fluorescence quantitative PCR results showed that the expression of CYLD mRNA in the TSA group (1.16 ± 0.16), Genistein group (1 ± 0.09), TSA+Genistein group (1.45 ± 0.09) were higher than the control group (0.47 ±0.10) (F=36.373, P=0.000). ④ Expression of CYLD protein in TSA group (0.53 ±0.04), Genistein group (0.52 ± 0.03), TSA+Genistein group (0.87 ±0.05) were also significantly higher than the control group (0.26 ± 0.02), (F=132.629, P=0.000).Conclusion:Trichostatin A and Genistein can inhibit keloid fibroblast proliferation and promote apoptosis in acting alone or in combination.The mechanism of TSA and Genistein in inhibiting the proliferation and promote apoptosis of KFb may be related to the up-regulation of tumor suppressor gene CYLD. |
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