| Psoriasis vulgaris (PV) is a common clinical, unexplained chronic inflammatory skin disease. Its occurrence, development process are involved in a variety of chemotactic factor, relate to the genetics of the internal cause and the external environment, such as infection, nutrition and mental state. PV belongs to one of many autoimmune diseases and easy to recrudescence. At present, studies suggest that PV is an immune disordered disease associated with Thl cells. IFN-gamma (IFN-γ) inducible protein10(IP-10) is a member of CXCL chemokine family and also known as CXCL chemokine ligand10. Protein10kDa and chemotactic lymphocytes factor are induced by IFN-γ. IP-10not only has strong function of recruitment of neutrophils and other inflammatory cell, but also can promote a variety of cells to release inflammatory factors and promote keratinocyte proliferation. IP-10can mediated Thl inflammation as a chemokine. IP-10expression in PV lesions of patients is sustained upregulation. After successful treatment, the expression of IP-10is decreased. IP-10can promote the occurrence and development of PV. IFN-γ has an important relationship with PV. The concentrations of IFN-γ in PV patients was significantly higher than that of normal people in serum or in cell culture supernatant and IFN-γ plays an important role in keratinocyte proliferation and apoptosis in immune skin disease. IFN-γ plays a key role in keratinocyte proliferation and can promotes keratinocyte proliferation.In recent studies, it has been found that a variety of diseases were associated with genetic and epigenetic changes. Epigenetic regulation mechanism plays an important role in the development of autoimmune diseases and tumors. Epigenetics is involved in the pathogenesis of PV. However, the specific pathogenesis of PV has not been understood now. Epigenetics includes DNA methylation, histone modification and small molecule RNA regulation. Although epigenetic changes do not alter the sequence of A T C Q it makes an important Influence on gene transcription, proliferation, differentiation, apoptosis and other cell processes. HaCaT cell line is commonly used for study of the antipsoriatic drug activity. This study about cultured human HaCaT keratinocytes was induced by IP-10and IFN-γ to explore the effects on proliferation and inflammatory and the epigenetic changes. Then compared characteristics in PV lesions cocultured HaCaT cells with IP-10or IFN-γ. IP-10and IFN-γ make an important effect on keratinocytes. It is suggested that psoriatic keratinocytes model may be established through co-cultured keratinocytes with IP-10or IFN-γ. Perhaps, IP-10and IFN-γ play an important role on the occurrence and development of PV.Aim1. To detect cell proliferation and inflammatory under epigenetic change in HaCaT cells induced by IP-10and IFN-γ.2. To observe the proliferation and inflammation of psoriasis under epigenetic change by comparison of PV lesions with normal tissue. At the same time, to compare characteristics in PV lesions with cocultured HaCaT cells induced by IP-10or IFN-γ. Finally, the role of IP-10and IFN-γ in the development PV is studied.Materials and methodsClinical data and cases20cases outpatients with PV samples were collected in our hospital from October,2011to April,2012and were diagnosed by histopathology. Simultaneously20healthy resected foreskin samples cases were collected in Department of Urology from January to February,2012. This study was permitted by Ethics Committee of our hospital informed consents from all patients.HaCaT cells are preserved in Molecular Cell Biology Res Center Medicine of Zhengzhou University.MethodsThe experimental samples were divided into two kinds:cells and tissues1) Cells:The cultured HaCa T cells were divided into3groups:IP-10(20ng/ml)group; IFN-γ (50u/L) group and control group treated with neither IP-10nor IFN-y. All groups were cultured for48h.2) Tissues:psoriasis cases and forskin samples1. Western blottingAfter tissues were bruised by using homogenizer, the total protein was extracted from each group of HaCaT cells and tissues from psoriasis cases and forskin samples. The SDS-PAGE was performed onto the nitrocellulose membrane(NCM). The expression of DNMT1, NF-κBp65, HDAC1, Cyclin D1, IL-6and P16(β-actin as internal control) were detected by Western blotting.ImmunostainingAll sample slides were fixed with4%paraformaldehyde. Respective NF-κB and IL-6primary antibodies, followed by the SP kits were incubated on the slides. At the same time, PBS was substituted for the primary antibody as the negative control.3. Methylation specific PCR (MSP)The genome DNA was extracted from lesion samples from psoriasis cases, forskin samples from healthy cases and each group of HaCaT cells. DNA methylation modification were followed by, methylation-specific PCR reaction and detection products by DNA electrophoresis.4. Statistical analysisStatistical analysis was performed using SPSS17.0software. The date shown as (x±s) of immunoblotting, histochemical staining and MSP were analyzed with ANOVA among groups, a=0.05was considered as the level of significance.ResultsWestern blottingHaCaT cellsCompared with the control group, the expression of DNMT1, NF-κBp65, HDAC1, Cyclin D1or IL-6in IP-10and IFN-γ groups was up-regulated and the expression of P16was down-regulated, P<0.05.TissuesCompared with the control group, the expression of DNMT1, NF-KBp65,HDAC1, Cyclin D1or IL-6in PV group was up-regulated and the expression of P16was down-regulated, P<0.05. ImmunostainingHaCaT cellsThe immunostaining of NF-κBp65and IL-6was intenser in IP-10and IFN-γ groups, when compared with the control group, P<0.05.TissuesThe immunostaining of NF-KBp65and IL-6was intenser in PV lesion group, when compared with the control group, P<0.05.MSPHaCaT cellsThe MSP of P16showed that the methylated p16was higher in IP-10and IFN-γ groups than that in the control group, P<0.05.TissuesThe MSP of P16showed that the methylated p16was higher in PV lesion group than that in the control group, P<0.05.Conclusions1. Western blotting analysis shows that the expression of DNMT1and HDAC1in IP-10and IFN-γ cells and the lesions groups are promoted than in the corresponding control groups, suggesting that epigenetic change is existed in HaCaT cells after induced by IP-10and IFN-γ and PV lesion tissues.2. In IP-10and IFN-γ cells and in psoriatic lesions groups, the expression of CyclinD1, NF-KBp65and IL-6are significantly increased, suggesting that IP-10and IFN-γ may enhance proliferation and inflammation. IP-10and IFN-γ may down-regulate the expression of P16.3. MSP displays that P16methylation level in IP-10and IFN-γ cells and in psoriatic lesions groups are higher than that in the corresponding control groups. While non methylation is decreased significantly.4. IP-10and IFN-γ can promote proliferation and inflammation via epigenetic change. Suggesting that IP-10and IFN-γ may participate in the mechanism of psoriasis. |
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