The Study On Regulation Of MAPK Signal Transduction Pathways To The Protective Effect Of Skin Photoaging

Objective:To study the in vitro UVA irradiation of skin fibroblasts, the cells change and related cytokine changes, testing the initiating MAPK signal transduction receptor expression of EGFR and MAPK signaling pathways downstream genes c-jun, c- fos expression changes. By screening out the high specificity of the c-junsiRNA after UVA irradiation of skin fibroblasts transfected cells, the gene downstream of MAPK reduced c-jun in the skin photoaging MMPs, collagen.Method:1. Take a young adult foreskin fibroblast cell culture passage, the cell groups, with the first 5 passages to do the experiment,using different measurement of UVA (5 J/cm2, 10J/cm2,20J/cm2) irradiation, the establishment of control Groups (0 J/cm2), established fibroblast aging model of light, light microscopy and electron microscopy cell morphology, RT-PCR and enzyme-linked immunosorbent assay (ELISA) measured before and after irradiation in vitro cultured skin fibroblasts theⅠ,Ⅲprocollagen mRNA expression and protein expression. MTT cell viability determined after 24h culture.2. With UVA (5 J/cm, 10J/cm2,15J/cm2) irradiation, the control group,24 hours after irradiation detected by realtimePCR MAPK signal transduction pathway downstream genes c-jun, c-fos, and final MMP gene-1, MMP-3 in the mRNA expression. Enzyme-linked immunosorbent assay (ELISA) determination of cell supernatant MMP-1, MMP-3 expression, weasternblog detect c-jun, c-fos expression.3. For the early experiments, c-jun increased with radiation dose, design five c-junsiRNA, realtimePCR, and westernbolg from the genetic level and protein level of its screening, screening out the most efficient silencing c-junsiRAN, and optimize the transfer dyeing conditions.4. Radiation dose exposure to 15J/cm2 fibroblasts in vitro, realtimePCR detect MMP-1, MMP-3 mRNA,Ⅰ,Ⅲprocollagen mRNA,2 hours after the transfection of c-junsiRNA,24 hours after transfection,48 hours After the enzyme-linked immunosorbent assay (ELISA) detected in the supematants were MMP-1, MMP-3,Ⅰ,Ⅲcollagen content.5 to (5 J/cm2,10J/cm2,20J/cm2) irradiated fibroblasts, the establishment of the control, detection MAPK signal transduction pathway initiating the expression of EGFR receptors to detect TGF-β1, IGF-1, KGF, VEGF several cytokines in the changes before and after irradiation.Results:1. With the UVA radiation dose, fibroblasts, the OD value of MTT decreased, UVA 10 J/cm2, UVA 20 J/cm2 irradiation group OD was significantly decreased, compared with the control group there was significant difference (p<0.01). TypeⅠmRNA RT-PCR products were integrated with the UVA light dose increased the absorbance decreased, UVA 10 J/cm2, UVA 20 J/cm2 irradiation group were significantly lower (p <0.05, P<0.01), UVA 20 J/cm2 Group inhibition of collagen synthesis is more obvious in the control group and the other two groups, there was a significant difference (P<0.01).Ⅲcollagen content and mRNA expression compared with control group, UVA 20 J/cm2 irradiation was significantly lower, there was a significant difference (P<0.01). Supernatant collagen typeⅠand UVA radiation as the dose decreased in a dose dependent manner. UVA 10 J/cm2, UVA 20 J/cm2 irradiation group,Ⅰcollagen content was significantly reduced compared with control group (P<0.05, P<0.01), that fibroblasts were treated with UVA 10 J/cm2, UVA 20 J/cm2 dose, can inhibit collagen synthesis, and the dose of UVA 20 J/cm2 group more obvious inhibition of collagen synthesis, and the control group and the other two groups, there was a significant difference (P<0.01); and 5 J/cm2 dose group and control group, no significant difference (P> 0.05). TypeⅢcollagen content compared with control group, UVA 20 J/cm2 irradiation groupⅢcollagen were significantly reduced, there was a significant difference (P<0.01), that fibroblasts in the UVA 20 J/cm2 irradiation dose can be inhibit collagen synthesis, while UVA 5 J/cm2, UVA 10 J/cm2 dose group compared with the control group,Ⅲcollagen showed no significant difference (P>0.05).2. With the increased dose of UVA radiation, fibroblasts increased expression of c-junmRNA, compared with the control were significantly different, (p<0.01). c-fosmRNA no difference between groups (P>0.05). MMP-1, MMP-3 mRNA expression with radiation dose also increased, a significant difference. Protein and gene expression have also shown consistency. C-jun as the dose increases, the difference between the groups p<0.01, and c-fos expression in no significant changes (P> 0.05).3, design a 5 c-junsiRNA, by realtimePCR, and westernbolg from a common genetic level and protein level of its most efficient screening silent c-junsiRAN, silencing efficiency of more than 80%. Sequence: GCAUUCUUGUCACAAUAAATT.4 to 15J/cm2 radiation dose exposure of fibroblasts in vitro,2 hours after the transfection of c-junsiRNA,24 hours after transfection, MMP-1, MMP-3 mRNA, with the control group (transfected with negative sequence group) reduced compared to mRNA expression, including MMP-1 (P<0.01), MMP-3 (P<0.05).Ⅰ,Ⅲprocollagen mRNA, compared with the control group (transfected with negative sequence group)Ⅰprocollagen (P<0.01),Ⅲprocollagen (P<0.05). After 48 hours, enzyme-linked immunosorbent assay (ELISA) detected in the supernatants were MMP-1, MMP-3,Ⅰ,Ⅲcollagen content showed the same results with the mRNA expression of MMP-1 (P<0.01), MMP-3 (P<0.05),Ⅰcollagen (P<0.01),Ⅲcollagen (P<0.05).5. UVA irradiation in cultured skin fibroblasts showed consistency EGFRmRNA and protein were higher and increased with radiation dose,5 J/cm2 (p<0.05),10J/cm2,20J/cm2 (P<0. 01). UVA irradiation in cultured skin fibroblasts lead to TGF-β1, IGF-1, KGF secretion decreased, UVA 10 J/cm2, UVA 20 J/cm2 irradiation group was significantly decreased compared with control group, there was significant difference (P< 0.01). VEGF secretion increased, UVA 20 J/cm2 irradiation group compared with the control group, there was a significant difference(P<0.01)。Conclusion:1.UVA irradiated cultured dermal fibroblasts, leading to cell senescence, proliferation activity; of typeⅠ,Ⅲcollagen synthesis inhibition.2. UVA irradiation in cultured skin fibroblasts activation of MAPK signaling pathway, its downstream genes was significantly increased c-jun, c-fos changed little, the terminal gene MMP-1, MMP-3 are regulated.3. The realtimePCR, and westernbolg gene level and protein level from the common to the five c-junsiRNA screening, screening out the most efficient silencing c-junsiRAN, silencing efficiency of more than 80%.Sequence:GCAUUCUUGUCA CAA-UAAATT, the name JUN-h-825.4.UVA irradiated fibroblasts in vitro after 2 hours, the c-junsiRNA transfected fibroblasts, to some extent MMP-1, MMP-3 can reverse the downward,Ⅰ-type,Ⅲcollagen synthesis also reduce the decline.5. UVA irradiation in cultured skin fibroblasts, EGFR expression was significantly increased in the supernatant of cytokines, TGF-β1, IGF-1, KGF decreased secretion; VEGF secretion increased。Keywords:ultraviolet; fibroblasts; skin photoaging; MAPK signaling pathway; siRNA.