| BackgroundUVB irradiation is the most powerful wavelength which causes sunburn, photoaging and skin tumors. UVB can cause the generation of free radicals and relevant reactive oxygen species (ROS), the cytokine production and secretion, the local and systemic immune suppression, DNA damage and mutation, and contribute to photo-aging and photo-carcinogenesis.Proteomic techniques have been used as a powerful tool for the qualitative and quantitative measurement of diversity of total proteins related to specific cellular responses. Hundreds of proteins could be separated on a 2D-PAGE and then identified by mass Spectrometer. Therefore, the proteomic scheme was implemented to search globally for the differentially expressed proteins.Life science has entered the post-genome era, and proteomics is the study of the whole protein, which is expressed activity at a specific time and space in cell, tissue or organism. It makes a quantitative, dynamic and overall study on organism at the protein level, thus to gain a complete understanding of physiology, disease processes and the extensive regulatory network at deeper level.RNA interference is the reverse genetics method has become a good tool to study gene function. Compared in terms of gene knockout technology, RNA interference design of the whole process easier,actived rapid, the effection is obvious, opened up a new way for gene therapy.EGCG is the main active component of green tea has anti-inflammatory, antioxidant, anti cancer, immune regulation, and a wide range of pharmacological effects. We have confirmed that EGCG can reduce UV-induced skin cells (including human keratinocytes, human fibroblasts, etc.) light damage, reduce inflammation cytokine secretion and light product formation, but on the UVB irradiation caused epidermal cell acute injury and EGCG intervention Changes of the protein group and the NPM1 light injury in the present study little. We use proteomics two-dimensional gel electrophoresis and mass spectrometry technology to evaluate the influence of the intervention of EGCG and UVB radiation on differentially expressed protein in human epidermal keratinocyte line HaCaT, in order to further clarify and explore the biological activity and anti-optic effect mechanism of EGCG. We observed and analyzed the apoptosis and the subsequent changes in proliferative activity by silencing NPM1 gene expression by specific siRNA, in order to identify the function and mechanism of NPM1 gene occurred in human skin, light damage and repair.ObjectiveTo identify, by a proteomic strategy, the new molecular targets of proteins expression in HaCaT cells in response to UVB and EGCG exposure .And then to evaluate the effects of different dosages of UVB irradiation and intervention of EGCG on proliferation, expression levels of NPM1 protein in human eternal keratinocyte-HaCaT cells. Further clarify and explore the biological activity of EGCG has anti-photodamage and mechanisms, and the protection of NPM1 in pohtodamage,for the development of natural sunscreen provides a theoretical basis and practical applications and experimental data for reference.Materials and methods1. Cell culture: HaCaT cells were cultured in RMPI-1640 medium with 10% calf serum and cells were plated in 3.5 and 10 cm dishes with an equal number.2. Preparation of ferulic: Prepare EGCG with DMSO, the concentration of stock solution is 50mg/ml, and the EGCG concentration is 50μg/ml.3. UVB irradiation: The time-effect and dosage-effect of UVB irradiation were conducted according to the experiment design. Ferulic was added into the medium after UVB irradiation.4. The proteins were harvested and seperated in two-dimensional gel electrophoresis(2-DE). The differential protein expressions between the two groups of cell were analyzed using image analysis software.5. The differentially expressed protein spots were selected, digested in-gel with trypsin, and measured by matrix assisted laser desorption time-of-flight mass spectrometry (MADI-TOF-MS).6. The data obtained from peptide mass fingerprinting (PMF) were used for protein database search .7. Western blot assay was used to measure NPM1 protein expression at 24h after 30mJ/cm2UVB irradiation.8.Constructed NPM1 the siRNA target sequence ,the real-time quantitative (RT-PCR)was used to measure NPM1 mRNA expression after disturbance.9. MTT assay was used to cell activity after siRNA interference,EGCG and 30 mJ/cm2UVB irradiation.10.Flow cytometry was used to cell proliferation after siRNA interference,EGCG and 30 mJ/cm2UVB irradiation.11.Statistical analysis: The experimental data were analyzed with SPSS. P values less than 0.05 were considered to be statistically significant.Results1.There were 42 differential protein spots were identified between the control group and EGCG group, and we choose 16 spots for Peptide quality fingerprint spectrum identification. Among them, 9 protein spots present high expression in HaCaT cell, while 4 protein spots present low expression after EGCG treatment.2.The treatment of EGCG can significantly inhibit the protein expression in HacaT cell induced by UVB irradiation. 23 proteins down-regulated by UVB were all up-regulated b, while the 30 up-regulated proteins were all down-regulated due to the treatment of EGCG.3.The designed specific fragments of NPM siRNA were transfected into HaCaT cells and then we detected the expression of NPM mRNA by the Real-time PCR. Then, we select NPM-915-siRNA for the following experiment which has highest silence efficiency after 48h.4. Compared with control group, UVB irradiation significantly suppressed HacaT cell proliferation and induced apoptosis, which is similar by siRNA interference, while pre-treatment of EGCG can make improvement of it.Conclusions UVB radiation can cause a large number of proteins differentially expressed in HaCaT cells. EGCG significantly inhibited changes of differentially expressed proteins caused by UVB. UVB induced HaCaT cell damage, which is characterized by inhibited cell growth activity. Treatment of EGCG after UVB irradiation can protect HaCaT cells from UVB damage process, and the detailed mechanisms may be concerned with regulation of NPM gene expression. These results provide new evidences to understand the process of epidermal cell injury induced by UVB and further study the mechanism of anti UV-induced damage at the molecular level. |
PDF Full Text Request