| Atmospheric ozone molecule is combinated by an oxygen molecule and an oxygen atom which comes from oxygen molecule decomposed production by solar radiation. Ozone layer is blue and irritant trace gas in atmosphere and is the critical composition of stratospheric air. Although Ozone layer is not thichkness, it can provid protection for human and other creature by the absorption of 99% high intensity solar ultraviolet radiation. Because of some nature factor and influence of human activities on the environment, the ozone layer above the earth was destoryed and then large area ozone hole presented. It is undoubtedly that ozone hole will significantly reduce ozone's effect of blocking solar ultraviolet radiation,and result in more ultraviolet radiation reaching earth surface. In september 2006,the Antarctic ozone hole reached 29.5 million square kilometers, with a total area of more than three of China. Moreover, according to scientists assessment that in the next 10 to 20 years, deterioration of the ozone hole is still possible. Although in the global, human are taking the necessary measures to try to reduce the continued destruction of ozone layer,such as the use of freon succedaneum, even in the future we may repair the ozone hole, WHO estimates that earth animals, plants and plankton, etc. still will be receive more and more ultraviolet radiation in particular ultraviolet B(UVB) in the next 40-100 years. This is not only harmful to human health itself, but also for the human living environment. According to their different wavelength,ultraviolet radiation from the sun can be divided into three partment:UVA(wavelength:315nm～400nm), UVB (wavelength: 280nm～315nm), UVC (wavelength:100nm～280nm).Because of the prevention of atmosphere, all UVC and approximately 90% UVB can be absorbed by ozone, water vapor, oxygen and carbon dioxide,UVA is more less affected by the atmosphere. Therefore, the UV radiation reaching earth surface combine UVA and a small amount of UVB. Meanwhile, the absorption fucution of ozone layer is mainly in the region of 310nm-315nm, namely UVB wavelength range. That is, the destruction of the ozone layer will lead to UVB of reaching earth surface rapidly increase, so the effect of UVB damage to the living being give rise to more and more human attention on it.Skin that cover the body surface is the first line of defense against outside injury, it will receive more external environment radiation than the other organs,such as ultraviolet and visible light radiation.95% UV irradiation reaching skin was absorbed by keratinocytes. It is undoubtedly that the increase of environment UVB will induce more damage to keratinocytes, such as causing skin erythema and inflammation. Therefore, this study selected the immortalized human epithelial cells (HaCat cells) and 310nm UVB to investigate the damage mechanism. Previous studies showed that, UVB at least has four ways to destroy cell normal functions, to affect cell survival conditions, such as lead to cell necrosis or apoptosis.1:The direct damage to cell DNA;2:Activation of sphingomyelinase which can lead to sphingomyelin degradation, result in the increase level of ceramide and their derivatives;3:Activation of cell surface death receptors such as CD95, etc.; 4:Create free radicals and lipid peroxidation through the cell membrane and mitochondria membrane. Which pathways and signaling molecules involve in the damages of HaCat cell caused by UVB are still unknown. These are our focus in this study.This study was divided into three stages. First, we observed the changes of cytoactive, apoptosis,p38, p42/p44, p53, PARP and 14-3-3σafter HaCat cell exposed different UVB doses and/or in different times after particular UVB dose. According to these discovers of 14-3-3σexpression changes after exposed to UVB in the first experiment and the important role of 14-3-3σin apoptosis and cell cycle arrest, we concluded that it is necessary for us to construct stably 14-3-3σRNAi HaCat cell lines and 14-3-3σstably over expression HaCat cell line for the further studies。OBJECTIVE:1,To explore the roles of p38 signal transduction pathway in UVB induced apoptosis;2,To explore the roles of ERK signal transduction pathway in UVB induced apoptosis.3,To investigate the changes of 14-3-3amRNA after expose to UVB irradiation;4,To construct RNAi retroviral vector targeting 14-3-3σand establish stably transfected HaCat cell lines.5,To construct thel4-3-3σretroviral vector and establish stably 14-3-3σover expression HaCat cell lines.METHODS:The research methods consists of three parts.Part one1,HaCat cells were used in this study. With 1min,3min,5min,10min and 15min UVB irradiation respectively, the ratio of cell survival after expose to UVB 0h,3h,6h,9h,12h,18h,24h and 30h was measured by MTT reduction assay.2,After being irradiated by UVB radiation at the dosage of mock irradiation,1min,3min,5min,8min,10min respectively, apoptotic cells stained by Hoechst33258 were observed and counted through fluorescent microscope at the time points after irradiated 4h,8h,12h,24h. 3,After being irradiated by UVB radiation at the dosage of 5min UVB, western blotting was used to examine the expressions of p38,p42/p44,p53,PARP, and so on the time point after irradiated 12h.4,After being irradiated by UVB radiation at the dosage of mock irradiation, lmin,3min,5min,8min and 10min UVB irradiation respectively, cells were collected on the time point after irradiated 1h.With 5min of UVB irradiation, the cells were collected on the time points after irradiated 0h,1h,2h,4h,6h,12h.The RNA were extracted from above groups and reversed into cDNA by reverse transcription-PCR. The 14-3-3a mRNA expressions were detected by 1% agarose gel electrophoresis. Part two5,According to the 14-3-3σfull sequence searched by GeneBank and the principle of siRNA design,we searched three suitable RNAi target sites and chemically synthesized three pairs of hairpin siRNA of 14-3-3σ.And then three pairs of hairpin siRNA were inserted into pSUPER-retro-EGFP/neo plasmid.6,pSUPER-retro-EGFP/neo-si14-3-3σwere transformed into competent DH5a cells to amplification itself. And then the positive clones were confirmed by sequencing.7,The plasmids were transient transfected into HaCat cell by Lipofectamine TM2000 for 48h. The third hairpin siRNAi is the best one which were detected by western blotting.Therefore, the plasmids that contain the third hairpin siRNA were transformed into competent STBL3 cells for more amplification and then transfected into the packaging 293FT cells to generate, amplificate and depurate virus.8,HaCat cells were infected with the recombinant retroviral vector three times。After sustained pressure by G418 selection, we construct stably 14-3-3a RNAi HaCat cell lines.9,The expression of 14-3-3σwas detected by Western blotting and Real-time fluorescence quantitative PCR.Part three10,According to the analysis of 14-3-3σgene sequence,we found that 14-3-3σgene can be digested by Bgl II and BamH I,and can be placed into the sites after the CMV IE promoter. To amplification 14-3-3σgene, pLEGFP-N1 plasmid was selected as no-load plasmid in this study. The upstream primer and downstream primer contain there protective basic of 14-3-3σgene were then designed.11,Useing the human genome as a template and high-fidelity DNA ligase, we amplified 14-3-3σgene by PCR. The PCR products were identified by 1% agarose gel electrophoresis and reclaimed amplified DNA fragments by agarose gel DNA extraction kit.12,The productions of 14-3-3σsequence and pLEGFP-N1 plasmids were digested by Bgl II and BamH I. And then we used T4 DNA ligase to connect them. The connections were transformed into competent DH5a cells to amplificat connections. Then the positive clones were confirmed by sequencing.13,pLEGFP-N1-14-3-3σplasmids were transformed into competent STBL3 cells for more amplification.14,pLEGFP-N1-14-3-3σplasmids were transfected into the packaging 293FT cells to generated, amplificated and depurated virus.15,HaCat cells were infected with the recombinant retroviral vectors three times.After selected by sustained pressure of G418, we constructed stably 14-3-3σover expression HaCat cell lines.16,The expression of 14-3-3σwas detected by Western blotting and Real-time fluorescence quantitative PCR.RESULTS:Part one 1,At a fixed UVB irradiation dose, prolonged incubation of the cells following the irradiation resulted in decreased cell survival ratio, which, however, began to increased when the minimum rate was reached; Increased with UVB irradiated doses, the survival rate were taper off when HaCat cells were detched in same time. Further more, in the group with 15min UVB irradiated, there was no recover until 30h after irradiated.2,There were significant deviation in apoptosis ratio among different dosages UVB irradiated and different time points after UVB irradiated(P<0.05). In differents dosages, the apoptosis ratio is the highest when HaCat cells were irradiated by UVB in five minutes, moreover the ratio peak after HaCat cells incubated for 12h.3,There is no marked changes of p42/44 detected among the time points after UVB irradiation, meanwhile, the p38,p53 levels showed progressive increase after 4h, the expression of PARP also showed progressive increase, but the expression descended at the point of 12h after UVB irradiation, meanwhile cleaved PARP showed progressive increase and up to the peak at the point of 12h,and declined in 24h.4,The 14-3-3σmRNA expression level was up-regulated at 3min,5min,8min and 10min UVB irradiated groups.With fixed dosage of 5min UVB irradiation, 14-3-3σmRNA expression level was up-regulated in lh after UVB irradiation. Part two5,Design and synthesis three RNAi sequences to aim directly at different target sites of 14-3-3σsequence.And then selected the best one.it is RNAi-5'-ACCTGCTCTCAGTAGCCTA-3'.Sh-14-3-3σ-F3 5'GATCCCC ACCTGCTCTCAGTAGCCTA TTCAAGAGA TAGGCTACTGAGAGCAGGT TTTTT A3'Sh-14-3-3σ-R3 5'AGCTT AAAAA ACCTGCTCTCAGTAGCCTA TCTCTTGAA TAGGCTACTGAGAGCAGGT GGG 3'6,The RNAi sequences and pSUPER-retro-EGFP/neo plasmids were recombined,the positive clone were identified by colony PCR screening, sequencing result showed that the recombinant PSuper-retro-EGFP/neo-si14-3-3σplasmid was successfully constructed. PSuper-retro-EGFP/neo-si14-3-3σplasmids,packaging plasmid PIK and 293 FT cells co-cultured to produce recombinant retroviral vector of PSuper-retro-neo-EGFP-si14-3-3σ.7,Green fluorescence of the stable transfected HaCat cell lines could be observed under inverted fluorescence microscope.8,The results of Real-time fluorescence quantitative PCR showed that the sequence of RNAi-14-3-3a could effectively down-regulate the level of 14-3-3σ, the average inhibition ratio over 70%.9,The results of western blotting showed that the expression of 14-3-3σprotein was effectively down-regulated. Part Three10,14-3-3σfull sequence was successfully amplified using template of human genome. The molecular weight of amplified DNA fragment was proved correct by 1% agarose gel electrophoresis.11,The 14-3-3σsequence and pLEGFP-N1 plasmid were recombined, positive clone were identified by colony PCR screening,1% agarose gel electrophoresis, sequencing.All results showed that the recombinant pLEGFP-N1-14-3-3σplasmid was successfully constructed.12,pLEGFP-N1-14-3-3σplasmids,packaging plasmids PIK and 293FT cells co-cultured to produce recombinant retroviral vectors of pLEGFP-N1-14-3-3σ.13,Green fluorescence of the stable transfected HaCat cell lines could be observed under inverted fluorescence microscope. The result showed that exogenous gene can express in HaCat cell。14The results of Real-time fluorescence quantitative PCR showed that the exogenous sequence could effectively up-regulate the level of 14-3-3σ.The stably transfection cell has 2.71-fold expression in average than normal cells。15,The results of western blotting showed that the expression of 14-3-3σprotein was effectively up-regulated.CONCLUSIONS:1,UVB irradiation can induce HaCat cells growth inhibiting and apoptosis in a dose-and time-dependent manner;2σThe UVB induced apoptosis may through by p38 pathway, not by ERK pathway.3,14-3-3σmRNA expression was up-regulated by UVB irradiation.4,We have successfully constructed the RNAi retroviral vector targeting 14-3-3a and have established stably transfected HaCat cell lines.5,We have successfully constructed thel4-3-3σretroviral vector and have established stably transfected HaCat cell lines.
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