| Objective：1. To study the protection and mechanisms of trehalose on HaCaT cells against UVBirradiation．2. To observe the repairing activities of trehalose ointment to chronic UVB injuredmice and rats.3. To explore the fermentation conditions and permeability technique of trehalosesyntheses produced by Pseudomonas sp. A50.Methods：1. To establish UVB injured HaCaT cell model: choosing315nm UVB light sourceas the only light source, HaCaT cell was exposured to different doses of UVB.After48h reincubation, cell viability was measured by CTG method. Theappropriate irradiation dose for further work was defined as the dose causing50%cell death.2. To study the inhibitory effects of trehalose on UVB-induced HaCaT cell injury:before UVB irradiation, different doses of trehalose were added to HaCaT cells.Then cell viability was measured at48hours incubation after UVB irradiation toidentify the anti-ultraviolet effect of trehalose.3. To study the preliminary mechanism of trehalose anti-ultraviolet activity on UVBinjured HaCaT cell: using expression profile microarray, differentially expressedgenes between trehalose treated cells and control cells were identified and verifiedby qRT-PCR. Lentviruses with these genes were constructed and transduced intoHaCaT cells. The lentviruses-contained cells were treated with UVB irradiationand cell viabilities were measured to assess the relationship between genes andcell survival rate. γH2AX expression was detected by Western blot to assess therelationship between γH2AX expression level and UVB irradiation, trehalose orthese differential genes.4. To assess the inhibitory effects of trehalose ointment on chronic UVB injuredmice and rats: KM mice and SD rats were used in this study and both divided intosix groups randomly. The groups are UVB irradiated group, ointment base group,low-dose trehalose group, high-dose trehalose group, positive control group andnegative control group. Five animals were assigned to each group and each animal was shaved to exposure back skin. The total dosage of UVB irradiation was86.4J/cm2(KM mice) and118.8J/cm2(SD rats) respectively. The mice were killedafter30days irradiation and bloods were collected for blood test and plasmabiochemical tests and skin tissues were assigned to HE stains. The rats were killedafter46days irradiation and skin tissues were assigned to HE stains.5. To study the relationship between cell growth and dissolved oxygen and trehalosesynthase enzyme produced: Fermentation process of Pseudomonas sp. A50in5Lfermentation cylinder was observed in order to optimize the enzyme producingcondition. Using toluene as permeabilizing agent, the adding amount and time oftoluene was examined to explore the whole-cell biocatalyst method ofPseudomonas sp. A50cells.Results and conclusion:1. UVB injury model was established and the optimal dosage of UVB irradiationwas48mJ/cm2.2.0.5%(w/v) trehalose can protect UVB-irradiated HaCaT cells and the cell survivalrate was back to around75%of the original.3. Four significant differential expression genes, belonging to snoRNA, includingup-regulated uc021rnm.1, uc021qhm.1and uc002bro.1genes and down-regulateduc022boe.1gene were identified by expression profile microarray and qRT-PCR.Lentiviruses overexpressing uc021rnm.1, uc021qhm.1, uc002bro.1or siRNAtargeting uc022boe.1were transduced into HaCaT cells. Cell viability assay ofUVB treated recombinant cells showed the snoRNA uc021rnm.1was positivewith the recovery of the cells. Western blot analysis showed the down-regulationof γH2AX, a marker of double stranded DNA damage, which is upregulatedusually after UV damage, implying that trehalose rescued HaCaT cells from UVBinjury through alleviating DNA damage.4. Trehalose ointment made of oil-in-water formulations was white, uniform color,fine texture and smoothly. No oil-water separation and deterioration phenomenonwas observed after placing it under39℃or4℃for6months.5. In mice chronic photo injury repairing experiments, by visual observation, theback skin of mice in positive group and high-dose trehalose group were similar toof negative control. In low-dose trehalose group, slight skin swollen was observed.The back skins of mice in ointment base group and UVB irradiated group were seriously damaged and erythema, pigmentation, thickened and scaling symptomwere observed on mice back. Rats in positive group and high-dose trehalose grouphad slightly erythema on the back and in low-dose trehalose group, red andswollen was observed and in base group and negative group, seriously escharosiswere found over and there on the back skin.6. HE staining results of rats and mice skins were basically identical. Themicroscopic structure of mice skin in positive group and high-dose trehalosegroup, similar to negative group, is basically normal: bunchy collagen fiberswithout rupture, a little keratinized, slightly hyperplasia of sebaceous glands andthickening of epidermis. In low-dose group, thickening of epidermis andhyperkeratosis was observed. In ointment base group and UVB irradiated group, aseriously epidermal thickening and hyperkeratosis was observed and collagenfibers were ruptured and disarranged.7. Weak acidic environment and the carbon source added is benefit to trehalosesynthase production.8. The maximum enzymatic activity in5L fermentation cylinder of A50-producedtrehalose synthase was4.08U/L, better than shake flask fermentation.9. Adding0.5%(v/v) toluene to Pseudomonas sp. A50cell sediments, a whole-cellbiocatalytic system could be established. But the permeable agent andpermeabilization technology should be further studied. |
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