Effects Of Heat Treatment, UVB Radiation And Their Combination On HSP72and P53Expression In Human Melanocytes

Objective Through detection the protein and mRNA expression differences of HSP72andP53in human epidermal melanocytes in the heat treatment, UVB radiation and thetwo combined groups to analysis of possible mechanisms of melanocyte function underheat and heat combined UVB radiation treatment preliminary, aiming at providing arational for the use of heat and heat combined UVB radiation treatment in vitiligo.Methods (1) Melanocytes were isolated form fresh specimen of circumcision andcultured in melanocyte-specific medium. After two genarations purified melanocytes wrerharvested.(2) Melanocytes were selected from3-5generation as growth to60%-70%confluence and treated with heat (1h at42°C)for3d. The protein expression levels andchanges in trend of HSP72were observed at the time of4h,6h,24h after the last treatmentby Western blot, P53protein expression and trend at6h,12h,24h.Select the optimum timefor protein expression as the follow-up experiments time point to harvest cells.(3)Cellswere randomized: control group,heat treatment group (treated1h at42°C for3d),UVBradiation group(50mj/cm2for3d)and combined group(first42℃,1h,then50mj/cm2for3).(4)At the optimum time point according to (2), Western blot and RT-PCR techniques wereused to detect HSP72and P53expression changes between different groups.(5)The greyvalue from western blot was analyzed by Gene Tools. HSP72relative expression weredemonstrated by the ratio of grey value of target protein bands and β-muscle actin bands.RT-PCR results were demonstrated by2-ΔΔCtmethod, which is the target genes relative to acontrol group of genes changed in multiples, in order to reduce errors caused by differences in cell number and RNA extracted process and normalized the target genes. SPSS13.0statistical software package were adopted to analyze experimental data.Results (1) After heat treatment,the induction protein expression of Hsp72wasconspicuously increased at4h, peaked at6h and decreased at24h. P53protein expressioncontinued increase from6h to24h after heat treatment.(2) HSP72protein and mRNAexpression changes between different groups: Western blot analysis demonstrated thatHSP72protein expression in heat treatment group(2.022±0.058) and the combinedgroup(2.080±0.045) was higher than the control group(0.532±0.033，p<0.001). Nosignificant difference between the UVB-radiation group (0.546±0.021)and the controlgroup(p>0.05). Fluoresent real-time quantitative PCR showed that HSP72mRNA level inheat treatment group (6.769±0.895)and the combined group (7.474±0.467) wassignificantly upregulated compared with the control group(1.000,p<0.001). While therewere no significant upregulation in the group of UVB-radiation(0.856±0.086, p>0.05). Theresults of ANOVA of the factorial design showed that heat treatment and UVB-radiationhad no interaction effect both in protein and mRNA level(F=1.399,2.109respectively,p>0.05for all).(3) P53protein and mRNA expression changes between different groups:Western blot analysis demonstrated thatP53protein expression in heat treatmentgroup(0.512±0.016) and the UVB-radiation group(0.554±0.021) was higher than thecontrol group(0.316±0.024，p<0.001) and the combined group(0.850±0.020) was higherthan the singal treated groups. Fluoresent real-time quantitative PCR showed no significantdifference of P53mRNA level between heat treatment group（0.755±0.045）,UVB-radiation group（0.812±0.186） and the control group（1.000）(P>0.05). The level incombined group （1.173±0.106）was higher than the other three groups. The results ofANOVA of the factorial design showed that heat treatment and UVB-radiation hadinteraction effect both in protein and mRNA level(F=29.762,23.048respectively, p<0.05 for all).Conclusion (1) Heat treatment can induced the protein expression of HSP72and P53, thesignificant time is6h and24h after the last treatment respectively.(2) The heat treatmentgroup and the combined group can induced significant increase of HSP72protein andmRNA expression, and expression levels were similar between the two groups.HSP72mRNA and protein expression were not increased in UVB-radiation group, soHSP72expression was excited by heat.According to the role of HSP72protein,wespeculated that HSP72may trigger melanocyte proliferation activity and protectmelanocyte from UVB radiation damage.(3) The protein expression of P53is upregulatedin treatment group, UVB radiation group and the combined group compared with thecontrol group.Heat treatment and UVB radiation has a synergistic effect in increasing P53protein expression. No significant difference changes in the level of P53mRNA betweenthe heat treatment,UVB radiation and control groups, suggesting that the simple heattreatment or UVB radiation-induced P53protein increase is post-transcriptional levelchanges.But heat and UVB combined can synergistically increase P53mRNA. Based onthe role of P53in the formation of melanosomes after UV radiation, we supposed thatmelanogenesis after heat treatment is by way of P53pathway which is similar with UVB.