Study On Autophagy In Apoptosis Of Hsf Induced By Uva

The first part: the effect of UVA on apoptosis of HSFobjective:The purpose of the experiment is to culture and identify primary human skin fibroblasts(HSF),to obtain sufficient cell sources.From the morphology observation,the effect of different doses of UVA on the growth of primary HSF is observed.The relationship between the activity of HSF and UVA dose is analyzed.The further object is to explore the effect of UVA on apoptosis of primary HSF and detect the optimal dose and time of UVA inducing apoptosis of HSF.Methods:1.When 2-14 years old male circumcision foreskin was collected,after disinfection,the foreskin was separated from the dermis.The foreskin was cut up into pieces.In the meantime,the collagenase was joined,and the supernatant was abandoned after centrifugation.Then complete medium(containing 87%high glucose DMEM,10%FBS,1%GlutaMAX,1%NEAA,1%penicillin-streptomycin)was added to HSF,which was put in 37?and 5%CO2 incubator to culture.The cells were identified by immunocytochemistry and high purity cell sources were obtained.Through continuous generations,cryopreservation cells were preserved.The growth of cells was observed by direct counting.2.UVA-irradiated HSF model was established to observe the cell morphology.At1.5 x 105 cells/ml density cells were incubated in a 6-well plate.When cell fusion degree reached 80%,HSF was put in the SUV-1000 sun ultraviolet ray simulator under the beam light.There were 8 groups,including control group,4 J/cm~2 group,8 J/cm~2 group,12 J/cm~2 group,16 J/cm~2 group,20 J/cm~2 group,40 J/cm~2 group,80 J/cm~2 group.After irradiation,the cells continue to be cultured.The changes of cell growth at different time points under a microscope were observed and recorded.3.After irradiation of different doses of UVA,there were 5 groups:control group,4 J/cm~2 group,8 J/cm~2 group,12J/cm~2 group,16 J/cm~2 group.Each group was set with 3 compound holes.The cells were cultured for 4h,and then added to 20ul MTT and 150ul DMSO.The absorbance OD value was detected at 490nm with the enzyme marker.4.The detection settings of apoptosis were 5 groups:control group,4J/cm~2 group,8J/cm~2 group,12 J/cm~2 group,16 J/cm~2 group.After UVA irradiated HSF,cell was cultured for 4h.Meanwhile,Binding Buffer,5ul Annexin V-FITC and 5ul PI was added to HSF for detection by flow cytometry.Results:1.HSF adherently grew for 1-2 days in the form of a spindle shape.Cell identification found that the waveform protein staining was positive.Cultured primary cells were consistent with the characteristics of HSF.The direct counting method found the cells rapidly proliferated in the 3rd to 5th day,in a logarithmic phase and the cell fusion degree reached to 80%,slowly growing in the 6th-7th day,in a plateau stage.2.With the increase of UVA dose,the damage of UVA to HSF increased in a dose-dependent manner:the larger the radiation dose,the more intracellular particles,the more rounded the cells,the more the arrangement of the cells and the more the cytoplasmic refraction sexual decline.After UVA irradiated HSF for 4 hours,the cell morphology greatly changed.3.When the irradiation dose was greater than 8J/cm~2,with the increase of irradiation dose,the cell activity decreased.There was a significant difference(P<0.01),compared with the untreated control group.There was no statistically significant difference between the control group and the 4 J/cm~2group(P>0.05).There were significant differences among the other groups(F=219.01,P<0.01).When the dose of UVA was 8 J/cm~2,the cell activity was over 85%and the cells were in good condition.4.With the increase of UVA dose,the apoptosis rate of HSF gradually increased.There was no significant difference between the control group and the 4 J/cm~2 group(P>0.05),while the difference between the 12 J/cm~2 group and the 16 J/cm~2 group was significant(P<0.05).The differences among the other groups were statistically significant(F=106.61,P<0.01).The irradiation dose was greater than 8J/cm~2,which was significantly different from the control group(P<0.01).Conclusions:1.UVA irradiation may cause the morphology changes of primary HSF in a dose-dependent manner.2.After UVA irradiated primary HSF,cell activity was inhibited,and the higher the UVA dose,the more obvious the decrease.3.UVA may induce primary HSF apoptosis,and the rate of apoptosis may increase with the increase of UVA intensity.The second part:the effect of autophagy on apoptosis of HSF induced by UVA objective: The purpose of the experiment is to observe the changes of the cell activity when autophagy inducer RAPA and inhibitor 3-MA were added to primary HSF after UVA irradiation.The further object is to clarify the relationship between autophagy and apoptosis of HSF induced by UVA.Methods: 1.The experiment was divided into control group,RAPA group,3-MA group.The cells were preincubated with 100nmol/L RAPA and 50?mol/L 3-MA before UVA irradiation for 2h.After 8J/cm2 UVA irradiated,HSF were cultured for 4h and then the cell morphology was observed.2.Cells were inoculated in a 96-well plate at approximately 5000 cells per well,including RAPA group,control group and 3-MA group.There were three replicate wells in each dose.After two days,the 96-well plate was exposed to UV light and the cell viability was measured by MTT assay after 4 hours.3.According to the above method,the apoptosis of RAPA group,control group and 3-MA group was detected by flow cytometry.Results: 1.From the morphology observation,the HSF morphology of RAPA group was the best,followed by the control group,the worst in the 3-MA group.2.After intervention,the order of cell activity from high to low was RAPA group,the control group,3-MA group.RAPA group and 3-MA group were significant,compared with the control group(P<0.05).The differences between the groups were statistically significant(F=23.86,P<0.01).3.After intervention,the apoptosis rate from high to low in order was 3-MA group,control group,RAPA group.The differences between the groups were statistically significant(F=43.70,P<0.01).Conclusion: 1.Autophagy may protect primary HSF from UVA irradiation and promote autophagy to protect cell morphology and cell viability.2.Autophagy may inhibit primary HSF apoptosis after UVA irradiation.Inhibition of autophagy may promote primary HSF apoptosis after UVA irradiation.