Study Of The Effect Of DNA Methylation On UVA-induced Chronic Damage In Mouse Embryonic Fibroblast

Background:Skin is barrier for protect the body from its environment such as ultraviolet radiation(UVR)and other exogenous harmful factors such as chemical factors.The long wave ultraviolet radiation(ultraviolet A,320-400 nm)can cause chronic damage in skin,skin photoaging,inflammation and even the skin cancer.The DNA methylation would make change when cells is under the action of UVA,the UVA-inducing the methylation transferase(DNMT)will be involved in cell growth,cycle,cancer and other regulation.In mouse fibroblast cells,the expression of DNMT is affected by UVA,and the relationship between the changes of the expression of DNMT in cells induced by UVA are the innovation of this article.Objects:After different dose of UVA irradiation(25 KJ/m2,50 KJ/m2,100 KJ/m2,150 KJ/m2,200 KJ/m2)repeated three times(each time 24 h interval),fibroblast cells in mice is detected from the level of protein and mRNA level to the expression of DNMTs;After detected of different concentration of 5-az(including 0 mM,2.5 mM,5 mM and 10 mM)influence on cell ability of NIH3T3 cells,it is need to chooset the one of best expression on to conbine with UVA radiaion.Using 5 mM concentration of 5-az and associated with UVA(UVA dose is chosed 100 KJ/m2)establish a chronic injury model to detec impact on cell activity.We detected the expression level of induction of DNMTs(DNMT1,DNMT3 a,DNMT3b)in cells after 5-az processing joint with the action of UVA radiation,and in last,we dectected the expression cycle related genes(CCND1,CCND2,etc)and if inducing cell cycles change after test 5-az processing joint under the action of UVA radiation Methods:Using the method of fluorescence probe DHE staining detected the changes of intracellular ROS levels.using MTS essay detected the cells' ability of NIH3T3 cells after the radiation of UVA and the action of 5-az.Using quantitative real-time PCR method(QT-PCR)detected the expression level of DNMT 1,DNMT 3a,DNMT 3b,CCND1,CCND2,HO-1,mRNA.Western blotting electrophoresis was used to detect the expression level of DNMT1,DNMT3 a,DNMT3b,CCND1,CCND2,HO-1 and other proteins.Results: 1.After repeated long wave ultraviolet radiation(UVA),the cells' ability is decreased in NIH3T3 cells activity,and the decreasing of cells' ability is correlated with UVA dosageSeveral times of UVA radiation in NIH3T3 cells(three times of UVA radiation,each time interval for 24 h and dose same as the previous),its corresponding cells' ability is obviously showed a trend of decreasing.In low dose UVA radiation,the redue is not very obvious,but at high dose UVA radiation(at dasage of 100 KJ/m2)after compared with the group of control,the corresponding cell's morphology and cells' ability is gone to a significantly change.The change is big.In cell's morphology,after large doses(compared with the group of control),cells appear to obvious shrinkage state and there are a large number of cell debris.2.The influence of 5-az treatment with the combination of long wave ultraviolet UVA radiation effect the expression of metabolic activity in mouse embryonic fibroblast cells(NIH3T3 cel s)Compared with the untreated group,a separate deal with 5 mM compare to 5-az's treatment conbining with 100 KJ/m2 UVA radiation have obvious effects on cell activity.5-az reduced the expression level of DNMT1,DNMT3 a and DNMT3 b in cells after UVA radiation,blocking the cell to protect itself by regulating the expression of DNMTs.The expression level of CCND2 in single treatment is higher than in UVA combinaion with 5-az.3.Undering the irradiation of low dose UVA,after 24 hours treatment the cells with high dose irradiation,the cells' ability and the corresponding expression level of DNMTs and the impact on the oxidative stress changed.Compared with the control group,low dose of radiation,the survival rate of NIH3T3 cells under high doses of UVA radiation is higher than survival rate after only large doses of radiation.The DNMTs and the expression of HO-1 also expressed higher than a single high dose of UVA radiation.Conclusion:UVA plays a promoting role in the expresion of DNA methylation,its prmotion is mainly through the inducing of DNMT1,DNMT 3 to guidance change.DNMTs play a protected role in UVA radiated cells,in demethylation drug combine with UVA radiation,the expression of DNMT1 significantly decreased compared to the single effect factors.