| Objective:To study the effects of ubiquitin ligase Ring2 in DNA damage and cell cycle changes after expousing to benzo(a)pyrene in human brochial epithelial,and the role of ubiquitylation of histones in DNA damage repair.Methods:16HBE and 16HBE(si RNA-Ring2) cells respectively exposured with different concentrations(0,1,2,4,8,16 and 32μmol/L) Ba P for 24 hours and differnet time(0,1,2,4,8,12 and 24 hours) with 16μmol/L Ba P. To reflect the DNA damage with enzyme-linked immunosorbent and immunohistochemical method to detect BPDE-DNA adducts level. To reflect the changes of the cell cycle with flow cytometry and Western Blotting technology to detect the change of cell cycle and proliferating cell nuclear antigen(PCNA) level.Results:1. DNA damage detection:(1)ELISA ELISA test: Compared with normal control group, 16 HBE and 16HBE(si RNA-Ring2) cells were exposed to different concentrations of Ba P after 24 h,the BPDE-DNA adducts mean all with exposure increasing concentration increased significantly(P = 0.016; P = 0.001). Covariance analysis showed significantly higher(P <0.01) compared to 16HBE(si RNA-Ring2)cell group of estimate mean(1.627) and 16 HBE cell group(1.233). Compared with the control group, 16 HBE and 16HBE(si RNA-Ring2) cells with 16μmol / L Ba Pexposure concentrations at different times, BPDE-DNA adducts mean increased with the increasing exposure significantly(P = 0.003; P = 0.011). Covariance analysis showed 16HBE(si RNA-Ring2) cells group BPDE-DNA adducts estimate mean(1.838) was significantly higher than 16 HBE cell group(1.273)(P <0.01).(2)Immunohistochemistry: the 16μmol / L Ba P exposed 16HBE(Si RNA-Ring2) and16 HBE 4h after show, 16HBE(Si RNA-Ring2) intracellular green fluorescence ratio16 HBE cells was significantly enhanced after the cells were exposed to prompt interference generated BPDE-DNA adducts than normal cells after exposure increased.2. The cell cycle detection:(1)flow cytometry: Compared with the control group,16 HBE and 16HBE(si RNA-Ring2) cells were treated with different concentrations of Ba P exposure after 24 h, 16 HBE cell percentage of S-phase cells were increased with the increased exposure concentration increased significantly, 16HBE(si RNA-Ring2)cells is declining. Covariance analysis showed 16HBE(si RNA-Ring2) correction cell group mean(17.09%) was significantly lower than 16 HBE cell group(31.55%)(P<0.01). Compared with the control group, 16 HBE and 16 HBE after using 16μmol / L Ba P exposed at different times, 16 HBE cells were infected with the increase of the number of time and a significant increase in the proportion of cells in S phase(si RNA-Ring2) cells, 16HBE(si RNA-Ring2) cells are showing a downward trend.Covariance analysis showed 16HBE(si RNA-Ring2) correction cell group mean(13.07%) decreased significantly compared 16 HBE cell group(28.04%)(P <0.01).(2)Western blotting: 16 HBE different concentrations of Ba P exposure, you can see an increase in the expression of 8, 16 and 32μmol / L group PCNA, the rate increased significantly, statistically significant(P <0.01). 16μmol / L Ba P exposed 16 HBE cells at different time points(0,1,2,4,8,12,24h), with the increase of exposure time, the expression of PCNA also increases, and there is a significant time effect. PCNA compared 1,2,4,8,12,24 h each exposure point and negative controls, were significantly different(P <0.01). 16μmol / L Ba P exposed 16 HBE cells and 16HBE(Si RNA-Ring2) cells 4h altered expression of PCNA increased PCNA expression16 HBE cells, whereas expression 16HBE(Si RNA-Ring2) cells is reduced.Conclusions:1. The normal cells with DNA damage after exposure to Ba P has significant dose-response and time effect;2. Ubiquitin ligase Ring2 decreased expression of cell is more sensitive to DNA damage induced by Ba P exposure;3. Ubiquitin ligase expression Ring2 reducing cell DNA damage caused by exposure to Ba P may be more sensitive to shorten the S phase, which may be even more severe one of the reasons of DNA damage. |
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