Study On Bystander Effect Induced By UVC In V79 Cells

PrefaceIonizing radiation is confirmed as a damage of cell genetic material.For a long time,it is known that biological effects induced by radiation,such as gene mutation, chromosomal aberration,cell death,and carcinogenesis have been attributed to direct irreparable and mispaired DNA damage in target cells;while this effect did not occur in the unirradiated cells.There is dose-effect relationship between radiation dose and targeted cytological effect;that is the lower the radiation dose,the weaker the effect is. Hoever,more and more studies discovered that theradiogenic genetics alteration might also occur in non-irradiated cells near irradiated cells or the nomal cells in the culture solution.The nomal cells called bystander cells,and this phenomenon is bystander effect.There is not a final conclusion on the mechanism of the bystander effect induced by radiation.The effect is different from traditionary radiation radiation.Till now,It known that it may have relationship with the active substance signaling molecule secreted by the irradiated cells.Besides ionizing radiation,the target cells irradiated by UV also can induce bystender effect.In this study,the V79 cells are irradiated withUVC.First,we need find the optimal irradiation dose.Next,culture the nomal cells with the medium cllected in different time in the optimal irradiation dose.Then observe the type and the intensity of the bystander effect.Fortherrmore we should investigate the mechanism of the bystander induced by UVC with molecular biology technique.Material and Methods1.Cell culture:The Chinese hamster fibroblast cellline(V79-C3)was grown in high glueose Dulbeeeo's minimum essential mediurn(Gibeo)supplemented with 10%foetal bovine serum(Hyelone)and antibioties(Penieillin ands trePtomyein)at 37℃in an ineubator with atmosphere of 5%CO~2. 2.Target cell:When the cell cover 75%in the 75ml cell culture flask;digested with trypsinase; inoculated in six pore plate;l*10~6each;cultivated another 12 hours.Before irradiation, the media were discarded,cells were washed with PBS three times,then discard PBS,the eells were exposed to UVC.A set of 2 black lightlamps(PhiliPs),Providing UVC wavelength between 200nm-290nm,was used as a UVC source.Exposuxe doses were measured and calculated based on light with wavelength of 254nm.Cells were exposed from below at 9mWcm-2 plaeed the Petri dishes beneath the lamps by 5mm.Exposure time ranged from10s,20s,30s,60s,90s.3.Acquisition of the irradiated conditioned mediunnThe V79 irradiated with the optimal dose;fatch the culture fluid in 4h,8h 12,24h,filtered with 0.22μm micropore filter;stroed in low temperature for usage.4.Bystander cells:When the cell cover 75%in the 75ml cell culture flask;digested with trypsinase; inoculated in six pore plate;l*10~6each;cultivated another 12 hours;discard the media; cultivated another 24 hours with ICM.5.Methods:(1)Survival rate:MTT viability assay(2)Cellular morphology observation:photo microscope(3)Necrosis and apoptosis:flow cytometry(4)Chromosomal aberration:colchicine and Giema(5)ROS:flow cytometry(6)P53 mRNA:RT-PCR(7)P53 protein:immunohistochemistry(8)Screening of the bystander factor:2D-electrophoresis and mass spectrumResults1.Under the expose doses of 90,180,270,540,810mJ/cm~2;thesurvival rates are: 65.5±4.4%,50.4±3.5%,43.4±2.7%,38.8±2.9%and 29.4±1.6%.2.Apoptosis rates of the target cells:22.59±2.22%,48.89±3.67%,56.69±4.05%, 64.57±4.66%and 73.48±4.37%. 3.Under the optimal dose of 180mJ/cm~2:the survival rates of the bystander cells are 61.5±3.9%,78.8±3.3%,84.2±4.1 and 74.9±3.7%.4.The apoptosis rates of the bystander cells cultured with ICM are 20.78±2.38%, 13.45±1.99%,10.51±1.53%and 28.57±3.59%.5.The chromosomal aberration rate of the bystander cells cultured with ICM are 12.33%,8.67%,5.67%and 15.00%.6.The average fluorescence intensity of the bystander cells cultured with ICM are 38.38±3.05,25.69±2.37,16.30±1.35 and 48.35±2.32.7.The gradation of P53 protein in the bystander cells cultured with ICM are: 79.05±5.23,68.36±5.12,51.81±4.35 and 78.65±5.67.Conclusion1.In this study,the bystander effect induced by UVC,the optimal does is 180mJ/cm~2.2.In this study,the type of the bystander effect is apoptosis.3.In this study,the TNF-a convertingenzyme(TACE)in the ICM maybe one of the bystander factors.