Induction Of Bystander Effect Of UVC In V79 Cell Line

PrefaceIonizing radiation is confirmed as a damage of cell genetic material. In a long time, it is known that the 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. However, there is increasing evidence that radiation-induced genetic alterations might also occur in non-irradiated cells near irradiated cells. This phenomenon is termed the "bystander effect" . Besides ionizing radiation, the target cells that be irradiated by UV also can induce bystander effect. Experimental evidence indicates that the DNA damage of target cells is the cause of bystander effect. In the process of repairing the damages, or dying because of grave damage, cells can secrete or excrete some unknown factors, which is called bystander factors. These factors may affect bystander cells directly, or transfer some informations to other cells that can induce effect, thus they can induce bystander effect. Ultraviolet irradiation can make 2 consecutive pyrimidines on DNA transform to cyclobutane pyrimidine dimmer (CPD), CPD can prevent replication and transcription, thus induce cell death finally. UVC, as a kind of high quantum energy waves, whether can induce bystander effect is a critical issue.Material and Methods1．Cell cultureA Chinese hamster f靊roblast cell line (V79-C3) was grown in high glucose Dulbecco's minimum essential medium (Gibco) supplemented with 10% foetal bovine serum (Hyclone) and antibiotics (penicillin and streptomycin) at 37(?) in an incubator with atmosphere of 5% CO. 2．Establishing a cell necrotic modelIn preliminary experiment, cells grew to 75% confluent in 75 cm flasks, were trypsinized and transferred at densities according to different aims in 60-mm Petri dishes or other carriers for irradiation, and continue to be cultured. Before irradiation, the media were discarded, cells were washed with PBS once, and discard PBS, the cells were exposed to UVC.A set of 2 black light lamps (Philips), providing UVC wavelength between 200nm-290nm, was used as a UVC source. Exposure doses were measured and calculated based on light with wavelength of 254 nm. Cells were exposed from below at 9 mWcm(1W=1Js), place the Petri dishes beneath the lamps by 5mm. Exposure time ranged from 6 s, 22s, 89s, 178s, 222s. Medium was added in the dishes after irradiation, and continue to culture.3．Bystander effect induction and the strongest effect induced by theirradiated conditioned medium without cellsThe irradiated cells are incubated for 24 hours after the irradiation. The media of the cells that be irradiated by 2000mJ(?)cm of UVC were harvested at 4,8,12,24 hour, and they were added into normal confluent cells, at the same time fresh medium were added into irradiated cells. The bystander cells were continued to culture for another 24 hours.4．Indexes assays(1) Survival fraction: MTT viability assay.(2) Cell multiplication ability: Clonogenic assay.(3) The ratio of cells number of necrosis and apoptosis: Flow cytometry.(4) Analysis of chromosome aberration: The cells were treated with colcemid to prepare metaphase, the cells were harvested and underwent hypotension, fixation, and the cell suspensions were dropped onto cold clean slides. The slides were air dried and kept at room temperature until staining was performed. Results1．V79 cell line were irradiated by UVC, the doses as 50, 200, 800, 1600, 2000mJ/cm, the survival fraction assessed by MTT are 100%, 88．50(?)5．07%, 65．90(?)3．37%, 53．62(?)4．27%, 22．26(?)2．50%, 9．02(?)1．22%; in the similarity condition, the viabilities assessed by clonogenic formation are 89．28(?)6．07%, 64．14(?)3．15%, 50．69(?)2．61%, 21．72(?)3．45%, 8．64(?)0．99%;2．The majority of dead cells are necrotic, the necrotic fractions are 18．26(?)1．78%, 31．61(?)2．55%, 41．71(?)3．68%, 64．04(?)2．86%, 68．03(?)6．06%;3．The fractions of chromosomal aberration are 2%, 6%, 15%, 24%, 30%;4．The bystander cells cocultured with the media of the target cells, the survival fractions assessed by MTT are 54．91(?)2．05%, 77．03(?)3．92%, 84．73(?)1．36%, 96．7(?)5．65%; in the similarity condition, the viabilities assessed by clonogenic formation are 51．40(?)3．54%, 72．12(?)2．40%, 82．66(?)2．98%, 94．68(?)4．58%;5．The bystander cells cocultured with different time of the media of the target cells, the majority of dead cells are apoptotic, the apoptotic fractions are 39．49(?)3．58%, 36．86(?)2．99%, 8．80(?)2．53%, 6．32(?)1．59%;6．The chromosomal aberration fractions are 17%, 9%, 4%, 2%.Conclusions1．The DNA strains of the cells irradiated by UVC were damaged, the type of cell death is necrotic.2．The media of the dying cells were ascertained could induce bystander effect, and the type of cell death of the bystander cells is apoptosis, and the apoptosis fraction decreases according to the sequence of the media of the target cells.3．The most bystander factors or the strongest effect induced by UVC are produced in the first 4 hours after UVC irradiation.