Construction Of A Co-culture Experiment Model With Fluorescent Tags And Its Application In Detecting The Bystander Effect Of Radiation-induced DNA Damage

Objective：To establish a new experimental model for the research of the cell’s DNAdamage bystander effect induced by ionizing radiation, and explore the bystandereffect of DNA double strand breaks and repair induced by the gamma-ray radiationand the low dose of alpha-particles.Methods：Construct a pDsRed1-N1-Hspc300plasmid which can express a red fluorescentprotein localized in cell cytoplasm, and a stable expression cell line was generated bytransfecting this plasmid into the human fibroblasts cells (HFS). One of the cells wasused as target cells by irradiated with gamma rays or alpha particles, and the otheracted as the radiation bystander cells. By mixing this two population of cells andco-cultured, then collected the cells at different time points. The confocalimmunofluorescence microscope was used to view and count the number of theγH2AX foci in the irradiated target cells and the bystander cells. And the radiationbystander effect of DNA damage and repair (mainly the damage of DNAdouble-stranded breaks) was investigated.Results：1. A co-cultured cells experiment cell model has been established for the studybystander effect of DNA-double strand breaks (γH2AX foci) generated by theirradiated target cells. 2. After2Gy60Co γ-ray irradiation,the irradiated target HFS cells and thebystander cells HFS-RFP were mixed and co-cultured. DNA double-strand breaks (γH2AX) were generated in both irradiated target cells and bystander cells in a similartendency and amount. γH2AX in both cells reached the maximum value at30minutes after irradiation and mixed, then reduced slowly and there is a significantdifference between the irradiation group and the control group(P<0.05) at every timepoint.3. Under alpha particle irradiation, directly irradiated target cells HFS-RFP andthe bystander cells HFS had a similar trend in the occurrence of the γH2AX foci，which in both types of cells reached a peak at1h after irradiation and mixed, andthere is still a significant difference between the time points of0h and24h (t <0.05).There are two peaks of the formation of γ H2AX foci, at1h and8h after irradiation,respectively.Conclusions：1. A new research model of radiation bystander effect has been established.2. The gamma-ray irradiation can induce the cell bystander effect of DNADSB,and the bystander effect is the same as that in the irradiated target cells,and itsdamage can be well repaired.3. Alpha particles irradiation can induce the DSB bystander effect, its changingtrend is similar to the directly exposed cells,and a secondary injury of DSB appearedafter irradiation in both target and bystander cells.