Protective Effect Of EGF On X-ray Induced Chondrocyte Apoptosis And DNA Damage

Objective: To observe the protective effect of epidermal growth factor(EGF)on X-ray induced chondrocyte apoptosis and DNA damage,in order to provide experimental and theoretical basis for in vivo experiments and clinical drugs.Methods: The articular cartilage of the hind limbs of the newborn SD rats was taken,and the primary rat chondrocytes were cultured by enzymatic digestion.The obtained primary chondrocytes were divided into four groups: X-ray irradiation group,X-ray irradiation with EGF group,non-irradiation group,non-irradiated with EGF group.Irradiation using a dedicated X-ray machine to produce a knee joint chondrocyte X-ray injury model with a radiation dose of 16 Gy.AO/EB staining was performed to observe the apoptosis of chondrocytes.The ?-H2 AX focus method and comet assay were used to test the DNA damage of chondrocytes,and the results were analyzed by Image Pro Plus software and CASP software.Result: 1.The rate of viable cells of primary chondrocytes cultured by enzymatic digestion was greater than 90%;2.Observation of chondrocyte apoptosis: the apoptosis rate of chondrocytes was significantly increased in X-ray irradiated group compared with non-irradiated group(P<0.01).The difference was statistically significant.After the addition of EGF,it was found that the apoptosis rate of chondrocytes was significantly lower in the X-ray irradiation with EGF group than in the X-ray irradiation group(P<0.05).The non-irradiated with EGF group was compared with the non-irradiated group.The apoptotic rate was also significantly decreased(P<0.01)?These differences were both statistically significant.Compared with the non-irradiated group,the apoptotic rate of chondrocytes in the X-ray irradiation with EGF group was still significantly increased(P<0.05),and the difference was statistically significant.3.?-H2 AX focus method was used to detect DNA damage: the average number of ?-H2 AX focus per cell was significantly increased in the X-ray irradiation group compared with the non-irradiated group(P<0.01),and the difference was statistically significant.The average number of ?-H2 AX focal points per cell was decreased in the X-ray irradiation with EGF group compared with the X-ray irradiation group(P<0.05).The average number of ?-H2 AX focus per cell was also decreased in the non-irradiated with EGF group compared with the non-irradiated group(P< 0.05).These differences were both statistically significant.The verage number of ?-H2 AX in the X-ray irradiation with EGF group was significantly increased compared with the non-irradiated group(P<0.01),and the difference was statistically significant.4.The DNA damage was detected by comet assay: the tail length,comet length,tail moment and Olive tail moment measured by CASP software were used as the experimental indicators of comet.The above four indexes were significantly increased in the X-ray irradiation group compared with the non-irradiated group.(P<0.01),the difference was statistically significant.After adding EGF,X-ray irradiation with EGF group compared with X-ray irradiation group,the tail length(P<0.01),the comet length(P<0.05),the tail moment(P<0.01)and the Olive tail moment(P<0.01)were decreased.The tail length(P<0.01),the comet length(P<0.05),the tail moment(P<0.01)and the Olive tail moment(P<0.01)were also reduced in the non-irradiated with EGF group compared with the non-irradiation group.The differences were both statistically significant.The tail length(P<0.01),the comet length(P<0.05),the tail moment(P<0.01)and the Olive tail moment(P<0.05)were increased compared between the X-ray irradiation with EGF group and the non-irradiated group.The difference was statistically significant.Conclusion: This study demonstrates that EGF can reduce the apoptotic rate and DNA damage of chondrocytes after X-ray irradiation,but can not completely reverse the chondrocyte damage caused by X-ray irradiation.