The Effects Of Erythropoietin To The Cell Proliferation And Apoptosis Of Irradiation Damaged Rat Submandibular Gland Cells

Objective:To culturing submandibular gland cells of rat in vitro and exploring the effects of Erythropoietin to the cell proliferation and apoptosis of irradiation damaged rat submandibular gland cells,and aim to providing research foundation for the prevention and treatment of salivary gland irradiation damage.Method:The bilateral submandibular glands were obtained from Wistar rats of 7 days old in a sterile environment,application of tissue-explant technique in culturing.The cells were purified by enzymatic digestion and differential adhesion,the cell phenotype was identified by cytokeratin-8(CK-8)antibody and ?-Amylase antibody immunohistochemical staining,and PAS staining was used to test the cell function of secration hepatin.The HE staining technique method,Hoechst 33342 staining and scanning electronic microscopy(SEM)were used to observe morphological characteristics of P2 cells.Added various concentration(5?50?500?1000IU/ml)EPO to the normal culturing submandibular gland cells,use the cck-8 method to test cell proliferation rate,and the inverted phase contrast microscope was used to observe the morphological change of cells.Research the impact of EPO to the normal culturing cells' growing.And then,0,2.5,5.0,7.5,10 Gy irradiation dose was added to the P2 cells respectively,through cell viability test to screen out the appropriate irradiation dose which induce the submandibular gland cell reversible damage.The experiment was divided into five groups,there were control group,irradiated only group,add EPO before irradiated group,add EPO after irradiated group,add EPO both before and after irradiated group respectively.The CCK-8 technique method was used to test cell proliferation and the flow cytometry Annexin-V/FITC fluorescent light staining was used to analyse the cell apoptosis.Observe the prevention and treatment effect of EPO to the irradiated submandibular gland cells.Result:The biological characteristics of cultured submandibular gland cells in vitro: The inverted phase contrast microscope was used to observe that a large number of cells have climbed out around the tissue blocks at the fourth day,the most are polygon and short spindle shape,and the pseudopod extend crosslinking.The 7th to 8th day,the most tissue blocks have full of cells,and stratified growth began to appear.And now,the enzymatic digestion method was used to disperse cells as the first subculture,the purified cells was continue to culture 48 hours,afterwards,we can observe that the most cells adherence,and about7-8 days overgrowed polygon shape cells ranged in paving stones sample.The cytoplasms were purple red in PAS staining,and cytoplasms were brown yellow,nucleus were bluish violet.It indicates that the CK-8 antibody and ?-Amylase antibody were positive stained in the immunohistochemical staining.The cells were polygon and angle shape in HE staining,and the blue stained nucleus were oval,the cytoplasms were pink stained.Through the fluorescence microscopy,we can observed that the big oval nucleuswere blue stained,lighter than the cytoplasms which were connect,and in the SEM,we can saw the cells have different lengths of pseudopodia,and the nucleus were darker-stained.Secretory granules were visible in some cells.The effects of EPO to the cell proliferation of normal submandibular gland cells: After the administrating of EPO to normal cells24 h,48h,72 h,we found that there were no significant change in cell proliferation and also the cell morphology.Appropriate irradiation dose selection: After 24 h of the irradiation effect,we observed that the cell viability rate decrease with the irradiation dose increase.Particularly,when the irradiation dose was 7.5Gy,the cell viability decrease 39%(P<0.05),which has statistical significance.The effects of EPO to the cell proliferation and cell apoptosis of irradiation damaged cells: When added into 7.5Gy irradiation once,after24 h,we found that there were no significant change in cell proliferation and also the cell apoptosis rate.Conclusion:Primary rat submandibular gland cells can be successfully cultured by tissue-explant technique,and passaged in vitro.There were no significant effects of EPO to the cell proliferation and apoptosis of irradiation damaged rat submandibular gland cells,and its mechanism needs further study.