The Model Establishment And Evaluation Of Radiation-induced Injury Of Submandibular Gland Epithelial Cells In Rat

Objective:Different doses of SD submandibular gland epithelial cells were irradiated to establish a suitable radiation injury model.Methods:The submandibular gland cells were isolated from the submandibular glands of newborn 3d SD rats by tissue block method and enzymatic digestion.The submandibular gland epithelial cells were obtained by trypsin differential detachment and passage and purified,cultured and passaged;Identification of keratin(CK7)by immunocytochemistry applied to P2 cells;The P2 generation rat submandibular gland epithelial cell suspension was prepared,and the cell suspension was irradiated with different doses(0Gy,1Gy,3Gy,5Gy,7Gy,9Gy)using a medical electron linear accelerator;The cell proliferation activity of each irradiation dose group was detected by CCK-8(Cell Counting Kit-8)technique;The apoptosis of each irradiation dose group was detected by Annexin V-FITC/PI flow cytometry;The enzyme-linked immunosorbent assay was used.The content of salivary amylase α1(AMY1)in the supernatant of each group was determined by ELISA method.The glycogen secretion and E-cadherin of each group were detected by glycosaminoglycan PAS staining and immunocytochemistry.Results: The primary SD rat submandibular gland epithelial cells were polygonal on the6 th day and arranged in a cobblestone-like manner.On the 14 th day,the cells were paving stone-like;the P2 generation rat submandibular gland epithelial cells were positively expressed by CK7,which proved that the cells were epithelial sources;After 72 h,there was no significant difference between 0Gy and 1Gy cells in the group.The cell morphology was uniform and the cell density was about 90%.The cell morphology was uniform in the 3Gy group,and the cell density was slightly decreased.The cell density in the 5Gy group was further reduced,but the cell morphology was acceptable.The cells in7 Gy and 9Gy group had large heteromorphism and a large number of apoptosis in cells.CCK-8 assay showed that the proliferation activity of each group increased first and then decreased in 1-7 days,and the proliferation ability of cells decreased with the increase of irradiation dose.There was no significant difference in the proliferation activity between0 Gy and 1Gy group.The difference of cell proliferation activity between the remaining groups was statistically significant.The cell proliferation activity of the 5Gy group decreased to about 53.95% of the 0Gy group.The results of flow cytometry in each group showed that there was no significant difference in apoptosis rate between 0Gy and 1Gy groups,and the differences among the remaining groups were statistically significant.the apoptosis rate of 5Gy group increased significantly to 12.65±0.72%;The concentration of AMY1 in the supernatant of each group decreased with the increase of irradiation dose,the difference was statistically significant(P<0.05).The concentration of AMY1 in the 5Gy group decreased to about 59.9% in the 0Gy group.The glycogen staining results of the cells in each group showed a large amount of deep red glycogen particles in the cytoplasm of the 0Gy and 1Gy groups,and the deep red glycogen particles in the cytoplasm of the3 Gy group.A slight decrease,the red glycogen granules in the cytoplasm of the 5Gy and7 Gy groups were significantly reduced and the staining became lighter,while the cytoplasm of the 9Gy group had almost no red glycogen granules;The immunocytochemistry results of E-Cad in each group showed 0Gy.The cytoplasm of the1 Gy group was dark brown,and the cytoplasm of the 3Gy and 5Gy groups became light brown.The 7Gy and 9Gy groups showed very few brown particles in the cytoplasm.Conclusion: In this study,we compared the proliferative activity,apoptosis and secretory function of submandibular gland epithelial cells in SD rats with different irradiation doses,and obtained the damage of rat submandibular gland epithelial cells induced by radiation,while 5Gy may be the radioactive injury cytology of submandibular gland epithelial cells in SD rats.The model is suitable for the dose of radiation.