| Objective: To study whether human amniotic mesenchymal stem cells(h AMSCs)pretreated with hypoxia can protect and repair salivary gland epithelial cells from radiation injury in mice.Methods:(1)3D The submandibular gland cells of SD rats were separated by enzyme digestion,and purified by trypsin differential digestion and differential adhesion method;(2)keratin 7(CK-7)was identified by immunohistochemistry in P2 generation;(3)human amniotic mesenchymal stem cells were identified by flow cytometry in tissue engineering laboratory of Zunyi Medical University;(4)The experiment was divided into 4 groups;blank control group(submandibular glandular epithelial cells without radiation treatment group),5Gy radiation control group(submandibular glandular epithelial cells treated with radiation but not co-cultured with h AMSCs),hypoxic co-culture group(hypoxic pretreatment h AMSCs and submandibular gland epithelial cells co-cultured for 7 days after irradiation)and normoxia co-culture group(h AMSCs and submandibular gland epithelial cells co-cultured for 7 days after irradiation);(5)P3 generation of rat submandibular gland epithelial cells using electron linear accelerator 5Gy irradiation was performed;(6)After co-cultivation for 7 days according to the predetermined group,the cell supernatant of each group was collected separately,and the content of salivary amylase ? 1(AMY1)in the submandibular gland epithelial cells of each group was measured using enzyme-linked immunosorbent assay(ELISA);CCK-8(Cell Counting Kit-8)technology was used to detect the proliferation of cells in each group;Annexin V-FITC/PI flow cytometry was used to detect the apoptosis of cells in each group;Quantitative real-time-PCR was used to detect the cells in each group AQP5 m RNA expression level.Results:(1)The submandibular gland epithelial cells of P2 rats were uniform in shape,arranged like paving stones,and adhered to the growth.The mouse submandibular gland cells CK7 was positively expressed,which proved that the cells were derived from epithelial cells.(2)The shape of P3 h AMSCs is relatively uniform,with long fusiform,spiral shape and adherent growth.Flow cytometry identification showed that h AMSCs highly expressed CD44,CD73,CD90 and CD105,and lowly expressed CD11 b,CD19,CD34,CD45 and HLA-DR.(3)Corresponding examination of cell function after co-cultivation for 7 days according to the predetermined group.Determination of salivary ?-amylase content: the blank control group was significantly higher than the co-culture group,and the 5Gy radiation control group had the lowest content(P<0.05).The amylase content of the culture group was higher than that of the normoxic co-culture group,but the difference was not statistically significant.(4)Cell proliferation activity cck-8 showed that the blank control group had higher cell proliferation activity than the co-culture group,the 5Gy radiation control group had the lowest,and the hypoxia pretreatment group was higher than the normal oxygen group(P<0.05).(5)Flow cytometry detection of cell apoptosis showed that the blank control group had the lowest apoptosis and the 5Gy radiation control group had the highest(P<0.05).The hypoxic co-culture group had an increasing trend compared to the normal oxygen co-culture group,but the difference was not has statistical significane.(6)Quantitative real-time-PCR detection of AQP5 m RNA in each group of cells showed that the expression level of the blank control group was the highest,the expression level of the 5Gy radiation control group was the lowest,the co-culture group was centered,and the differences between the groups were statistically significant(P<0.05),the hypoxic pretreatment of h AMSCs showed a tendency to increase compared with the normoxic co-culture group,but the difference was not statistically significant.Conclusion: HAMSCs co-culture can protect and repair submandibular gland epithelial cells from radiation injury;co-culture with hypoxic pretreatment has better protection and functional repair than that with normal. |
PDF Full Text Request