| Objective:To investigate the ability of human amniotic mesenchymal stem cells exosomes pretreated with hypoxia for functional recovery of salivary gland epithelial cells after radiation injury.Methods:（1）The submandibular gland tissues of 3-day-old SD rats were isolated by enzyme digestion,and the primary cells were purified by differential digestion and differential adhesion of trypsin.（2）When submandibular gland cells were subcultured to P2,the Cytokeratin 7（CK-7）was identified by immunohistochemistry.（3）The Human Amniotic Mesenchymal stem cell of P3 generation was provided by Tissue Engineering Laboratory of Zunyi Medical University,and the cell phenotype was identified by Flow cytometry.Then,the h AMSCs of P3 generation were divided into hypoxic and normoxic groups,which were treated with Hypoxia（1%O2）and normoxic preconditioning for 48 hours.Then h AMSCs-exo was extracted from the culture supernatant of h AMSCs by modified ultra-high speed centrifugation,and its morphology was analyzed by transmission electron microscope,its size and concentration were analyzed by NTA,and its specific protein CD9 and CD63 were detected by Western blot（WB）.（4）The submandibular gland epithelial cells of P3generation rats were irradiated by 5Gy with electron LINAC.（5）The experiment was divided into 4 groups:Blank Control Group（submandibular gland epithelial cells were not irradiated）,Radiation Control Group（submandibular gland epithelial cells were irradiated with 5 Gy）,Normoxia Plus Medicine Group（submandibular gland epithelial cells were irradiated with 5 Gy for 3 days）,Hypoxia Plus Medicine Group（submandibular gland epithelial cells were irradiated with 40μg/m L h AMSCs-Exo for 3 days）.（6）Submandibular gland epithelial cells were cultured in the above-mentioned groups for 1-3 days:the proliferative activity of the cells in each group was detected by cck-8 at 1,2 and 3 days,and supernatant of each group was collected on the 3rd day of Culture,the content of Salivaryα-amylase was measured by Elisa,and the expression of AQP5 m RNA was detected by Q-PCR.Results:（1）The submandibular gland epithelial cells of SD rats grew in a uniform shape,with paving stone-like arrangement and adherent growth.The cells expressed CK7 positively,indicating that the cells were epithelial origin.（2）HAMSCs were swirling and adherent growth.Flow cytometry showed high expression of CD71,CD44,CD29,low expression of CD34 PE,CD45,HLA-DR.（3）HAMSCs-Exo was obtained by modified ultra-high speed centrifugation under hypoxia and normoxia.The samples were observed by transmission electron microscope as round or oval membrane vesicles,shaped like"tea saucer",and the sample size was about30-200nm.According to NTA test,the average particle size of h AMSCs-Exo under hypoxia and normoxia was 81 nm and 84 nm,respectively,mainly concentrated in the range of 30-150 nm,which was in line with the development characteristics of exosomes.The concentrations of h AMSCs-Exo were 1.80-1010/m L and 2.67-108/m L respectively.The average concentration of exosomal protein measured by BCA was0.41μg/L and 0.16μg/L respectively.Western blotting showed that both hypoxic and normoxic exosomes groups were positive for CD9 and CD63.（4）Compared with the Control Group,the cell proliferation activity of the radiation-treated group decreased significantly at 1-3 days（p<0.05）.Compared with the Radiation Control Group,the cell proliferation activity of normoxic and hypoxic group was significantly increased2 and 3 days after treatment with exosomes（p<0.05）.Compared with normoxic group,the proliferative activity of Hypoxia Group was higher only on the 1st day,and increased on the 2nd and 3rd days,but there was no statistical difference.（5）On the third day,the contents ofα-Amy in each group were determined by Elisa:compared with the control group,the contents ofα-Amy in radiation control group were significantly increased,while those in normoxia and hypoxia groups were significantly decreased（p<0.05）.Compared with the control group,the content ofα-Amy in normoxic group and hypoxic group was significantly lower（p<0.05）.Compared with the normal oxygen group,the content ofα-Amy in the hypoxic group increased,but there was no statistical significance.（6）The results of Q-PCR showed that AQP5 m RNA expression level was significantly lower in the radiation control group than in the blank control group,and the difference was statistically significant,the AQP5 expression level of normoxia plus group and Hypoxia plus group was significantly higher than that of normoxia plus group,but the difference was not statistically significant.Conclusion:Hypoxia preconditioning with human amniotic mesenchymal stem cells exosomes can repair the submandibular gland epithelial cells of SD rats with radiation injury;However,this study has not revealed its significant advantage over normoxia-treated h AMSCs-Exo in the functional recovery of submandibular gland epithelial cells. |
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