| Background:The salivary gland,also known as the parotid gland,is the organ that secretes saliva.Saliva is closely related to swallowing,digestion,taste,language,protection of oral mucosa and prevention of dental caries.Dry mouth is a subjective feeling caused by the decrease of objective saliva secretion,often caused by radiation therapy of malignant tumors,autoimmune diseases(Sjogren's syndrome)and so on.At present,it is only symptomatic treatment for the subjective feeling of dry mouth,but it cannot fundamentally relieve the suffering of the patient.In recent years,the rise of cell therapy and tissue engineering has brought dawn to the treatment of xerostomia.Adipose stem cells have also attracted attention as a possible seed cell.This article will explore the feasibility of transforming rat adipose-derived stem cells into acinar-like cells.Objective:To culture rat adipose-derived stem cells(ADSCs)in vitro,it was explored whether it has the ability to transform into a secretory acinar-like cells and thus become potential seed cells in parotid tissue engineering in the microenvironmental experiments simulated by submandubular gland cells(SMGCs)lysates.Method:The abdominal groin of 3 weeks old SD rats was obtained in a sterile environment.ADSCs were extracted and subcultured.The morphology of the cells was observed by microscopy and HE staining.Cell growth curves were drawn by CCK8 method.It was identified as having a multi-directional differentiation ability by osteogenic induction and adipogenic induction.The submandibular gland cell lysate was prepared,and the third generation adipose-derived stem cells were divided into the control group and the experimental group.The experimental group was treated with submandibular gland lysate.Morphological observations were made each time the fluid was changed.For the control group,the 7-day induction group,the 14-day induction group,and the 21-day induction group were subjected to CK8 anda-Amylase.For the control group,the 7-day induction group,the 14-day induction group,the 21-day induction group were subjected to CK8 anda-Amylase immuno:fluorescence staining,and the amylase assay kit was used to detect the amylase content in the cell supernatant at different times and analyzing conversion efficiency.Results:Successfully cultured SD rats with ADSCs:the primary cells were changed for half a second after 24 hours,and a small number of cells were adherent,mostly oval.After 48 hours,the number of adherent cells increased,the shape became irregular,and the morphology gradually became uniform after 4 days.It was polygonal or fusiform,and the arrangement was relatively loose.After passage,the cells began to grow rapidly,and they were passaged in about 2-3 days.There was no significant change in morphology when they were passaged again.After HE staining,the cytoplasm was red,the nucleus was blue and the nucleus was full,and the cells were long-spin type.The OD value was measured by CCK8 method and the growth curve of ADSCs was drawn.It can be seen that the whole cell growth was "S" type,which was consistent with the previous literature conclusions.After the induced ADSCs were induced by adipogenesis,a large number of lipid droplets of different sizes appeared inside the test on the 21st day,and were stained red by oil red "O".After osteogenic induction,opacity may occur around the 14th day.The region is called the"calcium nodule".The alkaline phosphatase detects that the cell cytoplasm is dark brown and the cell morphology is irregular.Successfully induced the transdifferentiation of ADSCs into acinar-like cells with secretory function:As the induction time gradually increased,the morphology of the cells with long spindle type gradually changed,and the number of cells with polygonal or "pebble-like",gradually increased.Secretory vesicles resembling lipid droplets appear in the cytoplasm of the cells,but they are more numerous and smaller in size than before.Immunohistochemical analysis showed that the cells were positive for a-Amylase antibody and Cytokeratin-8 after induction,while the control group was negative.The supernatant of the induced cells was detected by the rat amylase(AMY)enzyme-linked immunosorbent assay kit,and it was found that the conversion rate of adipose-derived stem cells increased continuously with time,but the rate of transformation was continuously decreased.Conclusion:Rat primary ADSCs with multi-directional differentiation ability can be obtained by enzymatic digestion and subcultured.Adipose-derived stem cells can be transformed into acinar-like cells that secrete a-Amylase and their transformation efficiency gradually increases with time. |
PDF Full Text Request