| Objective:radiotherapy is one of the effective methods for the treatment of head and neck malignant tumors.However,because the salivary glands are located in the area of head and neck,the salivary glands can be exposed to radiation,which can lead to the reduction and degrade of the function of the salivary glands.The saliva secretion decrease and saliva composition change may lead to a series of complications,such as rampant caries,swallowing and and vocal difficulties,oral mucosal lesions,resulting in a decline in the quality of patients’ lives.Although many researchers have done a lot of research on the mechanism of radiation salivary gland injury,there is still no definite conclusion and the effective methods found to prevent and treat radiation salivary gland injury.Protein kinase c(PKC)is widely found in nerve cells,smooth muscle cells and salivary gland cells.It can regulate the open of large-Conductance Ca2+-Activated K+ Channels(BK)and inhibit K+ transmembrane transport.It also plays an important role in cell apoptosis.In this study,the submandibular glands of rats and salivary gland cells were irradiated with a single dose of 15Gy respectively.Then we observe the protein expression level and the distribution in rat submandibular gland tissue and salivary gland cells at different time after irradiation.Methods:48 male Wistar rats,200 g to 250 g,were randomly divided into two kinds of groups,the control groups and irradiation groups.The submandibular gland region of the irradiation group rats was irradiated with one single dose of 15Gy X-ray after rats being anethetized,while the rats of control groups were anesthetized without irradiation.Total body weigh and submandibular gland weights were determined after irradiation.All rats were sacrificed at 1,3 and 30 days after irradiation and the bilateral submandibular glands were removed.One side of the submandibular glands was fixed immediately in 4%paraformaldehyde.After rinsing,the specimens were dehydrated in gradient ethanol solutions and embedded in paraffin.The histological changes of the submandibular glands after irradiation were observed under electron microscope after HE staining.The expression of PKC in submandibular gland of rats after irradiation was determined by immunohistochemical staining.The other side of submandibular gland tissue was ground on ice with lysate,then the supernatant was extracted and the expression level of PKC in submandibular gland tissue of each group was determined by Western Blot.HSG(human salivary gland)cells were cultured in glass-bottom dishes and irradiated with a single dose of 15Gy x-ray.The cells were fixed with 4%paraformaldehyde after 24 hours and 48 hours.The localization of PKC in salivary gland cells was observed by immunohistochemical staining.Results:(1)Immunostaining of PKC was mainly detected in acinar cells of rats submandibular glands.On the 1st,3rd,30th day after irradiation,the expression level of PKC in the acinar cells of submandibular glands in the irradiation groups showed an increasing trend,which were higher than that in the control groups after irradiation,respectively.(2)PKC is mainly expressed in cytoplasm in human salivary gland cell line after irradiated,and the expression level of PKC was higher after irradiation.Conclusion:PKC was mainly distributed in the cytoplasm of rat submandibular gland cells and human salivary gland cell line.The dyfunction of salivary gland after irradiated may be related to the increase expression of PKC.The increased expression of PKC may inhibite BK channel opening and promote cell apoptosis. |
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