Effect Of Hypoxia On Autophagy And Apoptosis Of Goat Temporomandibular Joint Disc Cells Before And After Serum Deprivation

Part 1:Effects of serum deprivation on apoptosis and autophagy in goat temporomandibular joint disc cellsObjective:To simulate the low blood supply state of the physiological TMJ disc,and to explore the relationship between autophagy and apoptosis of the disc cells of the temporomandibular joint by detecting the changes of autophagy and apoptosis in the TMJ disc cells of goats after serum deprivation.Methods:The TMJ disc cells of goat in 3-6 months old were isolated and cultured,and P2 generation cells were taken for experiment.The morphological changes of cells before and after serum deprivation were observed under the inverted microscope.The morphological changes of cells were observed by Hematoxylin-Eosin staining.Apoptosis was observed by fluorescence staining Hoechst 33258 and monodansylcadaverine fluorescence staining was used to detect the lysosomal activity of the cells.MTT absorbance values were measured and growth curves were drawn.The autophagy rate and apoptosis rate of cells were detected by flow cytometry.The changes of autophagy related genes,Beclinl and LC3? were detected by real time RT-PCR.Finally,the autophagy inhibitor 3-methyladenine was added to explore the relationship between autophagy and apoptosis by observing cell apoptosis.Results:After the serum deprivation,the TMJ disc cells of the goat were shrunken and formed from polygons.The results of Hoechst 33258 staining showed that there was an obvious apoptosis in cells after serum deprivation.monodansylcadaverine staining showed that serum deprivation could induce autophagy.The results of flow cytometry showed that 12h cells showed obvious autophagy after serum deprivation,36h reached its peak and then gradually decreased.The PCR results of autophagy related genes Beclinl and LC3-? were consistent with the results of flow cytometry.The flow pattern found that after 24h,the cell apoptosis was obvious after the serum deprivation,and the apoptosis rate increased gradually with time.After adding the autophagy inhibitor 3-MA,the apoptosis rate was further increased,which was in accordance with the results of MTT.Conclusions:The level of apoptosis and autophagy in normal culture are low.Serum deprivation can induce apoptosis and autophagy in goat TMJ disc cells.After the addition of autophagy inhibitor 3-MA,the apoptosis rate of cells increased farther,indicating that autophagy was beneficial to cell survival.Part 2:Effect of hypoxia on apoptosis and autophagy in goat TMJ disc cells.Objective:To study the effects of hypoxia on autophagy and apoptosis of TMJ disc cells in goat and its role in the survival of temporomandibular joint disc cells.Methods:The TMJ disc cells of goat in 3-6 months old were isolated and cultured,and P2 generation cells were taken for experiment.The rate of autophagy and apoptosis was detected by flow cytometry.The expression of autophagy related genes,Beclin1 and LC3-?,was detected by PCR.Results:Both serum and serum deprivation decreased the autophagy rate and apoptosis rate under hypoxia condition compared with normoxic condition.After hypoxia deprivation,autophagy induced by serum deprivation was inhibited,and the apoptosis rate of cells decreased.Conclusions:Hypoxia can reduce the apoptosis and autophagy of TMJ disc cells in goat.Hypoxia is beneficial to the survival of articular disc cells and the protection of cells.