Molecular Mechanism Of Ketamine Induced Granulosa Cell Apoptosis Through The Hippo Pathway

OBJECTIVE:In this study,human ovarian granulosa cells were identified by in vitro culture,and the study system of human ovarian granulosa cells was established.To investigate the effect of ketamine on granulosa cells by morphological changes of DNA apoptosis and DNA fragmentation.At the same time,the expression of YAP、TAZ in Hippo signaling pathway was studied,and the molecular mechanism of apoptosis induced by ketamine was discussed.Method:1.The follicular fluid was collected by lysis +precoll+trypsin digestion..The granulosa cells were identified by immunohistochemical staining of folliclein and the cell proliferation was detected by MTT assay.2.The morphological changes of cells were observed by light microscopy,TUNEL assay and flow cytometry were used to observe the morphological changes of cells in DMEM / F12 medium treated with ketamine at 0,0.3125 g / l,0.625 g / l and 1.25 g / The apoptotic status of each group was detected by Annexin V-FITC / PI double staining.3.The mRNA and protein of granule cells were collected and extracted from ketamine cells.Real-time PCR was used to detect the mRNA content of YAP and TAZ in Hippo signaling pathway.The content of YAP and TAZ protein were detected by Western Blot.Result:1.The cell isolated and purified from the Follicular fluid are mostly granulosa cells,and cells grow well.Folliclein immunohistochemical staining in the positive reaction of cells are greater than 90%.2.ketamine induced apoptosis of human ovarian granulosa cells.With the increase of ketamine concentration,the apoptotic cells increased gradually.TUNEL assay and flow cytometry Annexin are showed that the number of apoptotic cells increased with the increase of ketamine concentration.The results of V-FITC / PI double staining showed that the number of apoptotic cells was significantly increased when the concentration of ketamine was 0.625 g / l and 1.25 g / l,and the apoptotic rate increased with the increase of ketamine concentration.(P <0.05).(1)The expression of YAP and TAZ mRNA in granulosa cells were significantly different from those in granulosa cells induced by ketamine(P <0.05).The expression of YAP and TAZ mRNA in granulosa cells were significantly different(3)The expression of YAP and TAZ protein in granulosa cells were significantly different from those in normal ovarian granulosa cells(P <0.05),and the expression of YAP and TAZ mRNA was significantly different from that of normal ovarian granulosa cells Group YAP,TAZ protein expression decreased,and normal ovarian granulosa cell expression levels were significantly different.Conclusion:1.Using lysis method +precoll+trypsin method can be obtained high purity,stable growth of human ovarian granulosa cells.2.ketamine through the induction of human ovarian granule cell apoptosis increased,the cell morphology occurred apoptosis-related changes,cell shrinkage,intracellular DNA fragmentation,the number of apoptosis with the increase in ketamine concentration increased.3.The expression of YAP 、 TAZ decreased,the cell proliferation was decreased and the apoptosis was increased,suggesting that the Hippo signaling pathway may be an important signaling pathway for the mechanism of apoptosis of granulosa cells induced by ketamine.