NSC Combined With OEC Transplantation On Apoptosis Of Cochlear Cells In SNHL Model Rats

Objective:To observing the effects of NSCs and OECs transplantation on the expression of neurotrophic factor,apoptosis and antiapoptotic protein in the cochlea of SNHL model rats,and to explore the protective mechanism of cell transplantation on the cochlear cells injured by SNHL.Methods:(1)Cell culture: Fetal mice were selected as the tissue source of transplanted cells to culture,observe and identify NSCs and OECs.(2)Animal modeling: Gentamicin was used to model rats with SNHL,and the rats that meet the requirements were randomly divided into experimental group and control group,with 10 rats in each group.The experimental group was transplanted with NSCs and OECs,and the control group was replaced with artificial external lymph.(3)Cell transplantation: NSC and OEC were injected into the cochlea through a round window.The weight changes of rats in each group were recorded after operation.ABR was used to detect the changes of hearing threshold and auditory otomotor reflex,and samples were taken for detection.(4)He staining of cochlear sections.(5)NSC and OEC were detected by immunofluorescence staining.(6)TUNEL method was used to detect the number of apoptotic cells and MOD.(7)Immunohistochemistry was used to detect neurotrophic factors and apoptotic immunopositive cells.(8)Western blot was used to detect BDNF,NT-3 and Bax,Bcl-2,caspase-33 protein expression.Results:(1)NSCs and OECs were positive for Nestin and p75 NTR respectively.The cells with high purity,good growth and condition were obtained.(2)The threshold of auricular reflex was improved in the experimental group,the change of auricular jitter was enhanced,but not in the control group.(3)compared with the control group,the ABR threshold of the experimental group did not change significantly(P > 0.05);compared with the control group,the ABR threshold of the experimental group increased(P < 0.05).(4)The results of immunofluorescence staining showed that the NSCs labeled differentiated cells in the experimental group could express Nestin,the OECs labeled differentiated cells could express p75 NTR,but no fluorescence labeled cells were found in the control group.(5)He staining results: in the control group,degeneration and necrosis of inner and outer hair cells,partial necrosis of SGCs,atrophy of nucleus,obvious damage,vacuole like degeneration of cells,decreased density,loose and disordered arrangement,and decreased overall pathological damage in the experimental group.(6)TUNEL positive cell number test results: compared with the control group,the number of TUNEL positive cells in the experimental group decreased significantly(P < 0.05),the proportion of apoptosis index decreased,and the mod value decreased(P < 0.05).(7)Results of immunohistochemistry: compared with the control group,the number of BDNF and NT-3 positive cells and IOD in the experimental group increased significantly(P < 0.05),while the number of Bcl-2 positive cells and IOD in the anti apoptotic factor increased,the number of Bax,caspase-3 positive cells and IOD in the pro apoptotic factor decreased(P < 0.05).(8)Western blot results: compared with the control group,the expression of BDNF,NT-3,Bcl-2 protein in the cochlear tissue samples of the experimental group increased,and the expression of Bax and Caspase-3 protein decreased(P < 0.05).Conclusion:(1)NSCs combined with OECs transplantation can protect the cochlea of SNHL rats and reduce the damage of cochlear cells.(2)NSCs combined with OECs transplantation may be involved in the protection of cochlear cells by up regulating the expression of neurotrophic factors.(3)NSCs combined with OECs transplantation may be involved in theprotection mechanism of SNHL neural cell apoptosis by up regulating Bcl-2,down regulating Caspase-3 and Bax expression pathway.