The Study Of Homing And Hearing Effect In SD Rats After Noise Exposure With Intravenous Transplantation Of BMSCs

Noise induced hearing loss is a kind of sensorineural deafness caused by noise exposure for a long time,usually leads to the cochlea hair cells injury or auditory neuropathy.Mammalian hair cells cannot spontaneously regenerate after injury,and it will cause permanent hearing loss.Application of stem cell transplantation for cell replacement therapy is a major treatment strategies to restore the hearing.Bone marrow mesenchymal stem cells(BMSCs)have the characteristics of self-repair,self-renewal,high proliferation and multiple differentiation potential.Study at home and abroad,BMSCs have potential of promoting angiogenesis and various cells differentiation in the cochlea,has opened up a new train of thought for the treatment of noise deafness.This topic was to study the inner ear homing phenomenon and the changes of hearing by transvenous implanting BMSCs to the normal and noise deafness rat.This study will be divided into three parts.Part Ⅰ:Isolation,culture,identification and marking of SD rat BMSCsObjective: To establish a culture system of SD rat BMSCs,and to identify the cells effectively and mark stability to lay the foundation for using BMSCs vein transplantation in treatment of noise deafness experiments.Methods: We Isolated and cultured rat BMSCs through the whole bone marrow adherent method and stable subcultured to P3 or P4.Flow cytometric method was used to identify the the expression of four markers on the cell surface,such as CD29,CD90,CD45,CD11 b.And BMSCs stained by red Pkh26 which is a red fluorescent marker.Results: After the removal of suspended cells through total medium change on24 h,we can see that part of cells adhere to and flat out on the bottom of the bottle.Cells are fusiform,polygonal and irregular.Cells subcultured to P3 or P4,it showed uniform distribution and uniform long shuttle as fibroblast-like cells.The cells expressed CD29 positive rate is 91.15%;CD90 positive rate was 93.12%;CD45positive rate was 1.40%;CD11b positive rate was 0.83%.The BMSCs dyed by Pkh26 appeared to be red fluorescence In the inverted fluorescence microscope.Dye evenly distributed in the surface of cell membrane.Cell outline clear and the mark rate was100%.Conclusion: The whole bone marrow adherent culture method can establish stable BMSCs cell culture system.Through the cell morphology and fluid cytology identification,cells were identified as BMSCs which we need.It can be stable tagged by Pkh26 fluorescent.Part Ⅱ:Establishment of animal model of noise-induced hearing loss and study of hearing restorationObjective: To establish an animal model of noise-induced hearing loss,and to transplant BMSCs through tail vein,and to observe the change of ABR hearing threshold to provides the basis for the study of auditory function improvement after BMSCs transplantation.Methods: Experimental animals were divided into three groups A,B,C.Group B and group C were exposed to center frequency of 4000 Hz narrow band white noise at 120 d B SPL for 9 hours continuously.Group A and group C were accept cell transplantation of BMSCs labelled by Pkh26 through the rat tail vein path.ABR thresholds of each group rats were recorded before and after noise stimulation and at the 2nd,4th,and 6th week after transplantation.The changes of auditory function was comparatively observed between groups rats.Results: Rats in group A threshold to an average of 3.44±3.52 d B n HL;Group B rats normal threshold was 3.13±3.10 d B n HL,2d after noise stimulation threshold was49.06±9.35 d B n HL,2w,4w,6w were 46.88±11.53 d B n HL;Group C rats normal threshold was 2.19±2.56 d B n HL,2d after noise stimulation threshold was50.31±10.72 d B n HL,2w was 45.31±10.72 d B n HL,4w was 40.00±8.56 d B n HL,6w was 32.81±9.12 d B n HL.After statistics analysis,there were no statistical difference between group B and group C at 2w after transplantation(P > 0.05).4w,6w after transplantation,the difference between group B and group C were statistically significant(P < 0.05).Conclusion: 1.The noise stimulation can make rats produce permanent hearing threshold shift and have established a stable animal model of noise-induced hearing loss.2.4w and 6w after transplantation,the ABR thresholds of rats that have accepted cell transplantation of BMSCs is reduced.There has a certain degree of auditory function improvement.Part Ⅲ:The homing phenomenon of BMSCs in the inner ear and the study of hair cells morphological changesObjective: To track and verify BMSCs cell homing function and its distribution in the inner ear,and to observe the morphology and number changes of hair cells to confirm hair cells repair effect of noise-induced hearing loss after BMSCs transplantation.Methods: 1.All the animals were to be put to death at 6w after cell transplantation.Making cochlear organizations frozen sections and immunofluorescence staining and to observe the survival and distribution of BMSCs in the cochlea under the laser scanning confocal microscope.2.By making cochlea basilar membrane stretched preparation and silver nitrate staining,comparatively observed the morphology and number changes of hair cells under inverted phase contrast microscope.Results: 1.The cochlea of group C have found stem cell survival,and group A have not seen cell distribution or only a small number of cells survived.The BMSCs are mainly distributed in the spiral ligament,stria vascularis,basement membrane,bony spiral lamina and more obviously in spiral ganglion region.2.Cochlea hair cells of group A rats have no significant loss,neatly arranged and the cilia are clear.There are no phenomenon of messy,lodging and falls off.Group B appear different degree of apparent lacking.The hair cells disordered arrangement,damaged and the size of cell is different.There are phenomenon of lodging,disorganized and fusion disappearance.Hair cells in the group C rat cochlea appear arranged neatly and cell loss is less.In comparison,morphological abnormalities such as loss of hair cells,ciliated lodging and disordered arrangement of group C is mild than group B.Conclusion: 1.Venous transplantation of BMSCs can be migrated to the inner ear of noise-induced hearing loss rat by homing function and distributed in different level in the spiral ganglion area,basement membrane,spiral ligaments and stria vascularis.2.Strong noise stimulation can cause the damage and the lack of hair cells,mainly concentrated in outer hair cells;BMSCs transplantation can promote the regeneration and repair of hair cells.And it could relieve hair cell damage.