Degenerative Changes Of Auditory Function And Age-related Expression Of NGFR TrkA In The Cochlea Of The Senescence Accelerated Mouse, Transfection Of Human-β NGF Plasmid Vectors Into BMSCs Of Mouse

Objectiv: The purpose of the study was to analyze age-related hearing lossin the senescence accelerated mouse and the expression of nerve growth factorreceptor TrkA (NGFR TrkA) protein in the cochlea of the senescence acceleratedmouse of different mouths, and transfection of human-β-NGF plasmid vectorsinto BMSCs of mouse to provide the theoretical basis for the molecularmechanism and gene therapy of presbycusis.Methods: The3,5,7th month as analyzing time points were chose. The auditorythresholds and the expression of NGFR TrkA protein in the cochlea were studiedin the senescence accelerated mouse/prone8(SAMP8) at3,5,7months. The auditory thresholds was monitored by auditory brainstem respons (ABR). Theage-related expression of NGFR TrkA protein was analyzed by the optical densityof immunohistochemical staining. Put the BMSCs of mouse into four groups: A isthe group of recombinant plasmid encapsulated in liposomes, B is the liposometransfection group, C is the group of liposome parcel empty plasmid transfection,D is negative control with10%fetal bovine serum of DMEM/F12nutrientsolution. According to the Plasmid Extraction Kit, the recombinant plasmidcontaining NGF was extracted from Escherichia coli. The extracted plasmidwhich was identificated by restriction endonuclease HindIII and XhoI doubleenzyme digestion was transfected into bone marrow stromal cells. The BMSCswere fixed by4%paraformaldehyde after48hours of the transfection, then theexpression of NGF protein in cells were analyzed by the optical density ofimmunohistochemical staining.Results:1. The ABR of SAMP8mouse of each group was:3months SAMP8mouse: Left ear31.667±2.582, right ear32.500±2.739;5months SAMP8mouse:Left ear54.167±2.041, right ear53.333±2.582;7months SAMP8mouse: Left ear58.333±2.582, right ear58.333±2.582. Compared with the SAMP8mouse of3months, the ABR in SAMP8mouse of5months showed a remarkableincreasement (P＜0.05). Compared with the SAMP8mouse of5months, the ABRin SAMP8mouse of7months showed a remarkable increasement (P＜0.05).2. NGFR TrkA expression was observed in the cochlea of mouse in each group.The major expression is in cytoplasm and nucleus of spiral ganglion cells and hair cells. The optical density (OD) of NGFR TrkA immunohistochemical staining inthe mouse cochlea spiral ganglion cells of each group was:3months SAMP8mouse:0.5262±0.0148;5months SAMP8mouse:0.4758±0.0129;7monthsSAMP8mouse:0.4505±0.0148. Compared with the SAMP8mouse of3months,the optical density (OD) of NGFR TrkA immunohistochemical staining inSAMP8mouse of5months showed a remarkable significant reduction (P＜0.05).Compared with the SAMP8mouse of5months, the optical density (OD) ofNGFR TrkA immunohistochemical staining in SAMP8mouse of7monthsshowed a remarkable significant reduction (P＜0.05). The optical density (OD) ofNGFR TrkA immunohistochemical staining in the mouse cochlea hair cells ofeach group was:3months SAMP8mouse:0.6311±0.0200;5months SAMP8mouse:0.5487±0.0179;7months SAMP8mouse:0.5183±0.0187. Comparedwith the SAMP8mouse of3months, the optical density (OD) of NGFR TrkAimmunohistochemical staining in SAMP8mouse of5months showed aremarkable significant reduction (P＜0.05). Compared with the SAMP8mouse of5months, the optical density (OD) of NGFR TrkA immunohistochemical stainingin SAMP8mouse of7months showed a remarkable significant reduction (P＜0.05).3. The brown or yellow granules were discovered in BMSCs of each group.Compared with other groups, the optical density (OD) of NGFimmunohistochemical staining in group A showed a remarkable significantincreasement (P＜0.05). Conclusion: The expression level of NGFR TrkA protein decreases when theSAMP8develop a progressive hearing loss. This indicates that NGFR TrkAprotein probably has relationship with maintaining functional status of the cochlea.The NGF plasmid vectors can be successfully transferred into BMSCs of mousecells by Lipofectamine TM2000and the transfected NGF gene can also expressin BMSCs. The success of these studies can provide the premise and thefoundation for the next experiment of cochlear implant with β-NGF modifiedBMSCs in mouse.