Construction And Application Of Prestin Overexpressing Viral Vector In Outer Hair Cells Of The Cochlea

Noise-induced hearing loss(NIHL)is a sensorineural hearing loss caused by long-term exposure to a noisy environment.It is one of the most common occupational diseases.There is currently no effective treatment for NIHL.The root cause is cochlea.Outer hair cells(OHCs)are irreversibly dead due to noise damage.Prestin protein is an important sensory function protein specific to cochlea OHCs.It is the molecular basis of OHCs electrical movement and cochlear amplification effect.The compensatory increase makes up for hearing loss.ObjectiveThe recombinant Prestin adeno-associated virus-2(AAV-2)vector was constructed by cochlear hair cells to provide tools for the study of cochlear Prestin protein regulation.A new non-invasive inner ear gene transfection system was constructed.MethodsThe guinea pig Prestin gene was used as template for PCR amplification and extraction.AAV-2-Prestin vector was constructed by genetic engineering technology.The expression level of Prestin was detected by Western blot 1d,3d and 5d after transfection of cultured cochlear hair cells.The constructed recombinant Prestin adeno-associated virus type 2 vector was mixed with the in situ gel and transfected into the inner ear of guinea pigs through a round window method of tympanic puncture and tympanic administration.The cochlea tissue virus transfection distribution was observed by immunofluorescence method.Immunofluorescence was used to detect the expression and distribution of green fluorescent protein and Prestin,and WB was used to detect the expression of Prestin in the basement membrane.ResultsAfter constructing pAAV-EGFP-Prestin proviral plasmid to transfect HEK-293 T cells,weak green fluorescent protein expression was seen on day 1 and green fluorescent protein expression peaked on day 3;after AAV-2-Prestin virus was transfected into HEI-OC1 cells,The expression levels of Prestin protein were significantly increased in different groups at different transfection times(F = 414.755,P﹤0.001).The expression of Prestin protein was significantly higher in groups 3d and 5d than in group 1d(P﹤0.001),and the expression of Prestin protein in group 3d was significantly higher than that in group 5d.(P﹤0.001).Guinea pig cochlea began to express green fluorescent protein on the 7th day after administration.The green fluorescent protein was distributed in the cochlear vascular lines,vestibular membrane,basement membrane and Corti organs.Basement membrane plaques showed the expression of green fluorescent protein in outer hair cells,which was not seen inside.In hair cells,the expression level of green fluorescent protein increased significantly at 14 days;the expression level of Prestin protein in outer hair cells began to increase on day 7 after cochlea administration,and the increase was more significant after 14 days(P﹤0.05).No expression of Prestin protein was found in inner hair cells..WB detected the expression of Prestin protein in the basement membrane of each group,and the results of analysis of variance showed statistical differences(F= 3644.6729,P﹤0.05).Comparison between the groups found that there was no significant difference between the control group and the 4-day group(P﹥0.05).The expression level of Prestin protein in the 7-day group was significantly higher than that in the control group and the 4-day group(P﹤0.05),and the expression of Prestin protein in the 14-day group was significantly higher than the other groups(P﹤0.05).ConclusionRecombinant adeno-associated virus-2-Prestin vector was successfully constructed with good transfection efficiency and persistence.A new non-invasive inner ear gene transfection system was successfully constructed.