Fundamental Research Of Math-1/NGF-b Gene Treating The Noise-induced Hearing Loss

Objective:Ttranscription factor Math-1/NGF-b gene inducing the differentiation ofsupporting cells to hair cells, and it can be the treatment of noise induced hearing loss.1)In this study,we use the stationary noise of110dB on adult guinea pigs to manufacturedeafness model;2) we build adenovirus in the experiment, respectively, carringMath-1/NGF-b Math-NGF-b, and empty virus to transfect deaf guinea pigs.Then weobserved and compared the treatment effect;3) We get observation of the three treatmentresults and comparison with normal hair cells from the morphological observation by usingthe electron microscope to discuss whether there is the possibility that usingMath-1/NGF-b gene therapy to cure noise induced hearing loss.Methods: In this experiment we use normal adult guinea pigs, weighing between250-300G to manufacture noise deafness model; experimental group transfected withMath-1/NGF-b，Math-1, NGF-b adenovirus control group transfected with empty virus.First, in the steady-state noise environment, manufacturing deafness mouse model, and theABR test. Secondly, building3overexpressing vector carrying Math-1/NGF-b, Math-1,NGF-b three sequences, and its virus titer reach1012/ml. Then in the experimental group,guinea pig right ear transfected with Math-1/NGF-b, Math-1, NGF-b, while the controlgroup transfected with empty virus,at the same time we conduct the same operation in theguinea pig left ear but no transfection. Last, respectively, in the first four days, one week,two weeks, ABR test and scanning electron microscopy test and RT-PCR test andimmunohistochemical examination were be done to observe the the guinea pig hearingrecovery, and the morphological appearance of hair cells and the difference between thethree different treatments.Results:1. After uncontinuously exposed under noise for24hours, we successfullymanufactured deafness model of guinea pigs, which hearing threshold shift reached45-60dB after the ABR test.2.Scanning electron microscope suggest that the first back toback4of the Guinea Pig cochlea hair cells have disappear, atrophy, fusion to varyingdegrees.3. The amplification curve of RT-PCR sugested the Math-1/NGF-b, Math-1,NGF-b be amplified well. Virus titer: three viral vector titers reached1012/ml or more. Virus in vitro transfection experiments suggested that the virus infected and expressed well.6.3days after transfection, ABR test prompt hearing has been restored to about10dB.7.1week after transfection ABR test prompt hearing recovery about25dB.8. two weeks aftertransfection, the ABR test prompt hearing recovery reached a peak of about30dB.9.Electron microscope show, injury in transfecting Math-1/NGF-b mice cochlear hair cellsignificantly relieve compared with the control group. Injury in transfecting Math-1/NGF-bmice cochlear hair cell relieve better than Math-1used alone or NGF-b used along.Conclusion:1. Noise-induced hearing loss in guinea pig model is successful, in the firsteight hours after noise exposure, the instant ABR test show guinea pig’s hearing hasdecreased (15±2dB), but some recovery can be achieved in one day,they can be normal in3days. But after a total time of24hours’ uncontinuous noise exposure guinea pigs hearingthreshold shift can reach45-60dB, and ABR test show its hearing can not be restored in the3days.2. Under scanning electron microscopic examination, each back of hair cellsobviously injured.With the third, fourth back,the injury is most obvious, a first, a secondback, followed.2.Successfully constructing adenovirus which carring Math-1/NGF-bMath-1, NGF-b and connecting EGFP.In vitro the infection experiments was successful.3.We success to transfect the adenovirus vector of over-expression in noise-inducedhearing loss guinea pigs throuh the round window membrane,.Guinea pig’s hearingdamage is small (15dB).4.In the treatment group intervention, ABR prompted guinea pighearing recovery.5. Electron microscope test show that under therapeutic intervention, thetreatment group guinea pig hair cells was significantly lighter than the control group.6.Math-1expressing in the injury hair cells is a key factor in the regeneration of haircells,and the treatment seem to be more efficient when Math-1was together with NGF-b.7.1week after transfection, ABR test shows deafness in guinea pigs begin to restore andthe recovery reach to peak in two weeks.8.Under electron microscopy we found theobvious ladder like newborn hair cells.9.Immunohistochemistry prompted, two weeksafter transfection, MyoVIIa positive cells appearing, suggesting the growth of newbornhair cell.