The Study Of The Inhibitory Effect Of SiRNA-mediated Myelin Protein Zero Gene Expression In The Rat's Cochlea

OBJECTIVE:An effective virus vector was selected.And the auditory neuropathy animal model was established to study the inhibitory effect of siRNA-mediated myelin protein zero gene expression in the rat's cochlea.Then to investigate the change of the pathological regeneration of the auditory nerve's demyelination.METHODS:1.After many kinds of virus were injected in the rat's cochlea,the effect of the transfection of the adenovirus carrier to the internal ear cell was inspected by the fluorescence microscope and the light microscope.According to result of the effect of the transfection,the most effective virus carrier was designated.Then according to principle of RNA interference technique,adenovirus carrier was injected through round window from the route of below and behind of the ear.2.The perioperatively auditory brainstem response(ABR)was examined by Intelligent Hearing System Smart EP2.22 system,and at the same time the measured numerical result was statisticsed and analysised.Then the change situation of the incubation period and responds threshold between the preoperative and the 1,3,7,14 days after the operation were observed and compared separately.3.The clean level SPF Wistar male rats were divided into seven groups stochastically:Normal control group,blank control group,negative control group,1 day's group,3 day's group,7 day's group and 14 day's group.All animals take the surgery ear by the right ear,the left ear(opposite side)takes the internal comparison. After the viral carrier was introducted,the rat's cochlea spiral ganglion organization's protein and RNA of every group was withdrawed by the Trizol Reagent.Then the expression situation of the MPZ protein of the internal ear organizes was examined through Western Blot,and the expression situation of the MPZ mRNA of the internal ear organizes was examined by semi-quantitative RT-PCR.RESULTS:The effect of the transfection of the adenovirns Ad5 in the rat cochlea was the best.So it was decided to the viral carrier.The first day after the operation,various waves' incubation period of ABR were all extended,while the respond threshold were reduced.The cochlea auditory nerve myelinic membrane protein zero protein and mRNA had all slightly reduced at the first day,then all maintained the lower level until the 14th days.CONCLUSION:The Ad5-siRNA carrier could effectively knockout the myelinic protein zero(MPZ)gene in vivo to achieve the purpose of reducing the content of the MPZ protein.That induced the auditory nerve's demyelination.The auditory nerve's demyelination lead to the extending of the various waves' incubation period of ABR and the reducing of theâ…¢wave's respond threshold.That was similar with the performance of the auditory neuropathy in the clinical.Some new foundation about the research that studied the relationship between the auditory neuropathy and the auditory nerve's demyelination leaded by quiescence of MPZ gene was provided.