| In the hearing system of mammals,cochlear hair cells,as the terminal cells of sensory nerves,can convert sound stimulus into electrical signals and transmit it to nerve centre by means of auditory nerve so as to keep normal auditory function.Various factors such as excessive sound stimulus,aging,ototoxic drugs, infection and autoimmune diseases can lead to the irreversible damages of the cochlear hair cells,and thus cause sensorineural deafness permanently.Cochlear gene therapy,which is a reasonable method for rebuilding Corti's structure to recover auditory functions,can be carried out by making use of the key genes which can convert non-sensory epithelial cells into hair cells.It was newly reported that the Atohlgenes overexpressed in the inner ears of the adult deaf guinea pigs caused by aminoglycoside antibiotics can induce hair cell regeneration,moreover,part auditory functions recovery.On the facets of the mechanism of injury and the pathological changes of cochlea,noise deafness and drug deafness are different completely.At present, studies showed that the damages of inner ears caused by pulse noises were mainly mechanical injuries which were followed by and vascular and metabolic injuries. Wherein,the OHC was affected by the mechanical injuries firstly and the effect on IHC occurred later.Thanks to the locations support cells and nerve fibers lain in were deep,therefore,the injuries of them were not serious.All these phenomenons seem to indicate that the status of the remnant support cells and nerve fibers of the hair cells damaged by noises was better than that of the drug deafness.At present,whether the Hath1 therapy on the basis of this can realize hair cells regeneration and auditory recovery or not has not been reported.In this study,we imported Hath1 genes into the cochlea of the deaf guinea pigs caused by noises by making use of adenovirus vectors,and studied primarily the efficiency of gene transfer and the biological effects of gene therapy by means of auditory function detections and morphological means.The study included three parts as follows:Part one:Constructing the model of deaf guinea pigs by means of high strength pulse noiseThis part aims at to construct the model of deaf guinea pigs caused by noises, which will be used in the experimental study of the regeneration of cochlea hair cells.The guinea pigs were exposed with normal hearing in the pulse noises 200 times continuously.One week later,we detect the auditory functions of guinea pigs by means of auditory brainstem response(ABR),Compound Action Potential(CAP)and Cochlear Microphonic(CM).The results showed that the waveforms of ABR can not be educed or the threshold was very high(≥95 dB SPL)when we use maximum output stimulus in the frequency range of 4KHz,8KHz,16KHz and 20KHz;as such CAP in the frequency range of 1KHz,2KHz,4KHz and 8KHz;while the CM can not be educed in the frequency range of 1KHz,2KHz,4KHz,8KHz and 16KHz.The thresholds of the ABR and the CAP were unable to resume and the CM can not be educed after four and eight week's exposure.It was showed by SEM,cochlear stretched preparations and frozen sections that a majority of inner and outer fair cells lost,the support cells were intact,and a majority of afferent and efferent nerve fibers in the cochlea survived all right.Hair cell count showed that among the guinea pigs whose threshold of ABR in each frequency were no less than 95dB SPL,the loss rate of inner and outer hair cells reached to 91.4%and 97.2%respectively.The results mentioned above showed that it was the continuous exposure in the pulse noises with high strength led to guinea pigs to develop very serious sensorineural deafness;the loss of IHC and OHC were more than 90%,but the supporting cells,afferent and efferent nerve fibers in the cochlea survived all right and the basic frame of Corti's were intact.Moreover,the damages of auditory function and hair cells were irreversible.Therefore,the animal models adopted in the study were the idea models to study the regeneration of hair cells.Part two:The observations of experiment that transfecting the cochlea of guinea pigs with the genes carried by adenovirus.The precondition of gene therapy is to transfect target cells with target genes effectively.This part aims to seek for effective methods to transfer Hath1 genes accurately into the remnant support cells after noise injury.The guinea pigs with normal hearing transferred Ad-Hath1-EGFP/Ad-EGFP by means of approaches of intact round window membrane and perforating on the cochlea scala tympani.The results showed that,in the facet of transduction efficiency,the latter means was obviously excellent than the anterior one;however, in the facet of protecting auditory functions,the anterior means showed advantages.In the cochlea of normal animals,the target genes expressed mainly in the range of support cells and positive cells almost did not expressed in the range of inner and outer hair cells;except for the target genes can be expressed in support genes,it was also expressed in the spiral ganglion cells,mesothelial cells of scala tympani and vestibular bands. In order to ensure a high efficiency in gene transfection,we perforated on the cochlea scala tympani to transfer Ad-Hath1-EGFP into the cochlea which was damaged by noise.Fourteen days later,the remnant support cells in the cochlea expressed Hath1 widely and the transfection efficiency of it was higher than that of normal animals,and the positive cells derived from second turn almost reached to 100%..The expression sites of genes were same with that of normal animals group but the expression at the spiral ligament was stronger than that of normal animals group.The results above showed that the genes were transferred into cochlea from intact round window did not lead to cochlea injuries and the auditory functions can be protected better,however,the transduction success rate was relatively low (50%).Also importing the genes by means of perforating on the cochlea scala tympani had a high transduction success rate(92%),but it damaged the places which were adjacent with perforated position on the cochlea and therefore the high frequency hearing loss lightly.After the guinea pigs were damaged by noise, it had a high transduction success rate(80%)and the Hath1 genes can be expressed in the support cells,spiral ganglion cells,spiral ligament,mesothelial cells of scala tympani and vestibular.Moreover,it can be expressed continuously and smoothly after two weeks of gene transduction.Part three:The observations of in vivo experiment that overexpression Hath1 genes into cochlea to treat noise deafnessThe objectives of this part are to observe the biological effects of Hath1 genes on the cochlear of the guinea pigs with noise deafness.In addition,on the basis of the two parts above,we want to study primarily the regeneration,the repair and the auditory recovery of cochlear hair cells of adult mammals which were damaged by noise.Forty-five guinea pigs with NIHL were divided into three groups at random namely Ad-Hath1-EGFP group(30),Ad-EGFP group(5)and blank group(10), and to detect auditory functions(ABR,CAP,CM)after four and eight weeks of genes transduction.At the same time,various morphologic methods were adopted by us to observe the changes in the cochlea.The result showed that,after four weeks of gene transduction,the ABR threshold of each frequency of Hath1 treatment group,which averagely reached to 85dB SPL,was all lower than that of control ears(right ears),the Ad-EGFP group and the blank group,and the high frequency hearing was better than that of low frequency.CAP threshold at the frequency of 4kHz was 75dB SPL and it was 85 dB SPL at the frequency of 1,2,8 and 16kHz.CM could be educed at the frequency of 1KHz and the lowest threshold of it was 80dB SPL.It could not be educed at the frequency of 2KHz,4KHz and 8KHz and the input and output curves of CM were linear.Observed under the SEM,we found that,after four weeks of Hath1 gene transduction,the inner hair cells generated largely but the outer hair cells only had an island-shaped or flake-shaped resumption.The intact cochlea stretched preparations Pholloidin,Hoechst and the immunohistochemical results of NF showed that the nucleus of the inner hair cells arranged orderly in the range of IHC cells,but the nucleus of the outer hair cells were sporadic in the range of OHC cells and a great of cell nucleus of the support cells could be found outside the OHC range.In addition,afferent and efferent·nerve fibers showed distinctly.After eight weeks of gene transduction,the ABR threshold of each frequency of Hath1 treatment group,which was resumed and reached averagely to 75 dB SPL when it compared with the threshold at the fourth week,was all lower than that of control ears(right ears),the Ad-EGFP group and the blank group.CAP threshold at the frequency of 8kHz was 75dB SPL and it was 80 dB SPL at the frequency of 1,2,4 and 16kHz.CM could be educed at the frequency of 1kHz and 2kHz and the lowest threshold of it was 70dB SPL.But it could not be educed at the frequency of 4kHz and 8kHz while the input and output curves of CM were provided with primarily nonlinear characteristics.Observed under the SEM,we found that,after eight weeks of Hath1 gene transduction,the cochlea generated a great of inner and outer hair cells.TEM showed that the characteristic cilia structures and skin panels of hair cells occurred in the ranges of OHC and IHC.In addition,the specific markers of hair cells such as MyosinⅥand Prestin expressed too.The results mentioned above showed that the excessive expression of Hath1 in the cochlea which was damaged by noise can promote the generation of inner and out hair cells.The regeneration and the resumption of the inner hair cells were earlier than that of the outer hair cells and we presumed that the hair cells generated newly might derive from various support genes;the part functions of cochlea recovered and the auditory functions improved partly. |
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