Experimental Study For The Damaged Cochlear Of Newborn Rat By Transferring Gene Math1

In the mammals'hearing system, hair cells, the terminal cells of sensory nerves, can convert sound stimulus into electrical signals and transmit them to nerve centre make means of auditory nerve in order to keep normal auditory function. Various factors such as excessive sound stimulus, aging, ototoxic drugs (aminoglycoside antibiotics, antimalarial drugs, antitumor drugs, loop diuretics, et al), infection (rubella, influenza meningitis, mumps, et al) and autoimmune disease can lead to the irreversible damages of the hair cells, and then cause permanent sensorineural deafness. Resume of sensorineural hearing loss consists of amplification devices (hearing aids) and devices that stimulate auditory neurons electrically (cochlear implants), but various factors, such as deafness degree, course, age, education degree, could influence the effect of the clinical treatments. However, even after using hearing aids or cochlear implants, there are still some patients cannot improve listening, now we need a new treatment for more deafness patients in the clinical work. Cochlear gene therapy, which is an effective method for rebuilding Corti's structure to recover auditory functions, can be carried out by using the key genes which can convert non-sensory epithelia cells into cochlear hair cells. From the recent twenty years, there are many reports on Mathl which can increase cochlear hair cells in vitro and in vivo. At present, it has not been reported whether the Mathl therapy on the damaged hair cells can achieve hair cells regeneration or not. In this research, we imported Mathl genes into the in vitro Corti's, taken use of immunohistochemical staining of MyosinⅦa and the quantity of hair cells. The study included two parts as follows:Part one:Constructing the model of damaged Newborn Rat Cochlear in vitroTo study injury effects of neomycin sulfate at different concentrations on cultured neonatal rat Cochlear hari cells.The organ of Corti in the whole cochlea from neonatal (PO) SD rats was isolated and cultured for 12 hours. Neomycin sulfate of different final concentrations was added to surviving culture media. Forty samples were randomly selected into a control and 3 test groups (n=10 in each group). Samples in the test group were treated with neomycin sulfate at 1 mmol/L,2mmol/L or 4 mmol/L for 24 hours. They were then prepared for confocal microscope examination via immunofluorescence staining of Myosin Vila. The number of hair cells within a 100μm segment was counted on 10 arbitrarily selected images and compared statistically using the CHISS software. Samples in the control group received no neomycin treatment. When compared to the control group, the number of inner hair cells in the apical turn showed statistically significant differences in only samples trested with 4 mmol/L neomycin sulfate. However, the number of inner hair cells in the middle and basal turns was statiscally different from the control group in all neomycin treated samples. The number of outer hair cells in the apical trun was statistically different from the control group in samples treated with 2 mmol/L and 4 mmol/L neomycin sulfate. The difference in middle and basal truns was of statistical significance for all three neomycin sulfate concentratins.The results indicate toxic effects on Cochlear hair cells by neomycin sulfate, with outer hair cells being more sensitive than inner hair cells. The damage appears to start from the basal turn. Increasing neomycin concentration not only increases the severity of hair cell damage, but increasingly involves the middle and finally the apical turn, especially outer hair cells. No hair cell damage was seen in the control group. The ototoxic effects of neomycin sulfate to cochlear cultures are similar to those seen on cochlear hair cells reported in in vivo studies. This can serve as a model for in vitro hair cell regeneration research.Part two:The Observation of experiment that transfecting the Cochlear of Newborn Rat with the Mathl genes carried by adenovirus.The aim of this part is to investigate the effects of Mathl gene in cochlear hair cells which were damaged by ototoxic drugs. On the basis of the part one, we will regenerate or repair cochlear hair cells which were damaged by ototoxic drugs.The 40 cochlear samples were divided into normal group, Neomycin group, Neomycin+Ad-Mathl-EGFP group and Neomycin+Ad-EGFP group, each group had 10 samples. After Mathl gene was tranfected into the cochlear, by immunohistochemical staining of MyosinⅦa, we observed the change of the hair cells. The results showed that the targe gene expressed in the range of supporting cells undering the hair cells which were damaged by ototoxic drugs in Neomycin Ad-Math1-EGFP group, although the normal group, Neomycin group and Neomycin Ad-EGFP group did not show the same result, it concluded that Mathl gene could prompt supporting cells transdifferent into hair cells.