Effects Of γ-secretase Inhibitors (dapt), And Math1 Gene On The Development Of Rat Inner Ear Hair Cell In Vitro

Mammalian Corti's organ is a very complicated structure, which is responsible for hearing, a certain number of hair cells needs to take part in maintaining the normal hearing. Some factors such as over-stimulation, the use of ototoxic drugs, and aging can cause irreversible damage to the hair cell, and lead to permanent sensorineural hearing loss. The traditional view is that mammalian hair cells do not have the ability to regenerate, so far, the effect treatment for sensorineural hearing loss is still a big problem to the clinician.In latest years, with the development of inner ear sensory epithelium in-depth study of the molecular mechanism, many studies have shown that Notch signaling related gene family, mainly including Math1, Hes1, Hes5 genes, plays a key role in the formation of hair cell in the mammalian inner ear sensory epithelium of the directional differentiation, and participates in the regulation of lateral inhibition and chimera formation. Over-expression of Math1 genes in vivo or by addingγ-secretase inhibitors, is able to generate new hair-cell like cells in the cochlea. But the numbers of hair cells are often limited in vivo. How can we produce more hair cells? Can it produce more hair cells if combiningγ-secretase inhibitors with over-expression of math1 gene? That has not been reported. In this study, to observe the development of hair cells we culture the basilar membrane of the neonatal rat in vitro and we study the structure and arrangement of the Corti Organ in the postnatal P0, P7, P14 rat,and we also observe the effects ofγ-secretase inhibitors (DAPT), and ad-math1-EGFP on the development differentiation and regeneration of mammalian inner ear hair cell in vitro. By using a variety of morphological methods to study the change in hair cells under different conditions. The study comprises the following two parts.Part one:The comparison of the development for the hair cells in vitro against in vivoObjective: To compare the law of development between the hair cells in vitro and vivo.Methods: Inner ears were dissected from the heads of postnatal day(P)0, 7, 14 by using forceps under a stereomicroscope(Olympus, Japan),fixed and perfused; Some cochlear epitheliums of P0 were removed mechanically from the cochlear bone in Leibovitz's15(L15). Organs of Corti were separated from surrounding tissues but Spiral ganglion neuron cells remained attached to the cochlear epithelium. And they were put on the 6-well culture plate to promote the adherent. After cultured for 7 days or 14 days, the morphological changes of cochlear hair cells between cultured in vitro and P0,P7,P14 in vivo were analysed.Result: The hair cells of cochlear are regularly arranged in form of one row of inner hair cells and three rows of outer hair cells under scanning electron microscopy. The morphology and the arrangement of the hair cell maturated gradually within the postnatal time. P14 is an important stage in maturity. The head plates of the pillar cells were slowly widened. The microvilli on the head plate of the pillar cells and the microvilli on the Deiter cells were gradually decreased. The degeneration of Kinocilia on hair cells was nearly finished from the basic turn to the apical turn. The development of whole Corti organ was completely maturated. Hair cells growed in a good condition after 7 days culture,and were normally arranged in the three rows of outer hair cells and one row of inner hair cells. The microvilli were gradually to mature. When after cultured for 14 days, the morphology of hair cell was very poor, the majority of the Corti's organ only arranged one row of inner hair cells and one-two row of outer hair cells, many cells in Corti's fused, lost, or even cutis plate collapsed.Conclusion:1,Within the periods from P0 to P14 of Corti's of the rat, the degeneration of hair cells was nearly finished from the basic turn to the apical turn, from inner hair cell to outer hair cell. 2,The Corti's organ cultured in a good condition for 7 days in vitro, the hair cells and supporting cells were gradually matured; when cultured for 14 days, the state of all cells in the basement membrane were poor, the structure of different types of cells have been destroyed, can not be observed in vitro. Maybe, these was too long for these cells to adapt in vitro.Part two:Effects ofγ-secretase inhibitors (DAPT), and ad-math1-EGFP on the development of mammalian inner ear hair cell in vitroObjective: To study whether combined application theγ-secretase inhibitors (DAPT) and ad-Math1-EGFP were better to promote the mammalian inner ear hair cell development and differentiation than alone.Methods: The method of the preparation and treatment of explant culture was the same as the part one. They were randomly divided into normal culture group, DAPT group, ad-math1-EGFP group, DAPT and ad-math1-EGFP group, according to incubation time is divided into 4 days, 7 days and 9 days group. The culture medium was replaced every day. DAPT was dissolved in DMSO; the final DMSO concentration in the culture medium was less than 0.1%. Add DAPT to DMEM every day, transfect ad-math1-EGFP respectively in ad-math1-EGFP group by 1 day. Medium was changed every day. DM group was cultured by adding DAPT immediately, after 8 hours cultured and transfected ad-math1-EGFP 1 day, then cultured with the same method to DAPT group. Then use immunohistochemical staining and scanning electron microscopy and semi-thin section to observe the number of hair cells and organization of basement membrane morphology.Immunohistochemical staining and scanning electron microscopy: The normal group showed one row of inner hair cells and two to three rows of outer hair cells in apical turn, and three rows of outer hair cells in middle turn. And the number didn't change obviously. In DAPT or DM group, there was one row of inner hair cells and four to seven or eight rows of outer hair cells in apical turn, and three to five rows of outer hair cells in middle turn. Math1 group showed one row of inner hair cells and three to four row of outer hair cells in apical turn, three to five row of outer hair cells in middle turn. The hair cell which in nomal and math1 group arranged regularitily.In 7 days group and 9 days group, there were a few ectopic myosinVIIa-positive cells in GER cell in Math1 and DM group after culture7 days and 9 days. The number of the hair cell in both DAPT and DM group were tend to increase, and could be seen some inner hair cells outside the line, outer hair cells arranged irregularly and closely, the orientation of the hair cells had changed. Semi-thin sections: the normal group showed one row of inner hair cells and three row of outer hair cells; math1 group showed one row of inner hair cells and four row of outer hair cells; while DAPT group and the DM group showed one row of inner hair cells and five row of outer hair cells.Conclusion: 1,DAPT induces a lot of ectopic Myosin7a-positive cells in apical turn of postnatal organs of Corti, may cause the direction of stereocilia on hair cell.2,Over-expression of Math1 induces some production of extra hair cells in GER cell. 3,when DAPT and math1 use together can induce ectopic Myosin7a-positive cells in apical turn and some extra hair cells in GER cell in middle turn. Together, they have a cumulative effect on hair cell regeneration.