Mechanisms Of DAPT On The Development Of Rat Inner Ear Hair Cells In Vitro

In traditional view, mammalian hair cell in inner ear is a kind of terminal cellwhich cannot be regenerated after impaired. So far, the treatment for sensorineuralhearing loss is still a big problem to the otologists. In last few years, scientists athome and abroad find some ways of regenerating inner ear hair cells with hardworking, including the transplation of promoting hair cell regeneration genes, stemcells and microRNA into the organ of Corti, etc. These techniques are still immature,and being at experimental study phase.Notch signaling is one of the most important signals in animal development,participating in kinds of cell lines directional differentiation during the embryonicdevelopment. Recent studies find out that Notch signaling participates in theregulation of lateral inhibition, and Notch signaling related gene family plays a keyrole in the directional differentiation between hair cells and supporting cells.γ-secretase activates Notch signaling, γ-secretase inhibitor inactivates Notchsignaling, Notch signaling related gene family, mainly including Hes1and Hes5isreduced, at the same time, Math1expression is increased. Math1is different fromHes1and Hes5, math1can promote hair cell regeneration in mammalian inner ear.microRNAs are small, non-coding RNA molecules that express extensivelyamong eukaryotes, they regulate such major processes as gene expression to genecontrolling. Thus, understanding microRNA expression and expressional regulationwill help us figure out the relationship between gene group and gene expressionalregulation. Now we have found out that microRNA183family is highly expressed ininner ear hair cell specially, there are some basements indicate that microRNA183family has some relationship with hair cell development. In out study, we analysisthe difference of microRNA expression in membranes between neonate and adultrats, and with or without DAPT membranes, trying to find out importing microRNAs in hair cell regeneration.Part I: γ-secretase inhibitor (DAPT) on the development of ratinner ear hiar cell in vitroObjective To study hair cell changes in new born rats when the Notch signalis inhibited by DAPT in vitro.Methods Cochleae were dissected from new born rats. The Organ of Corti wasfreed from surrounding tissues and explanted inextenso onto the surface of6wellculture plates and randomly divided into two groups: an experimental group inwhich specimens were treated with DAPT (a Notch signal inhibitor) at5uM, and acontrol group in which specimens were treated with DMEM containing DMSO witha final concentration lower than0.1%. The Organ of Corti was incubated at37degree in a humidified atmosphere with5%CO2for5days with daily replacement ofthe culture medium. The number of hair cells and morphology of the basementmembrane were then examined after immunohistochemical staining, and analyzedby statistical software.Results There was an increase of hair cells in the experiment group comparedto the control group, and at the same time, the increase of hair cell in the Organ ofCorti was confirmed with statistical significance by statistical software.Conclusions The basement of membrane of rats can be cultured successfullyin vitro, and when deal with DAPT, the number of hair cell can be increased.Part II: The effect of DAPT on the differential factors ofcochlear hair cellObjectives To explore the mechanisms of hair cell increase after deal withDAPT from two points: the Notch signaling and microRNA.Methods RT-PCR was used to confirm presence of Notch signal on P0, andquantitative RT-PCR was used to determine changes in downstream genes involved in Notch signaling on P5. Purify the microRNA following the instructions from thecultured organ of Corti, then compare the microRNA expression patterns withmicroarray analysis between the experimental and control group; Predicting targetmRNAs with computer predicting system.Results There was an increase of hair cells in the experiment group comparedto the control group. Notch signaling was present on P0and Notch signaling genes(Hes1, Hes5and Math1) changed after DAPT treatment on P5. There are132microRNAs up-regulated in experimental group, and36microRNAs down-regulatedcomparing with control group on P5,168microRNAs with at least2fold changes,about22%in all. Some microRNAs which related with the development of inner earhave higher expression in experimental group than control group.Conclusions Notch signaling is present in new born mammals. Increasing haircells may be possible through inhibiting Notch signaling. MicroRNA expression ischanged in experimental group, microRNA may play important roles in mammalianhair cell development, because microRNA292、microRNA124、microRNA18a、microRNA130b、 microRNA99a、 microRNA96and microRNA183is higherexpressed in experimental group than control group.Part III: The microRNA expression patterns between newbornand adult ratsObjective To understand the microRNA expression patterns between newbornand adult rats.Methods Prepare the Organ of Corti enough, purify the microRNA followingthe instructions, then compare the microRNA expression patterns with microarrayanalysis.Results There are284microRNAs express in the newborn rat inner ear, and209microRNAs express in the adult rat inner ear. Most of the microRNAsexpression persists into adulthood, some decrease. The microRNA183familyexpression is higher in newborn rats than adult rats. Conclusions MicroRNA expression is abundant in the mammalian inner ear.microRNA may play important roles in mammalian hair cell development, and themicroRNA183family is high expressed in newborn rats, it may concern with theproficiency of hair cell increase.