The Expression Of Myosin6-dab2 Related To CME In Inner Hair Cells Of The Cochlea At KM Mice

Object: In some kinds of cells, Myosin6 and Disabled-2(Dab2) are important molecules involved in the process of CME. They can provide power for the vesicle formation and the vesicle transportation in the CME. In this study, we try to investigate the expression of these two proteins in cochlea. Then we use chlorpromazine to make a pharmacological inhibition of CME in the inner hair cell to investigate changes in the electrophysiological characteristic. Through the above experiment, we try to figure out the main location of CME in the inner hair cell, and emphasize its significance for maintaining the function of hair cells.Method: 1) We dissect the cochlea to separate the basilar membrane. 2) We employ the immunofluorescence technology to label these two proteins and to make stretched preparations of the basilar membrane in postnatal rats during different developmental periods. 3) The laser scanning confocal microscope is used to detect the fluorescent signal of Myosin6 and disabled-2 to locate the expression of these proteins. 4) We detect the fluorescence intensity of target area in postnatal four age groups(P2w, P5 w, P9 w, and P16m). After analyzing intensity mean value(IMV), which can reflect the fluorescence intensity, we compare the difference of protein expression in these four groups. 5) We employ the q RT-PCR to detect the expression level of m RNA. Then we set the postnatal 9 week mice as the control group, and discuss the postnatal m RNA expression level changing trend in cochlea.6) At the same time, mice in these four age group are detected for ABR to evaluate the auditory function in different age.7) We use whole cell patch clamp to detect the Ica2+ and the Δ Cm of the inner hair cell, and compare the data of normal cell with the data that obtained from the cell after chlorpromazine perfusion.Result: 1) In the basilar membrane, fluorescence signals show in both inner and outer hair cells, but not in other cells. That is to say, Myosin6 and Dab2 only express in hair cells. It is worth noting that Dab2 expresses strongly in the apical area of IHC, a place where the cuticular plate exists. As the same, there is a local area where it could be detected with high fluorescent of Dab2. 2) After detecting and comparing the expression of My osin6 in different age, we consider that the expression level of proteincan increase from postnatal 2 week to postnatal 5 week, and then keep steady from P5 w to P9 w, but distinctly decrease from P9 w to P16 m. The expression level of Dab2 protein in P5 w is higher than that in P2 w. There is no significant difference in the level of P5w、P9w and P16 m. 3) The P9 w group is set as the control group. According to the CT value of q RT-PCR, we adopt Livak’s method to calculate the relative expression of two mRNA. Then we compare the relative quantity(RQ) of other three groups with the control group. A similar trend can be det ected in the study of these two mRNA, which is that the RQs of these two m RNA in P2 w are lower than those in P9 w. And there were no statistical difference in RQs of these two mRNA between P5 w group and P9 w group,as well as between P16 m group and P9 w group. 4) The average ABR threshold is 35.28±15.21 dB in P16 m group, 13.33±5.34 dB in P9 w group, 11.75±6.33 dB in P5 w. 5) After perfusing chlorpromazine for 3min, we reveal that the Ica2+ is reduced compared with the normal cell, as same as the Δ Cm.Conclusion:By immunofluorescence labeling and laser confocal microscope, we consider that Myosin6 and its regulatory factor Dab2 express in hair cells of KM mice, and Dab2 expression is especially strong in the apical area of IHC. It assures us that CME might be an important endocytosis model for IHC to recycle vesicles. According to our study, this CME process probably mainly takes place in the apical area of IHC. The results confirm the former conclusion of other researches from the protein level. The CME of IHC is similar to that of the apical epithelial cell to some extent. The cuticular pla te and the nearby cytoplasm are much likely to be key parts for vesicle endocytosis. In addition, as in the growth of the age and in the maturity of hearing function early after birth, the protein expression of Myosin6 and Dab2 incline to increase. These two proteins may be associated with the auditory maturity. But the further study is still needed to discuss the protein expression change in the old mice. CME could be a very important mechanism to keep the electrophysiological characteristics normal. If it is interfered, the IHC cannot work normal to transmit the acoustic signal to the terminal of auditory nerve.