The Molecular Mechanism Study Of E-cadherin In The Acute Noise Trauma

E-cadherin is a Ca2+dependent adhesion protein in the epithelial cells. It isexpressed in many sensory cells and supporting cells and play an important role in Cortiorgan development and structure maintaining. Previous studies showed the expressionlevel of E-cadherin was increased in rat cochlear sensory epithelium after noiseexposure. It was still unclear the role of E-cadherin in mouse cochlear sensoryepithelium after noise and need to know the expression level was increased by hair cellsor supporting cells. Here, we use inducible conditional gene knockout technology,microdissection method, instrument of confocal microscope and TEM to investigate themolecular mechanism of E-cadherin in acute noise trauma.1. Establishment of E-cadherin conditional Knockout mouse modelThe purpose of Part1experiment is to get an E-cadherin gene knockout mouse model,only cut the E-cadherin gene at the hair cells. With the Cre/loxP technology,B6.129-Cdh1tm2Kem/J mice was mated with Tg (Atoh1-cre/Esr1*)14Fsh/J mice andPrestin-CreERT2mice. Both two kind of Cre mice were inducible to activate the Crerecombinase system only by injection of Tamoxifen. So the E-cadherin gene could becontrolled both in time and on space by the drug of Tamoxifen. After pairing the mice,we get two conditions E-cadherin gene knockout mouse model: Cdh1-loxP/Atoh1-cremice and Cdh1-loxP/Prestin-CreERT2mice. First, the mice were genotyped to screenhomozygous transgenic mice of Cdh1-loxP. Then, we test Cre/loxP recombinase activityby immunofluorescence staining of the resulting mice after Tamoxifen injection. Finally,we use the ABR method to get the hearing level of conditional transgenic mice. It canprovide a baseline of hearing threshold of the mice for further studying of noiseexposure effect. As a result, Cdh1-loxP (+/+) Prestin-CreERT2(+/-) mice was screenedout for the request of follow-up experiments. 2. Microdissection Method of hair cell-enriched samples from inner earThe purpose of Part2experiment is to establish a well-preserved RNA extractionmethod of hair cell-enriched tissue. The success of molecular analyses of cochlearpathology relies on the collection of high-quality cochlear samples. Isolating sensorycell-enriched samples from mammalian cochleae is always a significant challenge forresearchers. By using microdissection technique, the isolation of hair cell-enrichedsamples can improve sensory cell purity significantly. Unlike previously obtainedsamples, current samples contain defined cell populations with a relatively consistentratio of sensory cells/supporting cells, and the RNA is well-preserved. With this method,we were able to collect three types of samples from the mouse cochlea: sensorycell-enriched, OHC-enriched, and IHC-enriched. The enriched samples could be used indownstream analyses, such as qRT-PCR, RNA sequencing and microarray.3. Molecular mechanism of E-cadherin in acute noise traumaThe purpose of Part3experiment is to study the changes of E-cadherin around theOHC in acute noise trauma, analyze RNA level of E-cadherin changes in OHC-enrichedtissue, and locate E-cadherin protein in the Corti organ. Using cdh1gene knockoutmouse model, we observe E-cadherin on the reticular lamina after noise. CollectingOHC-enriched tissue, we test the RNA expression level of several adhesion molecularby qRT-PCR. With the methods of3D-reconstruction and immunochemical TEM, wefocus on the details of connection between OHC and Deiters cells after noise exposure.As conclusions:1. Expression of E-cadherin around OHC was increased by Deiterscells after acute noise exposure.2. E-cadherin of Deiters cells is connected to reticularlamina by phalangeal process.3. After acute noise, E-cadherin was overexpressedbetween Deiters cells to block the gap of OHC apoptosis for maintaining the integrity ofthe reticular lamina.