The Apoptosis-related Gene Differential Expression In The Cochlea Of GJB2Gene Conditional Knockout Mice

Deafness is the most important cause of human communicationdisorders diseases. There are12.2%congenital deafness pateints in Chinacaused by GJB2gene mutations. The positive detective rate of GJB2genemutations in under-age children with NSHL are about20.1%. Therefore,GJB2mutation is a major cause of human NSHL. Many studies haveshown that loss of cochlear cells appeared after birth in GJB2conditionalknockout mice(cCx26Pax2Cre)and apoptosis might be its cause. In this study,we explore the mechanism of cochlear cells apoptosis in cCx26Pax2Cremiceby PCR array and immunofluroscence.P10and P18were selected from cCx26Pax2Cremice as normal groupand the wild type ones (BALB/C) as control group. Total RNA wasisolated from cochlear membranous labyrinth and PCR Array wasperformed using mouse apoptosis PCR arrays. The results showed thatcompared with wild-type mice, significant up or down-regulation in geneexpression was detected in16genes in cCx26Pax2Creones at P10. Of the16genes,14ones were down-regulated. Among the14genes,9ones can beclassified as anti-apoptosis or pro-proliferation genes,5ones can beclassified as pro-apoptosis or pro-inflammation genes. The other2genesexpression was up-regulated, and their main role was to promote apoptosis.Compared with time-matched controls, significant up or down-regulation ingene expression were detected in4genes in cCx26Pax2Cremice at P18. Ofthe4genes,3ones expression down-regulated were anti-apoptosis genes.The expression of the other one gene among the4ones was up-regulated,which acted as pro-apoptosis genes. Bcl2l10and Tnfrsf10b expressionshowed significant down or up-regulation at both stages. Compared with P10, the expression of caspase-8was up-ragulated at P18in cCx26Pax2Cremice.Three developmental stages of P8, p12and P18were selected fromcCx26Pax2Cremice and the correspongding wild type ones (BALB/C) andimmunoflurorescence technique was used to observe the expression of DR5(encoded by Tnfrsf10b gene) in the cochlea. It was founded that DR5had astrong positive expression in cochlear supporting cell, hair cell, typeⅡfibrocyts in spiral ligament, spiral prominence, spairal limbus, spiralganglion neuron’s cytoplasm and membrane, nerve fiber in Rosenthal canaland tectorail membrane in cCx26Pax2Cremice cochlea. The intensity ofexpression increased with the age increasing. However, the expressionpattern of DR5was different in wild type mice. The positive expressionwas found in tectorial membrane, supporting cell, spiral ganglion neuronand spiral prominence in different developing stage of mice. But theintensity of the expression was lower than that of the correspondingdevelopment stages of cCx26Pax2Cremice. Compared with the wild typemice, the expression of DR5in nerve fiber in Rosenthal canal ofcCx26Pax2Cremice was much stronger. The difference of optical densityvalues showed statistical significance(P<0.05).It is speculated that after the GJB2gene knockouted conditionally,cochlear energy-intensive cells are short of glucose and ATP, whichproducing large number of hyperoxide and DR5overexperssion ensues.The overexpression of DR5triggered the death receptor pathway causingapoptosis in the organ of Corti of cCx26Pax2Cremice cochlea directly. At thesame time, caspase-8in death receptor pathway may activate themitochondria pathway indirectly and amplify the apoptosis. Theconsequent nutrition disturbance of spiral ganglion neuron happened and itsapoptosis is triggered by the same way as that of cells of organ of Corti.The final result of the above activated pathways is the wide range ofcochlear cells apoptosis and the profound hearing loss in cCx26Pax2Cremice.