The Study On Inner Ear Gene Expression Profile And Inner Ear Development Of Hypoxia Offspring Rats In Pregnancy

Objective: To study the effect of hypoxia in pregnancy on the development of the inner ear of mice and investigate the possible molecular mechanism of CIH on the development of the inner ear.Methods: To establish the SD rat model of hypoxia during pregnancy,(1)study the relationship between CIH and the development of inner ear dysplasia: The auditory brainstem response(ABR)method was used to detect the hearing response threshold in the normal group and the hypoxia group,and the offspring of the normal hearing group(CON group)and the hypoxia group(MH group)were screened out,then we studied the morphological characteristics of the hypoplasia of the inner ear.(2)CON group and MH group were studied by gene chip technology: We screened differentially expressed genes;pathway enrichment was used to analyze differentially expressed genes;GO classification was used to annotate gene function,and the effect of CIH on the expression of different functional groups in the inner ear of fetus was studied.(3)To study the expression level of Connexin26 and the methylation of GJB2 gene promoter region in CIH,analyze the relationship between GJB2 gene mutation and Connexin26 expression.Results:(1)After hypoxia 1h in the first day of hypoxia group,the arterial blood gas analysis was performed in the normal group and the hypoxia group.The partial pressure of oxygen and oxygen saturation were analyzed to confirm the success of the model.(2)In the normal group,155 rats were born,and the rats in hypoxia group were given a total of 153 offsprings.The uterus of intermittent hypoxia and offspring rats of low birth weight has a positive correlation,and hypoxia group had the phenomenon of catch-up growth and premature delivery.(3)The auditory brainstem response(ABR)was used to detect the auditory function in the newborn rats at the beginning of the first week.The incidence of deafness in the hypoxia group was statistically significant compared with that in the normal group(P < 0.05).21 were randomly selected from the 154 normal offspring rats as the nomal group(CON group),21 were selected from the 43 hypoxia offspring rats as the hypoxia group(MH group).(4)HE staining showed that the Corti organ was normal in CON group,and there was no defect in the hair cells.In MH group,the Corti organ showed support cells,such as the outer pillar cell,inner pillar cell,outer phalangeal cell.but the inner hair cells and outer hair cells were missing.(5)TUNEL showed that only a very small amount of cochlear positive cells in the CON group,but the inner hair cells,outer hair cells,stria vascularis cells in the corti organ of MH group contain brown granules and TUNEL staining is positive.Morphological experiments showed that the Corti organ of the inner ear of rats in MH group had the phenomenon of hair cell apoptosis.(6)The gene chip technology was used to screen the effect of hypoxia on the expression of genes in the inner ear of pregnant rats.On the basis of differential gene expression screening criteria(Pvalue≤0.05),we detected 23073 genes and found that 88 genes were up-regulated and 92 genes were down regulated.A meaningful part of the target gene,such as those participating in ion homeostasis genes: GJB2,GJB6,Slc7a7,CLDN14(Regulation:down);transcription factor POU4F3(Regulation:down);water channel protein: Aqp5,Aqp7(Regulation:down);the inhibition of apoptosis genes: Bcl2l10,Bcl2l1,Birc3,Birc5,Hells,Nol3,PAK7(Regulation:down);apoptosis genes: Bak1,caspase2,Ltbr,Casp12,Tsc22d3,Dapk1(Regulation:up).(6))GO classification analysis: the molecular function of the genes with active transmembrane transport activity,metal ion binding,cytokine receptor binding,voltage-gated potassium channel activity,oxygen transport activity,methylation of CpG binding,etc.;The differential genes involved in the biological processes are mainly involved in oxygen transport,small molecule metabolism,protein transport and so on;The genes of the cell components were enriched in the extracellular space,the cell surface,the receptor complex,the intrinsic components of the membrane and so on(8)Pathway enrichment analysis showed that the differentially expressed genes mainly in the transduction pathway of transcriptional dysregulation: such as JNK signaling pathway,κB signaling pathway,NOD like receptor signaling pathway,,and so on.(9)Western Blot showed that the expression level of inner ear tissue in the CON group of Connexin26 protein was higher than that in MH group(gray level ratio: group CON 1.059±0.294 &MH group 0.725±0.264,F=0.795,P= 0.041).(10)GJB2 gene mutation is closely related to hereditary nonsyndromic deafness,which is the main responsible gene for deafness.The GJB2 promoter region was analyzed and the 2 CpG islands were found for the first time.The methylation rate of GJB2 gene promoter in MH group was significantly higher than that in COH group.It is speculated that the increased methylation level of GJB2 gene promoter region may be the cause of the lower expression of Connexin26 protein in the inner ear.Conclusion:(1)Offspring rats in hypoxia group have low birth weight and premature birth because of the chronic intermittent hypoxia in pregnancy,which is closely related to incidence of deafness.(2)CIH can alter the inner ear gene expression profile of the offspring rats in MH group,and over 30 transduction pathways like JNK are deregulated.Differential expressed genes include genes which can down regulate and maintain ion balance,transcription factors,water channel proteins,genes of inhibiting cell apoptosis and genes which can up regulate and promote cell apoptosis.(3)In MH group the methylation level of the main deaf genes GJB2 promoter region increased,resulting in the decrease of Connexin26 expression and the hair cell apoptosis.