Study Of Pathogenetic Mechanism Of Microtia Induced By Retinoic Acid

Objective:To investigate the influence of retinoic acid on proliferation and relative gene expression of auricular cartilage. Analyze the specific molecular mechanisms of retinoic acid in the pathogenesis of the microtia induced by retinoic acid. And further by observating the impact of retinoic acid on development of rat embryos to accumulate experiences for the establishment of retinoic acid-induced animal model of microtia.Methods:1,To culture auricular cartilage cell in vitro with different concentration of retinoic acid in culture medium. To investigate the impact of retinoic acid on proliferation of chondrocytes by drawing growth curves using MTT method. By the method of real-time fluorescence quantitative PCR to investigate changes of relative gene expression and to explore the influence to the phenotype of chondrocytes caused by retinoic acid.2,To culture auricular cartilage cell in vitro. Retinoic acid was added to the medium in experimental group. After 48 hours the cells were taken to detect changes of genome-wide expression by using large DNA microarrays. The data obtained from array analysis were used to identify genes that were significantly regulated by retinoic acid over the control populations. By using annotation and statistics analysis to search the key genes in the process of teratogenesis induced by retinoic acid; by using hierarchical cluster analysis to analyze the expression patterns and correlated groups of different genes induced by retinoic acid; In order to predict the molecular mechanisms of teratogenesis induced by retinoic acid, Gene Ontology enrichment was performed to evaluate gene function, and pathway analysis was performed to study the interactions of retinoic acid induced genes.3,To observer the intervention of retinoic acid to rat embryos, the rat embryos were exposed to retinoic acid at gestation 13.5 day by intraperitoneal injection.and taken using caesarean section at gestation 20 day. To access the influence of retinoic acid on the development of rat embryos and accumulate experience for further study.Results:1.The intervention of retinoic acid can reduce the proliferantion of auricular chondrocytes in a time and dose dependent, in contrast with normal auricular chondrocytes.2.After 48-hour intervention of retinoic acid at 1*10-6mol/Lconcentration, real-time fluorescence quantitative PCR found that the expression of Col2A1 reduced by 53%, the expression of Col10A1 reduced by 43%, the expression of ALP decreased by 43%, Runx2 decreased by 29%. Using Prizm 4 software for T-test analysis, the expression of Col10A1 and ALP in the treatment group compared with the control group was significant statistical significance (P＜0.0001). The expression Col2A1 and Runx2 in treatment group compared with the control group also decreased, but not statistically significant.3. After 48-hour intervention of retinoic acid at 1*10-6mol/Lconcentration, there were 131 genes unregulated and 31 genes down-regulated in the treatment group compared with the control group detected by using large DNA microarrays. The primary functions of regulated genes were cell proliferation, differentiation, apoptosis, migration and cell cycle. By statistics analysis and annotation alot of candidate genes in teratogenesis were screen out including:Cyp26A1,Cyp26B1,RARRES3,RARRES1,NANOS1,SNAI2,MGP,DAPK-1,SOX4,PLK2,CCNA2 and ATP5H. Analyzed by hierarchical cluster and GO enrichment, the regulated genes had similar function, and the function of the significant changed genes were translational elongation and signal transduction, etc. Through pathway analysis, it was found that genes induced by ACR were taken in oxidative phosphorylation and ribosome pathway. The function of retinoic acid was found to have tissue specificity compared with the literatures.4. Intervention of retinoic acid in rat embryos may adversely affect embryonic development, including developmental stagnation, death and a variety of congenital malformations such as external ear abnormalities. Compared with gastric tube perfusion, intraperitoneal injection of the dose should be adjusted appropriately.Conclusions:1. Intervention of retinoic acid would reduce proliferation of auricular chondrocytes and may adversely affect the traits of auricular chondrocytes. Retinoic acid plays function in a time and dose dependent way.2. The molecular mechanisms of retinoic acid induced microtia were include disrupted signaling pathway, and altered the key gene expression in external ear development process.3. The embryos exposed to retinoic acid showed external ear abnormalities. However the way, dose and time for intervention of retinoic acid should be further investigated to establish stable rat model of microtia.