Establishment Of Bio-detection Method Of Environmental Hormone Impact On Fishes' Reproduction

More and more attentions have been focused on the issue of endocrine disrupting chemicals (EDCs) in the environment in the worldwide, recently. It is challenging to study EDCs in the environment for instrumental methods because of various low level EDCs. To assess the potential impacts, biomarkers become effective method for analyzing the effects of EDCs in the environment. Of these biomarkers, vitellogenin (VTG) has been used as an important biomarker for monitoring EDCs, and easiest and most automated assay are designed around ELISA or general RT-PCR to detect VTG in fishes. The former needs of specific antibodies, complicated procedures, and compared to the molecular level of gene expression quantitative methods are still a wide gap between sensitivity;The general RT-PCR method in quantitative gene expression There is also the shortcomings of poor accuracy, can not effectively reflect the dose-effect relationship. In this study by medaka fish primary hepatocytes exposed E2, using Q-RT-PCR detection, such as fish and Barracuda VTG gene expression changes, the establishment of higher expectations of the environmental sensitivity of estrogen effect of evaluation methods. The following achievements are obtained:(1) This study by medaka primary hepatocytes cultured in order to establish stability in the toxicology test model, with the traditional live exposure experiment compared with the experimental animal conservation materials, supplies and laboratory experiments cycle. For endocrine disrupting chemicals lay the foundation for toxicology studies.(2) This study successfully established a real-time quantitative RT-PCR and the culture of medaka primary hepatocytes in vitro combination of environmental effects of estrogen evaluation methods, and showed high sensitivity.(3) VTG-I, VTG-II, CHG-H, CHG-L and ERαin vitro medaka primary hepatocytes were exposed with estrogen showed good dose-response relationship, which VTG-I, VTG-II, CHG - H, CHG-L as an estrogen detection of biological markers of the first option.(4) In this paper, amplified so-iuy mullet VTG,β-actin genes, gene sequence determination, the establishment of E2 detection of liver cells exposed so-iuy mullet VTG expression of real-time quantitative RT-PCR method. The dynamic environment for hormone testing and analysis of fish on endocrine disruptors the sensitivity analysis provides a methodology based.