The Role And Mechanism Study Of Hedgehog Signaling Pathway In ? Radiation Induced Developmental Neurotoxicity In Zebrafish Embryos

Objective:To explore the role of Hedgehog signaling pathway in the development of zebrafish embryonic neurodevelopmental toxicity,in order to further elucidate the mechanism of ?-induced neurodevelopmental toxicity,and to provide scientific basis for the prevention and treatment of ?-radiation neurotoxicity.Methods:Taking the AB line zebrafish as biological materials and using biological irradiance meter to 5 hpf zebrafish embryos by different irradiation doses,in order to observe the neurodevelopmental toxicity of?-ray on zebrefish embryos:(1)Using the microscope to observe the morphology of embryos and measure the yolk sac?s diameter and embryo?s diameter of 24 hpf and 72 hpf zebrafish embryos;(2)Oberseving the zebrafish embryos? morphological changes of brain tissue by HEstaining;(3)Using RT-PCR to detect the mRNA expression of Hedgehog signaling pathway in zebrafish embryos by ?-ray;(4)Using western blot assay to detect ?-ray-induced on the changes of the expression of Hedgehog signaling pathway related proteins in zebrafish embryos.Results:1.5hpf zebrafish embryos were irradiated by 0Gy,0.25 Gy,0.5 Gy,1.0Gy,2.0 Gy ? radiation,The zebrafish embryos developing to 6 hpf,12 hpf,24hpf,36 hpf,48 hpf and 72 hpf were collected,observed,photographed and measured under a fluorescence microscope.The results showed,72 hpf zebrafish embryos on 0.5Gy irradiation appeared more obvious and typical developmental malformations,such as swelling yolk-sac,pericardial edema spinal curvature and so on.We observed a significant increased of embryos diameter and decrease of yolk bag diameter in 24 hpf and 72 hpf embryos at 0.25 Gy irradiation between control and irradiated embryos.2.5hpf zebrafish embryos were irradiated by 0Gy,0.25 Gy,0.5 Gy,1.0Gy,2.0 Gy ? radiation,The zebrafish embryos developing to 6 hpf,12 hpf,24hpf,36 hpf,48 hpf and 72 hpf were collected,HE staining,observed and photographed under a fluorescence microscope.The sections showed,the brain neurons of the unirradiated group were neatly,complete,clear and tightness.The brain neurons in the irradiated group showed varying degrees of dysplasia,neuronal disorganization,increasing intercellular space,cytoplasm concentration,and appeared in different degrees of inanition.3.5hpf zebrafish embryos were irradiated by 0Gy and 0.5Gy ?radiation,The zebrafish embryos developing to 6 hpf,12 hpf,24 hpf,36hpf,48 hpf and 72 hpf were collected and detected the mRNA expression levels of shh,smo and gli2 b in zebrafish embryos by RT-PCR.The results showed,shh,smo and gli2 b genes were expressed during the development of zebrafish embryos in a different degrees.After 0.5Gy ?radiation,the mRNA expression levels of shh were increased,while the mRNA expression levels of smo and gli2 b were decreased,and the differences were statistically significant compared with the control group.4.5hpf zebrafish embryos were irradiated by 0Gy and 0.5Gy ?radiation,The zebrafish embryos developing to 6 hpf,12 hpf,24 hpf,36hpf,48 hpf and 72 hpf were collected and detected the protein expression levels of shh,smo and gli2 b in zebrafish embryos by western blot.Unlike the results of RT-PCR,after 0.5Gy ? radiation,the protein expression levels of shh were decreased,the protein expression levels of smo presented a trend of up-regulation and down-regulation during the entire developmental period of zebrafish embryos.The gli2 b protein was up-regulated only in 24 hpf embryos,the gli2 b protein in the other groups was down-regulated,and the differences between the irradiated group and the control group were statistically significant.Conclusions:1.Low dose ?-radiation(0.25 Gy ~ 2Gy)could induce the neurodevelopmental toxicity in the zebrafish embryo,and the Hedgehog signaling pathway could regulate the process of zebrafish embryonic neurodevelopmental.2.Early period zebrafish embryo(5hpf)is sensitive to making early warning for the biology responses induced by low-dose ?-radiation(0.5Gy).shh/smo/gli2 b gene and its protein expressing in early zebrafish embryo can be regarded as a sensitive biomarker.