| ObjectiveTo explore the effects of low-dose radiation on neural development of zebrafish,and to explore whether mi RNA is involved,so as to provide evidence for the biological effects and regulatory mechanism caused by low dose rate radiation.MethodsTaking the AB line blue zebrafish as biological materials and using137 Cs radiation source to continuously irradiate zebrafish embryos that had been developing for 2 hours,in order to observe the effects of γ-ray on neurodevelopmental toxicity of zebrafish embryos under different dose rates and different irradiation times:(1)Using the microscope to observe the gross morphology of embryos and analyze the hatching rate,abnormality rate and death rate;(2)Observing the lateral nervous system development by FM1-43 staining;(3)Observing the morphological changes of brain tissue cells by HE staining;(4)Conducting behavioral tests to analyze the swimming speed and reaction time of zebrafish(5)Screening the differential expression of mi RNAs in zebrafish brain after radiation and analyzing bioinformatics by whole transcriptome sequencing;(6)Detecting the temporal expression of mi RNAs,Dicer and Drosha genes related to the neural development of zebrafish embryos affected by γ-ray with RT-PCR technology;(7)Detecting the spatial expression of mi RNAs,Dicer and Drosha genes related to neural development in zebrafish embryos affected by γ-ray with whole embryo in situ hybridization;(8)Identifing target genes corresponding to mi RNAs and pathways associated with neural development and radiation effects in humans.Results1.Irradiating continuously zebrafish embryos developed to 2 hours with the γ-ray of 0m Gy/h、0.1mgy /h、0.2mgy /h and 0.4mgy /h,the zebrafishs developing to 24 hpf,48 hpf,72 hpf,96 hpf and 120 hpf were collected,observed,photographed and measured the incubation rate,deformity rate and mortality of 120 hpf zebrafish under a fluorescence microscope.The results showed that the zebrafish embryos Under0.4m Gy/h irradiation,developed obvious and typical developmental malformations,such as yolk sac malformation,pericardial edema,tail curvature,spine distortion,etc.The irradiation group showed a decrease in hatching rate;The mortality rate increased in 0.2 m Gy /h and 0.4 m Gy/h irradiation groups;The malformation rate decreased after 0.1 m Gy /h irradiation,and increased after 0.4 m Gy /h irradiation.Compared with the control group,the differences were statistically significant(P < 0.05).2.Irradiating continuously zebrafish embryos developed to 2 hours with the γ-ray of 0m Gy/h、0.1m Gy/h、0.2m Gy/h and 0.4m Gy/h,the zebrafishs developing to 120 hpf were collected.After staining the nerve mother cells of zebrafish lateral line in each group with FM1-43,and then observed and photographed under fluorescence microscope.It can be seen that the lateral line system of the embryos of control and 0.1m Gy/h group has orderly arrangement of hair cells and clear structure.However,the lateral line system of the 0.2m Gy/h and 0.4m Gy/h irradiation group showed different degrees of developmental abnormalities,the number of hair cells decreased,and the arrangement was disordered and fuzzy.3.Irradiating continuously zebrafish embryos developed to 2 hours with the γ-ray of 0m Gy/h、0.1m Gy/h、0.2m Gy/h and 0.4m Gy/h,the zebrafish embryos developing to 72 hpf,96 hpf and 120 hpf were collected,After staining the brain tissue of zebrafish in each group with HE,and then observed and photographed under fluorescence microscope.The sections showed that the nerve cells in the brain tissue of the control group were arranged neatly,the structure was clear,and the intercellular space was free of edema and dense.After 0.2m Gy/h and 0.4m Gy/h irradiation,the nerve cells in zebrafish brain tissue showed different degrees of developmental abnormalities,the arrangement of neurons was disordered,the cytoplasm was concentrated,and the intercellular space was enlarged and void appeared.4.Irradiating continuously zebrafish embryos developed to 2 hours with the γ-ray of 0m Gy/h、0.1m Gy/h、0.2m Gy/h and 0.4m Gy/h,the zebrafishs developing to 120 hpf were collected and their swimming speed and reaction time were detected.It was found that the average swimming speed of zebrafish in 0.1m Gy/h group was significantly increased,while that in 0.2m Gy/h and 0.4m Gy/h groups was significantly decreased(P < 0.05).In the light stimulation experiment,zebrafish in the control group and 0.1m Gy/h group responded more quickly than those in the 0.2m Gy /h and 0.4m Gy/h groups.5.Irradiating continuously zebrafish embryos developed to 2 hours with the cumulative doses’ γ-ray of 7.5m Gy,15 m Gy and 30 m Gy,the zebrafishs developing to 72 hpf,96 hpf and 120 hpf were collected,Screening the differential expression of mi RNAs in zebrafish brain after radiation by whole transcriptome sequencing and ce RNA network conducting,and the target mi RNA was bioinformatics analyzed.The results showed that dre-mi R-196a-5p,dre-mi R-210-3p and dre-mi R-338 were significantly differentially expressed in zebrafish embryos at each developmental stage(P < 0.05).The mature sequences of dre-mi R-196a-5p,dre-mi R-210-3p and dre-mi R-338 were all highly conserved.6.Irradiating continuously zebrafish embryos developed to 2 hours with the γ-ray of 0m Gy/h、0.1m Gy/h、0.2 m Gy/h and 0.4 m Gy/h,the zebrafishs developing to 6 hpf,12 hpf,24 hpf,48 hpf,72 hpf,96 hpf and120 hpf were collected,Detecting the temporal expression of mi RNAs,Dicer and Drosha genes related to the neural development of zebrafish embryos affected by γ-ray with RT-PCR technology;The results showed that dre-mir-196a-5p,dre-mir-210-3p,dre-mir-338,Dicer and Drosha genes were expressed to different degrees in zebrafish embryos at each developmental stage.After irradiation,the expression of mi RNAs decreased at 6hpf.When 12 hpf,24hpf and 48 hpf rise;At 72 hpf,96hpf,and 120 hpf,the 0.1m Gy/h groups decreased,while the 0.2m Gy/h and0.4m Gy /h group increased.Dicer and Drosha gene expressions increased at 6hpf,12 hpf,24hpf,48 hpf,72hpf,96 hpf,and decreased at 120 hpf.The above differences were statistically significant compared with the control group(P < 0.05).7.Irradiating continuously zebrafish embryos developed to 2 hours with the γ-ray of 0m Gy/h and 0.4m Gy /h,the zebrafishs developing to 12 hpf,72 hpf and 120 hpf were collected,detecting the spatial expression of dre-mi R-210-3p,Dicer and Drosha genes was detected by in situ hybridization of whole embryo.The results were consistent with the results of RT-PCR,and there was obvious heterotopic expression in the whole head after irradiation.8.Target gene prediction and GO analysis of has-mi R-210-3p showed that has-mi R-210-3p actively participated in the growth and development process of organisms and the radiation response process.Conclusion1.Different low doses of radiation have different effects on the neurodevelopment of zebrafish embryos.2.The expression of mi RNAs(dre-mi R-196a-5p,dre-mi R-210-3p,dre-mi R-338)and mi RNA processing enzyme genes(Dicer and Drosha)was changed under low dose radiation.Combined with the results of bioinformatics analysis of hsa-mi R-210-3p,dre-mi R-210-3p may be involved in the radiation-induced biological process of neural development in zebrafish embryos. |
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