| Objective:1. To establish a mature,stable and efficient zebrafish cultivation platform in laboratory.2. To study the effects of NC and HCPT on the development of zebrafish embryos.3. To study the effects of NC on the heart development of zebrafish embryos.4. To study the effects of NC on SOD activity and MDA content of zebrafish embryos.5. To test the expression of vascular endothelial growth factor receptor 2 (VEGFR-2) gene in zebrafish embryos after treated by NC with different concentrations.Methods: 1. This study combined the ordinary cultivation system with zebrafish model organism cultivation system to bulid the zebrafish cultivation platform.2. The 6 hours post fertilization (6hpf) zebrafish embryos were randomly treated with NC and HCPT for 72h and observed death rate and malformation rate.3. Zebrafish embryos of 48hpf were treated with NC of various concentrations (5, 3.15, 2, 1.58, 1.12 mg·L-1) for 12h and 24h .Then effects of NC on the zebrafish heart development were assessed under the microscope.4. Zebrafish embryos of 6hpf were treated with NC of various concentrations(0.5, 1, 2, 3 mg·L-1)for 72h. And then determinated the SOD activity and MDA content.5. Zebrafish embryos of 6hpf were treated with NC of various concentrations(1, 2, 3 mg·L-1) for 72h. Then RT-PCR was performed to examine the expression of VEGFR-2.Results:1. A mature zebrafish cultivation system has been established. And obtained embryos and hatched juvenile fishes successfully.2. LC50 was 1.66 mg·L-1 and EC50 was 1.34 mg·L-1 in NC 6hpf test groups. LC50 was 2.51 mg·L-1 and EC50 was 2.02 mg·L-1 in NC 48hpf test groups. LC50 was 4.21 mg·L-1 and EC50 was 3.29 mg·L-1 in HCPT 6hpf test groups. LC50 was 7.70 mg·L-1 and EC50 was 6.18 mg·L-1 in HCPT 48hpf test groups. The death rate and malformation rate increased along with the increase of NC concentration. Both rates decreased when treated with later development stage embryos.3. Embryos treated with NC after 12 hours and 24 hours , cardiotoxicity could be observed at all test groups .when treated with 5 mg·L-1 and 3.15mg·L-1 NC, embryos mainly showed cardiac arrest and heart hemorrhage. NC of 2, 1.58, 1.12 mg·L-1 mainly cause the cardiac malformations to zebrafish embryos. NC could significantly decrease heart rate of zebrafish embryos( P <0.01) in dose-dependent way. But the cardiotoxicity was not increased significantly in a stage-dependent way.4. when compared with the control group, the SOD activity of 0.5 mg·L-1 was increase significantly( P <0.01). The SOD activities of 1, 2, 3 mg·L-1 groups were decreased significantly when compared with control group( P <0.01). MDA contents were increased significantly with the increased of NC concentrations( P <0.01) but the contents didn't show significant differences between 0.5 mg·L-1 group and the control group(p>0.01).5. Compared with the control group, VEGFR-2 expressions of test group were decreased significantly as the NC concentrations increased (p<0.01).Conclusions:1. The combination of the two systems established a foundation for the zebrfish drug safety evaluation platform.2.①Zebrafish embryos of 6hpf are more sensitive to NC and HCPT than embryos of 48hpf.②Both NC and HCPT can cause embryotoxicity.③NC is more toxic to the development of zebrafish embryos than HCPT. 3. NC of low concentration can increase the activity of SOD and decrease the content of MDA; NC of high concentration decrease the activity of SOD and increase the content of MDA.4. NC have cardiotoxicity on the heart development of zebrafish embryos.5. VEGFR-2 gene expression of zebrafish embryo is dose-dependently decreased as NC concentration increased. |
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