Zebrafish For Drug Safety Evaluation Of Biotechnology Platform For Building Research

Objective:To establish the cultivation system of zebrafish in ordinary laboratory, grasp initially the biological characteristics of zebrafish through the daily breeding and embryo reproduction, and to lay the foundation for establishing the experiment platform of drug safety evaluation with zebrafish. Methods:Established ordinary cultivation system of zebrafish using fish tank, submersible pump, temperature controller, filtering apparatus, and so on. In addition, obtained zebrafish embryos with the methods of paired spawning and artificial fertilization. Results:The cultivation system of zebrafish in ordinary laboratory has been established, and obtained the embryos of zebrafish successfully. Conclusion:This ordinary laboratory cultivation system of zebrafish has the fortes of low price, convenience, stabilization, and strong maneuverability, which sets a foundation for establishing the experiment platform of drug safety evaluation with zebrafish in the future, and can be used in the protophase study. Objective: To observe the whole developmental processes of zebrafish embryos and analyse the eligible endpoints in embryotoxicity test. Methods: Selected healthy and eugamic zebrafish to oviposit in pair, and observed the developmental processes of zebrafish embryos in morphology systematically with stereomicroscopy. In addition, the whole developmental processes would be photographed. Results: The embryogenesis of zebrafish can be divided into seven periods broadly: zygote period, cleavage period, blastula period, gastrula period, segmentation period, pharyngula period and hatching period. Some eligible toxicological endpoints in embryotoxicity study were suggested. Conclusion: The embryogenesis of zebrafish and the morphological characteristics of different developmental phases were grasped initially. The suggested toxicological endpoints have feasibilities. Objectives: To study the effects of different concentrations of colchicine on the activities of SOD and Na⁺-K⁺-ATPase, and the effects on the tissue structures in liver and gill of zebrafish. To explore the test methods for drug safety evaluation which using the activities of some enzymes and the pathology changes in zebrafish as observation indexes, laying the foundation for establishing the experiment platform of drug safety evaluation with zebrafish in the future. Methods: Based on the preliminary experiment, zebrafish were exposed to five different concentrations of colchicine for 96h, and the median lethal concentration (LC50) was calculated for evaluating acute toxicity. In addition, zebrafish were exposed to three different concentrations of colchicine (0.67, 1.70, 4.26mg·L^-1) for 21 days in chronic toxicity test, and the activities of SOD and Na⁺-K⁺-ATPase in the liver and gill of zebrafish were measured every interval of 7 days. After the exposure of 21 days, taking the gill and liver tissues of each group with hematoxylineosin (HE) stain to examine the injury of structures which caused by colchicine. Results: LC₅₀ of colchicine was 16.90mg·L^-1. With the increases of colchicine concentrations and exposure time, the activities of SOD and Na⁺-K⁺-ATPase in the gill of zebrafish were suppressed significantly, and the activity of SOD in liver was inceased, but the activity of Na⁺-K⁺-ATPase in liver presented the tendency of depression. The pathological results show that the colchicine would lead to impairment to varying degrees on the gill and liver tissue. Conclusions: The SOD and Na⁺-K⁺-ATPase in liver and gill tissue of zebrafish are sensitive to colchicine, and can be used as observation index for drug toxicity evaluation. Objective: To study the effects of different concentrations of colchicine on the tnf-αand sod-lgene expression during zebrafish embryogenesis. To explore the test methods of reverse transcription polymerase chain reaction (RT-PCR) which using the embryos of zebrafish as testing objects, the relative amount of target genes' messenger RNA (mRNA) as observation indexes. Methods: Zebrafish embryos were exposed to three different concentrations of colchicine(0 mg·L^-1, 10mg·L^-10 and 40mg·L^-1). Collected the embryos at 24h, 48h and 72h after fertilization to detect the expression of tnf-αand sod-lgene by RT-PCR. Results: During the experiment, compared with the control group, the tnf-a gene expression was. suppressed in every colchicine treatment group, and presenting the concentration-effect relationship; the sod-1 gene of zebrafish embryos was up-regulation in the lower concentration group(10mg·L^-1), and the higher concentration group(40mg·L^-1) inhibited its expression. Conclusion: We studied the effects of different concentrations of colchicine on the tnf a and sod-1 gene expression during zebrafish embryogenesis, initially established the test methods of RT-PCR which using the embryos of zebrafish as testing objects, and the relative amount of target genes as observation indexes, which accumulated data for establishing the experiment platform of drug safety evaluation with zebrafish in the future.