| Preliminary studies have shown that the ATP-competitive small molecule inhibitor SP600125 causes pre-mitotic arrest and leads to nuclear mitosis,which can efficiently induce fish cell polyploidization in vitro.On this basis,further study the effects of SP600125 on fish development;combining with SP600125 can effectively block mitosis,explore the feasibility of using it to prepare fish cultured cells in vitro and fish kidney tissue chromosome specimens.The specific results are as follows:1.SP600125 incubation of fertilized eggs significantly affected the process of embryonic development and led to high embryo mortality.At the same time,it was accompanied by the occurrence of deformed fish fry,which was mainly manifested by skeletal development abnormalities such as spinal curvature and tail deletion.Incubating crucian carp fry with SP600125 also caused a large number of deformities of the fry,mainly manifested as a curved spine or tail.2.Eight genes related to bone development were extracted from transcriptome data of SP600125 treated cells,and their expressions in embryos,fish fry and in vitro cultured cells were analyzed by q RT-PCR.The results showed that: SP600125 incubation could affect the expression of genes related to bone development,resulting in significantly upregulated m RNA levels of smad6,stat1,lef1,bmp2,and twist1.3.In vitro culture cells of fish was used as materials,and SP600125 was used for preliminary treatment(the cultured cells were pretreated with SP600125 in a final concentration of 50-150 μmol/L and processing time of 22-28 h),and the chromosomes are prepared by operations such as hypotonic,fixation,and dripping.The results showed that the in vitro cultured cells materials pretreated with SP600125 had many chromosome division phases and good chromosomal structure.4.Using fish living kidney tissue as the material,pretreated with PHA for 3 times(the treatment doses of 8-10 μg/g,12-15 μg/g,5-6 μg/g,with interval times of 12 h and 3 h,respectively),fish is injected with 0.8μL/g SP600125 for 1.5-2 h,then preparing of cell suspension from kidney tissue.After hypotonic,fixation,dripping and other operations,the chromosome division phase with clear images and good chromosome morphology could be obtained.These results provide an important theoretical reference for further adjusting the strategies and methods of SP600125-induced ploidy in fish.It is also indicated that SP600125 is suitable for the preparation of chromosomes from fish cells in vitro or fish tissues in vivo,and the prepared chromosome specimens can be used for the research practice of chromosome counting and karyotype analysis. |
