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In vitro kultivace zárodečných kmenových buněk jesetera
XIE, Xuan
The study in this thesis concerns a research area related to sturgeon germ cell biology including identification, purification and in vitro culture. The research objectives are: expounding fish spermatogonial stem cell characteristic and regulation of spermatogenesis; evaluation of the existent methods in fish germ cell purification and in vitro culture; investigation of in vitro amplification of sturgeon germ cells; development of an approach to purify germ cells in sturgeon; identification of type A spermatogonia (ASG) in sturgeon. Novelty and scientific originality. This work first time developed sturgeon germ cell growth in vitro and combined cell culture with transplantation. The application of monoclonal antibodies on recognition of sturgeon ASG represents a new, and currently only tool of germ cell identification. Important scientific problem solved in the field of research. This work of germ cell culture observed the effect of somatic cells and growth factors on the proliferation of sturgeon germ cells through evaluation of germ cell mitotic activity during culture, which contributed to the study on the regulation mechanism of germ cell self-renewal and differentiation. This thesis also developed a method using flow cytometry to purify sturgeon ASGs. A monoclonal antibody was also found that enable to identify ASGs of sturgeon specifically. Theoretical significance and applied value of the thesis. The thesis summarized the accumulated knowledge of spermatogonial stem cell and regulation of its self-renewal vs. differentiation in fish, compared available protocols and advances in enriching spermatogonia and reviewed development of in vitro germ cell culture conditions. The work of efficient enrichment of type A spermatogonia was achieved based on its physicochemical properties, which is applicable for commercial and endangered fishes without cell-labeling systems such as transgenes and cell antibodies. Monoclonal antibodies generated from Pacific blue fin tuna can also recognize antigens from sterlet ASGs, indicating some epitopes for ASGs are conserved between species. The research results present interest for characterization of ASGs in proteomics level. Implementation of scientific results. A serum-free culture condition for sturgeon germ cells was established. The germ cells under this in vitro condition can remain growth, survival and maintain their transplantability for more than 40 days. Therefore, this method is expected to provide a long term supply for germ cell transplantation and surrogate production. Moreover, based on the analysis of germ cell light scatter properties, a purified ASG population was isolated. No dye and transgenes are introduced during sorting, which is helpful for characterization of germ cell populations and downstream germ cell manipulation. The application of monoclonal antibody on sturgeon ASG can achieve the accuracy of the ASGs identification on in vitro culture.
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