| 研究実績の概要 |
In past one year, I used 2 bacteria (F. columnare and A. hydrophila) and established the infection study. Through several experiments, I found the appropriate concentration and infection time. Through qPCR, post infection, immune-related factors (e.g. interleukin-1 beta, interferon-gamma) were elevated comparing with non-infected medaka. Through this method, I found the expression of these factors were changed post infection, and this represents the immune system of the medaka was stimulated after the infection by these two bacteria.
I also established fibrinogen gamma subunit knockout medaka (Olfgg(Del4) and Olfgg(Ins4)), and novel phenotype was observed. There was no significant difference between WT and mutants in mortality. However, through blood coagulation test, I found mutants showed retarded blood coagulation. Meanwhile, mutants also showed bleeding occasionally, but most of them can be cured. Unexpectedly, some mutants showed the anemia. By observing the morphology, hematocrit and blood cell staining, I found the homozygotes showed smaller number of the red blood cells (RBCs) and more immature RBCs. I suppose that the anemia phenotype may cause by the minor bleeding or fibrinogen may involve in the erythropoiesis.
At the same time, I was also establishing the tissue specific fibrinogen knockout medaka through cre-loxp system.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
Last year, I found fibrinogen knockout medaka showed novel phenotype, anemia. In order to clarify the reason of the anemia, I carried out several extra experiments, like blood coagulation test, blood cell staining, anemia induction, etc. I found the anemia of the mutants was not caused by the severe bleeding and rapid blood cell production. At present, I’m trying to prove that if minor bleeding and long-term erythropoiesis and blood cell circulation caused the anemia or not.
As for the infection study, I already established the infection study using A. hydrophila. Through repeated experiments, I found the most appropriate conditions (medaka condition, bacterial concentration, infection period, etc.) for infecting medaka using A. hydrophila. Using wildtype medaka, post infection, several immune-related genes (e.g., IL-1 beta, IL-17, interferon-gamma, etc.) were elevated through qPCR. And now, I’m using wildtype and mutant medaka (thrombin, Factor XIII & TG2 KO medaka) to carry out the infection study. Using these mutants, I can clarify the function of these proteins in immunity.
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| 今後の研究の推進方策 |
In next year, I will continue to investigate the relationship between fibrinogen and erythropoiesis and immune system. In erythropoiesis, I will check the expression level of several erythropoiesis-related factors in fibrinogen KO medaka through qPCR. Also, I will also try to trace the life span of the erythropoiesis in mutant medaka to clarify if the long-term maintenance of erythrocytes in mutants is affected or not.
In immune system (infection study), I will also find out the appropriate infection condition for another bacterium, F. columnare. Using these two kinds of bacteria, I will infect the WT and mutant (fibrinogen, thrombin, Factor XIII, TG2 and TG1 KO medaka) to find out the difference between them. Through different mutants, I can clarify the possible relationship or mechanisms between coagulation factors and immunity through several experiments, like qPCR, blood cell and epithelial cell staining, cell culture.
As for the tissue specific knockout medaka, I will continue to obtain the fibrinogen gamma flox KI medaka, and mate with choriogenin- or epithelial-cre medaka to obtain liver and epithelial specific knockout medaka. Through this, I aim to clarify the function of fibrinogen in different tissues.
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