动物实验 z-bio 2021-02-21 688

　　Introduction: The most important cytokines that regulate the proliferation and differentiation of vertebrate red blood cells are erythropoietin (EPO) and KIT ligand (KITLG; or stem cell factor). As described in other vertebrates, the binding of EPO and KITLG to their cognate receptors can ensure erythroid cell production by triggering specific signaling events. In addition to its role in erythropoiesis, KITLG is also pleiotropic, affecting a wide range of tissues and cells, including hematopoietic stem cells (HSC) and germ stem cells. It is an important regulator and plays a role in the formation of the body and many processes in adult organisms. In mammals, the KIT signal is related to erythropoiesis and bone marrow production, as well as neurogenesis and pigmentation. Interestingly, there are two types of KITLG in the body: transmembrane (TM) and soluble KITLG. The former is very important for the regulation of stem cells in their niches, while the latter affects distant tissues. The binding of KITLG to its receptor KIT (a member of the type III tyrosine kinase family) leads to its own phosphorylation, which in turn triggers various signal cascades, including phosphatidylinositol 3-kinase, MAPK, SRC and JAK pathways. Therefore, future research depends on the understanding of the diverse functions of ligands (Kitlga, Kitlgb) and receptors (Kita, Kitb), as well as binding specificity. Similarly, there are two copies of the epo gene in the zebrafish genome: epia and epob. The zebrafish epoa seems to play a role similar to that of mammals, but the role of epob in hematopoiesis has not been reported. For simplicity, Epo/Epo will be used instead of Epoa/Epoa in this study. Despite extensive research, the role of Kit signaling in zebrafish hematopoiesis is still unknown. So far, only 2 studies have shown that Kitlg may play a role in zebrafish hematopoiesis. The first study showed a slight increase in HSC when Kitlgb was overexpressed, while the second study showed a decrease in the number of HSC when Kitb was down-regulated. Based on this, the researchers concluded that, as shown in previous studies, Kita and Kitlga are not involved in hematopoiesis, but only in the formation of melanocytes. As a model organism, zebrafish needs to understand the regulation mechanism of its hematopoietic system development and its evolutionary conservation. The purpose of this study is to better understand the importance of Kit ligands in zebrafish hematopoiesis, so as to provide important insights for further research on hematopoietic development. We established suspension culture conditions for the expansion of zebrafish erythroid progenitor cells, and studied the effects of two zebrafish Kitlg ligands in zebrafish hematopoiesis by in vivo and in vitro experimental methods.

　　Animal populations and embryos: The fish are kept in the ZebTEC aquatic system. In this study, transgenic reporter gene lines expressing fluorescent genes under the regulation of tissue-specific promoters (gata1: DsRed and lcr: EGFP), as well as mutant lines (kitab5/b5 or sparse) and wild-type (WT) animals were used. In vitro experiments use 6-month-old fish to ensure the optimal number and condition of bone marrow cells in the whole kidney.

　　 In vitro culture: Isolate the bone marrow cells of the zebrafish whole kidney as described above. The cells were separated using Biocoll (1.077 g/mL) density centrifugation, initially seeded at 3×106 cells per ml, and cultured in zfS13 medium at 32°C and 5% CO2. Add specific cytokines (Epo, Gcsfa, Kitlga and Kitlgb) at a final concentration of 100 ng/mL, and add dexamethasone (Dex) at a final concentration of 1 µM. Use the CASY cell counter and analyzer to count the cells. In the next few days, maintain 2×106 cells per milliliter, and replace one third of the medium containing fresh cytokines and Dex every other day to ensure optimal cell growth. In order to study the clonogenic potential of whole kidney bone marrow cells after adding different cytokines, we performed clonal analysis in semi-solid medium (methylcellulose).

　　Cloning and expression of cytokines: First, use Phobius to predict the protein structure and analyze the hydrophobicity of the amino acid sequence of zebrafish Kitlga/b. Two large hydrophobic regions (aa 1-24 and 206-224 of Kitlga, aa 1-31 and 185-209 of Kitlgb) were identified respectively corresponding to the putative signal peptide (SP) and TM domains. In order to obtain a Kitlga/b version without these two domains, sequence-specific primers were used to polymerize the Kitlg complementary DNA (cDNA) fragments aa25～182 (Kitlga) and aa31～187 (Kitlgb) in the adult zebrafish retina Enzyme chain reaction (PCR) amplification. The baculovirus expression system is used to prepare soluble Kitlga/b in large quantities. The amplified fragment was cloned into a modified pAc-GP67-B vector containing 6xHis, and a recombinant rod was produced by co-transfecting pAc-His-Kitlga/b and BD BaculoGold Bright baculovirus DNA into sf21 insect cells Virus. Virus-infected cells express GFP and secrete recombinant His-Kitlga/b outside the cell. Finally, a Ni2 + -NTA agarose column was used to purify the secreted protein and use it for cell culture experiments. Prepare EPO protein and mRNA as described previously.

　　Result: Kit ligand promotes the erythroid and myeloid expansion of whole kidney bone marrow cells: First, we cloned and expressed recombinant zebrafish Kitlga and Kitlgb. Kitlga and kitlgb lacking SP coding region, intracellular region and TM region were amplified from adult zebrafish retina, and recombinant Kitlga and Kitlgb were prepared in sf21 insect cells. The His-tagged purified protein was used in the following experiments. In order to detect the biological activity of Kitlg protein, an experiment with whole kidney bone marrow cells isolated from 6-month-old zebrafish was designed. In order to reveal the biological activity and potential synergy of Kitlg protein and other recombinant cytokines, cells were treated with various factors or their combinations and counted at specific time points. After 3 days of culture, the potential enhancement of the myeloid system was evaluated. Two Kit ligands have been added, which are used separately or in combination with Gcsfa and have been shown to support myeloid cell formation. Interestingly, compared to the untreated control group, the number of cells under all tested conditions increased. Consistent with previously published studies, Gcsfa significantly promoted the growth of whole kidney bone marrow cells, and this effect was further enhanced by adding any Kit ligand. The additional effects of these two kit ligand paralogs are often similar. However, this synergy is only statistically significant in Kitlgb. In order to confirm that Gcsfa and Kitlga or Kitlgb jointly promote bone marrow production, we conducted morphological identification of cultured cells. On day 3, the culture mainly consisted of monocytes and macrophages. Although the addition of Kitlga or Kitlgb to Gcsfa will increase the cumulative number of cells, the overall composition of cell culture and the ratio of different cell types remain similar regardless of the presence of Kit ligands.

　　 Next, the Kit ligand was tested for its activity during the expansion of erythroid cells. Cells were treated with Kitlga and Kitlgb separately or in combination with Epo. The number of cells increased after 7 days of culture. Consistent with previously published studies, Epo significantly promoted cell growth, and when treated with any Kit ligand alone, the number of cells also increased slightly. Surprisingly, when Epo was used in combination with Kitlga, we noticed a significant increase in the number of cells after 7 days of culture compared to Epo alone. Although the coordination effect of Kitlgb is not significant, in the presence of Epo, the number of cells is still increasing. In order to check the composition of the cell culture, we further performed morphological identification of the cultured cells. On day 7, the culture consisted mainly of erythroid cells stimulated by Epo. As expected, the addition of Kitlga resulted in an increase in the number of erythroid progenitor cells, which explains the increase in the number of cells in this culture. In order to maximize the expansion of erythroid progenitor cells, we decided to improve the suspension culture conditions of whole kidney bone marrow cells. Although there is no report on suspension culture of zebrafish or hematopoietic progenitor cells, we speculate that conditions similar to humans and mice may be effective. Since different steroids play an important role in self-renewal and erythroid progenitor cell proliferation in chickens, mice, and humans, Dex was introduced in the experiment and tested with Epo and Kitlga in different concentrations and combinations. The results showed that as previously found in other vertebrates, Kitlga, Epo and Dex have a synergistic effect in in vitro culture to expand erythroid cells.

　　 Both Kit ligands act synergistically with Epo to promote the expansion of erythroid cells in zebrafish embryos: In order to detect the function of the two Kit ligands in vivo, we injected cytokine mRNA(s) into 1-cell embryos. Compared with epo alone, when kitlga and kitlgb are used in combination with epo, it shows lcr:EGFP expression and hemoglobin enhancement at 72 hours after fertilization (hpf). It is worth noting that the two Kit ligands have observed a similar trend of increasing the number of erythroid cells bound to epo, but in all types of experiments, this effect is only more effective for kitlgb, and there are statistical differences. The qPCR analysis of injected embryos also showed that at 72 hpf, the expression of erythroid cell markers increased. However, when epo binds to any ligand, these changes are quite subtle. Finally, consistent with previous studies, it was observed that the number of melanocytes increased after Kitlga overexpression, but Kitlgb did not.

　　 The expression of two kit receptors gradually increased during embryonic development: In order to better understand the cooperation mechanism between Epo and Kit ligands, qPCR was used to analyze the expression of the two kit receptors in tissues and during development. Among all tissues analyzed, kita has the highest expression in the kidney and kitb has the highest expression in the retina. During the embryonic development of zebrafish, the highest relative expression was observed for kita 7 days after fertilization and 72 hpf after fertilization. It was found that during the early development of erythroid cells, the expression of the two receptors gradually increased.

　　 In short, in this study, we used in vivo and in vitro methods to explore the role of Kitlg in zebrafish hematopoiesis.