动物实验,红细胞生成素,红细胞 z-bo 2020-09-11 342

　　Background: Red blood cells (RBC) are the most abundant cells in the blood and are essential for the transmission of oxygen in the body. After birth, the site where red blood cells are produced is transferred from the fetal liver to the bone marrow (BM) and spleen. In humans, the bone marrow is the main site of stable red blood cell production. In contrast, in mice, except for the bone marrow, the spleen plays a small role in steady-state erythropoiesis (10%). Under stress conditions such as bleeding and acute anemia, the spleens of humans and mice play a major role in stress erythropoiesis. Hematopoietic stem cells (HSC) exist in the bone marrow. The cytokines and signals produced by stromal cells composed of endothelial cells, osteoblasts and macrophages regulate the differentiation of various blood systems including red blood cells. HSCS first differentiated into macronuclear erythrocyte precursor cells, then burst erythrocyte colony forming cells (BFU-Es), and then erythrocyte colony forming cells (CFU-Es). CFU-es is more mature than BFU-es, and the colonies formed when CFU is cultured in methyl cellulose are smaller than BFU. Erislopoetin (EPO) is a 30.4 kDa glycoprotein, mainly synthesized by the kidneys, and is the main regulator of red blood cell proliferation, differentiation and survival. Under hypoxic conditions, EPO production is upregulated by hypoxia-induced transcription factor (HIF). EPO receptor (EPor) is mainly expressed in CFU-ES and is gradually down-regulated during erythroid differentiation. Under the stimulation of the combination of EPO and EPORS, CFUES develops into primitive red blood cells, then develops into basal spheres, then develops into polychromatic red blood cells, and finally develops into basal spheres. In the final stage of red blood cell differentiation, reticulocytes are enucleated and red blood cells mature into circulating red blood cells. Anemia is caused by a decrease in red blood cells, decreased production of red blood cells, increased destruction of red blood cells, or shortened lifespan of red blood cells. The World Health Organization defines anemia as a female hemoglobin level lower than 12 g/dl and a male lower than 13 g/dl. In addition to patients with inherited hematopoietic diseases, patients with chronic diseases (such as kidney and heart disease, cancer, inflammatory bowel disease, rheumatoid arthritis and human immunodeficiency virus (HIV)) have the highest incidence of anemia. I will. However, since 1998, some serious side effects have been reported, including EPO-related pure erythropoiesis caused by neutralizing antibodies induced by long-term injection of EPO. There are also other side effects, such as high blood pressure, venous thromboembolism, stroke and the risk of death. The currently available Erythronspirator (ESA) is a variant of EPO produced by human cells or Chinese hamster ovary cells. New ESA (such as peptidyl erythropoiesis) has been developed to activate endogenous EPO production and EPO gene therapy through HIF stabilization and GATA1 inhibition. However, none of these new methods are more effective than the existing ESA. Our previous research found that the granulocyte colony stimulator (G-CSF) can recruit newly synthesized red blood cells into peripheral blood (PB) to promote the differentiation and proliferation of red blood cells in vitro and in vitro. Yes, this study further investigated the effects of G-CSF and EPO on red blood cell production. After treating mice with G-CSF and EPO, BM and splenic red blood cell production were compared with flow cytometry. We studied the stimulation of early erythroid precursor subsets and the temporal regulation of erythroid precursor cell recruitment. The mechanism of G-CSF promoting erythropoiesis was discussed. Method: B. Charcoal lethal toxin (LT) was provided by the Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan, and purified as described above. EGFP mice [C57BL/6J-Tg(Pgk1-EGFP)03narl] and C57BL6J mice were purchased from the National Laboratory Animal Center.

　　Bone marrow and spleen red blood cell production analysis: C57BL/6J mice (male, 10-12 weeks old), injected with IU/grhEPO or 55μg/kg recombinant human G-CSF twice a day for 3 days. The same amount of normal saline was given as a negative control. The BM cells were isolated as described above. Grind the spleen with the plunger of a 50 ml syringe, and then resuspend it with a P1000 pipette to form a single cell suspension. The cells were blocked in RPMI-1640 medium containing 5% bovine serum albumin at 37°C for 1 hour. Subsequently, rat anti-mouse fluorescein isothiocyanate-labeled CD71 antibody (BioLegend) and rat anti-mouse xenoglobulin (APC) labeled ter-119 antibody (BD immune cell assay system) were placed at 37°C Incubate for 1 hour. After washing with phosphate-buffered saline (PBS), resuspend the cells in 1 ml PBS and use a Beckman Coulter Gallios? Analysis by flow cytometer.

　　Flow cytometry analysis of red blood cell recruitment in EGFP mice: Enhanced green fluorescent protein (egfp) mice (male, 8 months old) were injected into the tail vein of 55μg/kg G-CSF 2IU/gEPO once a day or for 3 consecutive days. . 20, 40 and 60 hours after the first injection, peripheral blood was drawn from the tail vein. Incubate with rat anti-mouse APC bound ter-119 antibody at 37°C for 1 hour. Then it was treated with 5μmRNA selective dye F22 at 37°C for 30 minutes. After washing with PBS, use a Beckman Coulter Gallios? Analyze cells by flow cytometry. EPO and soluble P-selectin immunoassay: C57BL/6J mice (male, 10-12 weeks old) were injected with 55μg/kg G-CSF into the orbit, once a day, for 2 consecutive days for EPO immunoassay, 5 consecutive days Run for a day. The soluble p-selectin immunoassay was performed once a day. Animals treated with the same amount of physiological saline were used as negative controls. 22, 44, and 66 hours after the first injection, PB was collected for EPO immunoassay. The soluble P-selectin immunoassay was performed 0 days before the first injection and 1-5 days after the first injection. According to the manufacturer's instructions, the enzyme-binding immunosorbent assay (ELISA) is used for EPO and soluble P-selectin immunoassays.

　　Detection of hematopoietic parameters: C57bl/6J mice (10-10 week old males) were injected retro-orbitally with 55μg/kg G-CSF or 1 mg/kg recombinant mouse p-selectin, and were treated for 2 consecutive days every day. Two days after the first injection, hematopoietic parameters were measured using an automatic hematology analyzer.

　　Results: G-CSF can make the R4 erythropoiesis ability of BM and spleen surpass EPO: G-in C57BL/6J mice lasted 3 days to compare the erythropoiesis ability of G-CSF and EPO after administration of CSF Or EPO is injected into the tail vein. The process of red blood cell production in bone marrow and spleen was analyzed by flow cytometry. Using antibodies against red blood cell markers CD71 and Ter119, red blood cells are divided into four groups to distinguish early to late: primitive red blood cells (R1), eosinophils (R2), late eosinophils and polystaining. Sexual acid red blood cells (R3) and primary color eosinophils (R4). Consistent with the results of other studies, in mouse bone marrow, G-CSF enhanced the non-lymphocyte population than EPO, while EPO significantly increased the total number of red blood cells. The total number of red blood cells in the bone marrow of mice in the G-CSF treatment group was lower than that in the EPO treatment group, but the R4 population induced by G-CSF was significantly higher than that in the EPO treatment group. The spleen is a secondary organ for red blood cell production, not for granulocyte production. When explaining the process of mouse spleen differentiation, we found that G-CSF treatment did not increase the number of non-lymphocytes or total red blood cells. Compared with the effect of BM, EPO up-regulated the ratio of early red blood cells (R1 and R2) and down-regulated the late red blood cells (R3 and R4), while G-CSF up-regulated the early R1 and R2 and the number of late R4 red blood cells significantly reduced The ratio of R2 and R3 cells. In summary, these results indicate that G-CSF can promote red blood cell production in mouse bone marrow and spleen more than EPO.

　　G-CSF mobilizes newly synthesized reticulocytes than EPO: Using the EGFP transgenic mouse model, G-CSF mobilizes newly synthesized red blood cells to PB and red blood cells than EPO and found that peripheral blood induces them to enter at a higher speed. After using the same experimental strategy, 20 hours after G-CSF treatment, the mobilization efficiency of newly synthesized red blood cells (R1) showed higher than that of EPO treatment. The recruitment efficiency after the first injection of G-CSF and EPO is 40 and 60 hours. the same. Since G-CSF can promote the synthesis of R4 red blood cells in BM and spleen more than EPO, we hypothesized that G-CSF can promote the recruitment of reticulocytes more than EPO. In the newly synthesized red blood cell population, only reticulocytes show residual ribonucleic acid (RNA) expression. Therefore, the RNA selective dye F22 is used in combination with ter-119 to mature together with the reticulocytes in the newly synthesized red blood cells. Distinguish red blood cells. In the two phases of 20 and 40 hours, G-CSF tends to mobilize more newly synthesized reticulocytes than EPO. Can not achieve H-CSF-dependent erythropoiesis and erythropoiesis by inducing EPO and P-selectin pathways: G-CSF up-regulates the transcription factor HIF-1α and 5 consecutive Gs bind to the EPO promoter-CSF injection Can increase circulating EPO levels. We hypothesize that G-CSF can stimulate red blood cell production and mobilize red blood cells by increasing EPO synthesis. Bacillus anthracis (a type of LT that induces hypoxia and induces EPO secretion in mice) was used as a positive control. Consistent with previous studies, our results show that after long-term administration to mice, circulating EPO levels gradually increase. In contrast, after G-CSF treatment in mice, no increase in circulating EPO levels was found in all examinations. The results indicate that enhanced G-CSF-dependent erythropoiesis and red blood cell recruitment are not mediated by EPO induction. P-selectin is widely regarded as a cell adhesion receptor, which regulates leukocyte rolling by binding to the glycosylated part of the main ligand P-selectin glycoprotein ligand 1 (PSGL-1). Due to the interaction of p-selectin (hsc)-psgl-1 (interstitial), the same properties as p-selectin attach hscS to stromal cells, especially follicular endothelial cells. G-CSF induced PSGL-1 knockout mice to mobilize larger bone marrow-like cells faster than wild-type mice. Studies have also shown that administration of G-CSF can increase the level of circulating soluble P-selectin. These evidences indicate that P-selectin/PSGL-1 mediated binding between erythroid progenitor cells and stromal cells partially regulates the recruitment of progenitor cells and the release of G-CSF from the BM to the bloodstream. Indicates that the proposal is adopted. It induces precursor-matrix interactions through circulating soluble P-selectin and P-selectin/PSGL-1 to promote red blood cell recruitment. To reproduce the G-CSF-mediated induction of circulating soluble P-selectin, G-CSF was injected into C57BL/6J mice for 5 consecutive days. After G-CSF injection, the level of circulating soluble P-selectin increased significantly. We designed an experiment to test whether soluble P-selectin can increase the number of red blood cells in peripheral blood. Two doses of G-CSF can increase peripheral blood white blood cell (WBC) and red blood cell counts, but the same effect was not observed when the second dose of soluble P-selectin was used. This finding indicates that G-CSF mainly does not mobilize red blood cells to peripheral blood through the P-selectin pathway.

　　Conclusion: The combination of G-CSF and EPO has been used to treat MDS, aplastic anemia and anemia in HIV patients for many years, but the mechanism of G-CSF in promoting erythropoiesis is still unclear. This study shows that G-CSF promotes erythropoiesis and has nothing to do with the secretion of EPO and soluble P-selectin. In addition, G-CSF induces BM and spleen instead of EPO to produce R4 red blood cells. G-CSF treatment absorbs newly synthesized reticulocytes into PB instead of EPO.