再生障碍性贫血,动物模型 z-bo 2020-08-03 665

　　Aplastic anemia (aplastic anemia) usually refers to the primary myelogenetic syndrome of unknown cause. It mainly manifests as low bone marrow production, pancytopenia, anemia, bleeding and infection.

　　(1) Animal model of aplastic anemia caused by chemical toxicity

　　[Modeling mechanism] The toxic and side effects of chemicals can interfere with bone marrow production and cause aplastic anemia. For example, in the modeling method of adenine nephrotoxicity, erythropoietin (EPO) secreted by the kidney is a cytokine required for the differentiation and maturation of red blood cells and megakaryocytes. Adenine can cause kidney disease and secrete EPO. Failure then leads to impaired hematopoietic function of red blood cells and megakaryocytes. Busulfan has selective bone marrow suppression. A single lethal dose or multiple low doses can cause hematopoietic stem cells and bone marrow microenvironment to be inhibited. Cyclophosphamide is commonly used. The side effect of its anti-tumor and immunosuppressive agents is bone marrow suppression, which reduces the production of hematopoietic cells. Benzene can be metabolized in the liver and then converted into hydroquinone and phenol. Both hydroquinone and phenol can bind to bone marrow DNA. It inhibits its expression and leads to impaired hemoglobin synthesis.

　　[Modeling method]

　　1. Rats with adenine-induced renal anemia were given 300 mg/(kg·d) intragastrically every day for 6 weeks. Obtain an anemia model of renal failure.

　　2. Model mice with renal anemia induced by busulfan are injected with busulfan 10 mg/kg daily for 8-9 days. If the drug is stopped for 60 days, the hematopoietic function of the bone marrow may be slightly inhibited. An increase in the dose may exacerbate the inhibitory effect. A single dose of 20-35 mg/kg busulfan can significantly inhibit bone marrow regeneration in rats. A weekly dose of 15 mg/kg or 30 mg/kg of busulfan to rabbits can cause aplastic anemia in rabbits when the total amount reaches 120-150 mg/kg.

　　3. Cyclophosphamide-induced renal anemia mouse model mice intraperitoneal single intraperitoneal injection of cyclophosphamide 100 mg/kg or cyclophosphamide 50 mg/kg intraperitoneal injection, repeated injections or 50 mg/kg subcutaneous injection three days later , 4 injections every other day.

　　Four. Benzene-induced renal anemia model mice were injected subcutaneously with 0.5 mg/kg benzene daily for 2-3 weeks.

　　[Model Features] After the chemical model, peripheral red blood cells, white blood cells, hemoglobin and bone marrow granulocytes decrease, the proportion of immature granulocytes increases, and the proliferation ability of bone marrow hematopoietic cells is weakened. However, the disadvantage of the chemical method is that the modeling time is long and it is easy to cause permanent damage to the bone marrow. [Evaluation and application of the model] This model uses the side effects of drugs that induce aplastic anemia, similar to human aplastic anemia. This model has a high success rate and stable results, but the model has a long duration and is prone to permanent bone marrow damage. This model provides an excellent experimental method for the development of experimental research on aplastic anemia and the screening of therapeutic agents for aplastic anemia.

　　(2) Physical methods to induce aplastic anemia animal model

　　[Modeling mechanism] Radioactive substances invade the human body, cause DNA damage, interfere with DNA replication, and inhibit mitosis. The strong inhibitory effect on active dividing and proliferating cells such as hematopoietic stem cells can reduce hematopoietic stem and progenitor cells in animals and destroy bone marrow The proliferation capacity of hematopoietic cells in the medium.

　　[Modeling method]

　　1. Mice irradiated with 3-4 Gy dose of X-ray or 60Co-γ can reduce the number of hematopoietic stem cells, while 8-9 Gy can kill most hematopoietic stem cells.

　　2. Using 32P (32 phosphorus) to give mice an intravenous injection of 1.4 mCi/kg body weight can cause aplastic anemia.

　　[Characteristics of the model] This model uses mice exposed to lethal or lethal X-rays or Co-rays and is found to have severe hematopoietic dysfunction. Peripheral red blood cells, white blood cells, and hemoglobin were all significantly reduced. The existing bone marrow granule cell lines were all lower than normal. The proportion of immature granulocytes increased, megakaryocytes also decreased significantly, and the growth ability of bone marrow cells decreased. [Evaluation and application of the model] The radiation factor used in this model that can cause aplastic anemia is similar to the human radiation factor that causes aplastic anemia, and the success rate is high and stable results can be obtained. There is an advantage. However, it requires special equipment and protection. It provides an excellent experimental method for conducting radiation protection research.

　　(III) Immune-mediated aplastic anemia animal model

　　[Modeling mechanism] After co-injection of active lymphocytes from different strains of mice, it produces immune-mediated effects that directly inhibit bone marrow production and IL-2, IFN-γ and other negative hematopoietic regulators indirectly inhibit bone marrow production.

　　[Modeling method] DBM2 mice were selected as cell donors at 6 to 14 weeks, BALB/c mice and females at 8 to 10 weeks. Preparation of thymic lymph node cell suspension: donate blood to DBA/2 mice, remove the thymus and lymph nodes aseptically, such as the neck, maxilla and mandible, axilla, groin, mesenteric and a small amount of normal saline. Add, grind and filter to form a single cell suspension. .. Trypan blue live cells to confirm that the cell survival rate should reach 95% or more, and the cells should be counted to the required concentration. After irradiating BALB/c mice whole body with 60Co-γ rays 5Gy, mixed cells from DBA/2 mouse thymic lymph nodes were injected through the tail vein on the same day, and the input cell volume was 1 x 1000000/0.2 ml respectively. Two months later, the animals were sacrificed, the bilateral femurs of the mice were excised, the two femurs were cut open with scissors to expose the bone marrow cavity, and 2ml of PMI1640 medium was aspirated. Wash the bone marrow cavity repeatedly, collect all bone marrow cells and mix well, and count the bone marrow under a microscope. Nucleated cells, peripheral blood imaging and T cell subgroup determination, IFN-γ determination, histological examination and observation of bone marrow ultrastructure. [Characteristics of the model] Experimental mice in the aplastic anemia group gradually lost disorganized fur, weight loss and restlessness from the 8th day, and gradually died after the 20th day. The autopsy revealed thin organs, thymus and spleen. Lymph nodes atrophy, and some mice have bleeding spots in the small intestine. In the aplastic anemia group, the number of nucleated cells in the peripheral blood and bone marrow of mice was significantly reduced, the T cell subset Th was significantly reduced, Ts was significantly increased, and IFN-γ was significantly increased. The bone marrow hyperplasia of the experimental mice was very low, the nucleated cells were significantly reduced, the hematopoietic cells replaced a large number of fat cells, and the number of non-hematopoietic cells increased; the spleen was atrophied and the number of small spheres was small. Very few, very small in shape. Hematopoietic and stromal cells are significantly reduced, different types of cells are depleted of ribosomes, mitochondria are swollen and degenerated, showing vacuoles or myeloid degeneration, no obvious or disappearing, endoplasmic reticulum and ultrastructural evidence showing different degrees of nuclear membrane The swelling, sinusoidal relaxation of blood, leaching, bleeding and structural destruction.

　　[Evaluation and Application of Model] Experimental research on aplastic anemia has been carried out. The effects of drugs have been observed and effective drugs have been screened out. Therefore, the success rate of aplastic anemia is high, the results are stable, and the onset time is short. Has the advantage of focusing and delaying death. Provides a good experimental method. It is usually used to study the causes of aplastic anemia and observe the effects of drugs.