动物模型,白细胞疾病 z-bo 2020-08-10 410

　　1 Animal model of leucopenia

　　In the attenuation experiments to study the toxicity of chemotherapy drugs, it is often necessary to establish animal models of chemotherapy-induced leukopenia and bone marrow nucleopenia. Leucopenia (leucopenia) is a common clinical blood disease. When the number of white blood cells in the peripheral blood of the body is continuously lower than 4×1000000000/L, it is called leukopenia; if the total number of white blood cells in the body is significantly reduced, lower than 2×1000000000/L, the absolute value of neutrophils is lower than 0.5×1000000000 /L even disappears, it is called agranulocysis (agranulocysis). Clinically, the clinical manifestations of patients with leukopenia are mainly fatigue, dizziness, and are often accompanied by symptoms such as loss of appetite, weak limbs, insomnia and dreams, low fever, palpitations, and cold backache; while agranulocytosis is mostly caused by sudden onset and fear Cold, high fever, sore throat are the main ones. Agranulocytosis is a manifestation of the development of leukopenia to a severe stage. The etiology and pathogenesis of the two are basically the same. Clinically, leukopenia can be divided into two types: unexplained and secondary. Among them, the former are more common, while the latter are mostly chemical factors, physical factors, drug factors and certain diseases; or can be seen in various solid tumors after chemotherapy, a variety of blood diseases, serious infections and unknown causes.

　　1.1 Rat leukopenia model

　　(1) Reproduction method SD rats weighing about 150g are injected with cyclophosphamide through the abdominal cavity at a dose of 30mg/kg body weight for 5 days. The model rats were taken blood samples before administration, on the 3rd, 5th day, and on the 2nd, 5th, 8th, 11th, and 15th day after the drug was discontinued. The total number of white blood cells in the peripheral blood was observed. After the last observation, the animals were killed by anesthesia, and their femurs were taken, bone marrow cells were washed with hanks solution, and the number of bone marrow nucleated cells was observed under a microscope.

　　(2) Characteristics of the model After the injection of cyclophosphamide, the food consumption and water consumption of the model rats are reduced, the activity is reduced, the coat is loose and dull, the weight gain slows down, and the body is obviously thin; the body begins to recover significantly after stopping the drug for 15 days. The total number of white blood cells in the peripheral blood of the model rats was significantly reduced after 3 days of administration, and the total number of peripheral blood white blood cells could be reduced to the lowest value on the 5th day of administration; the total number of white blood cells did not increase significantly on the second day after the drug was stopped. On the 11th and 15th day, the total number of white blood cells showed a gradually rising trend; the low white blood cell level of model rats maintained for a long time, and there was no obvious recovery on the 15th day after drug withdrawal. Microscopic observation showed that the number of bone marrow nucleated cells in model rats was significantly lower than that in normal animals.

　　(3) Comparative medicine. Through multi-point dynamic analysis of the total number of white blood cells, changes in body weight, and the number of bone marrow nucleated cells in the animal body, the model rats showed low white blood cell, low bone marrow nucleated cells and low body weight. Clinically, tumor patients have the same chemotherapy toxicity. This model can be used for the prevention and treatment of clinical chemotherapy toxicity, drug screening or other prevention and treatment methods. Previous studies in this area have used mice to establish models, but because the peripheral blood leukocytes of the mice can exceed the basic level after the 6th day, the experimental observation time is often insufficient and the experimental results cannot be evaluated. In the model established by this method, the white blood cell of the model rat recovered slowly after the drug was stopped, and the low white blood cell level was still maintained on the 15th day after the drug was stopped, so it can meet the observation time of the experimental study.

　　1.2 Animal model of acute myelogenous leukemia

　　Clinically, acute myelogenous leukemia (AML) refers to the malignant transformation of one or more hematopoietic stem cells and progenitor cells in the body, loss of normal proliferation, differentiation and maturation, uncontrolled continuous proliferation, and gradually replace bone marrow and infiltrate blood To body tissues and organs.

　　1. Mouse granulomonoleukemia model

　　(1) Copy method BALB/c male mice weighing about 20 g and 8 weeks old. Centrifuge the culture solution of acute myelomonocytic leukemia cells at 1000 r/min for 10 min, remove the supernatant and wash with normal saline 3 times to adjust the concentration to 2×1000000000/L. The prepared cell suspension was injected into the tail vein of mice, 0.5ml/mouse. After vaccination, the survival days of model mice were observed every day, and blood was collected for detection of peripheral blood white blood cell count, percentage of immature myelomonocytic cells and percentage of bone marrow blast monocytes. The morphology of bone marrow leukemia cells was observed under transmission electron microscope.

　　(2) Model characteristics: The tail vein inoculation method is adopted, and the survival time of the model mice can reach (38.40±2.21) days; its peripheral blood white blood cell count and the percentage of immature myelomononuclear cells increase significantly on the 14th day of vaccination , Respectively, are (19.24±1.73)×1000000000/L and (6.24±0.85)%, and there is a gradual upward trend; the percentage of bone marrow blast mononuclear cells also increases significantly, reaching (41.3±4.90)%. The model making method is simple and easy to master, the model has a high success rate and good repeatability. During the experiment, the model mice have weakened immunity, so it is necessary to strengthen the control of animal experiments and feeding management conditions.

　　(3) Comparative medicine Clinical acute myeloid leukemia is characterized by two types of immature cells derived from primordial myeloid cells in the bone marrow or peripheral blood. The pathological characteristics of this model are similar to clinical human acute myelomonocytic leukemia, and it is suitable for clinical research on the pathogenesis and drug treatment of similar diseases.

　　1.3 Animal model of chronic myelogenous leukemia

　　Clinically, chronic myelogenous leukemia (CML) can be divided into chronic myelogenous leukemia and chronic lymphocytic leukemia. Chronic myelognous leukemia, referred to as chronic myelognous leukemia (CML), is a clinically onset and relatively slow-developing leukemia. It is a malignant proliferative disease originating from pluripotent hematopoietic stem cells in the bone marrow. It is manifested by the expansion of the myeloid progenitor cell pool and the excessive growth of myeloid cell line and its progenitor cells. Clinically, CML mainly manifests as fatigue, weight loss, fever, splenomegaly and abnormal increase of white blood cells. Chronic lymphocytic leukemia, referred to as Chronic Lymphocytic Leukemia (CLL), is a disease in which the body's lymphocytes proliferate and accumulate abnormally in the body accompanied by a weakened immune function. Because the lymphocytes of chronic lymphocytic patients have a very long life span and are often accompanied by immune response defects, it is also called immune incompetent lymphocyte accumulation disease. The main clinical manifestations are mainly lymphadenopathy, often accompanied by hepatosplenomegaly, anemia and bleeding. A few patients also have skin damage.

　　1. Immunodeficiency mice-human chronic myeloid leukemia model

　　(1) Replication method NOD/SCID mice, which are obtained by backcrossing SCID mice and NOD/Lt strains (TL, BL, NK cell defects, lack of circulating complement, differentiation and dysfunction of antigen presenting cells). scid/scid mice (NOD-SCID mice for short). The mice were first irradiated with 400cGy 60Co gamma rays, and then leukemia cells were isolated from the fresh bone marrow and peripheral blood of patients with chronic CML who had never received chemotherapy. The infusion of (8～14)×10000000 leukemia cells through the tail vein has been sublethal Measure the mice irradiated with radiation, and alternately inject PIXY321 (7μg) and c-kit ligand (10μg) into the intraperitoneal cavity. Mice were sacrificed within 30 to 90 days after CML cell transplantation, and Southern blot analysis and flow cytometry were performed with human bcr-specific DNA probes.

　　(2) Model characteristics About 70% of human CML cells in transplanted NOD/SCID mice are Ph- cells. Whether it is transplanted with bone marrow cells or peripheral blood cells from CML patients, the detection level of ph+ cells is very similar. Using this method to replicate the CML model, NOD/SCID mice are better than SCID mice. Due to the lack of functional NK cells in NOD/SCID mice, a smaller number of patients' CML chronic phase cells can successfully establish a model. If artificial hematopoietic stem cells or fetal liver cells are transplanted into NOD/SCID mice, the number of human myeloid leukocytes will increase significantly, the artificial hematopoietic stem cells will differentiate to the myeloid lineage, while the red lineage cells and lymphoid lineage cells will be significantly reduced; Such as the use of patient CML cells, especially the accelerated phase and blast phase cells are easier to transplant successfully.

　　(3) Previous studies of comparative medicine have found that tumor cells derived from the blast stage of CML patients can reproduce in SCD mice. Leukemia cells are mainly distributed in bone marrow and peripheral blood before they are transferred to tissues without hematopoietic function. similar. However, because the hematopoietic growth factors, adhesion molecules and extracellular matrix of mice are not exactly the same as those of humans, the established immunodeficiency mouse-human chronic myelogenous leukemia model still has certain limitations. However, NOD/SCD mice can still be used to prepare ideal human CML animal models.

　　2. Transfect mouse bone marrow cells with P210 retroviral vector and transplant normal mice

　　(1) Replication method Isolate male BALB/c mouse bone marrow cells pretreated with fluorouracil (5-Fu) (150mg/kg body weight), and transfect them with P210 gene retroviral vector, press (1～2)×100000 cells It was injected into a homologous female mouse that received a lethal dose of radiation. Mice with an incubation period of more than 20 weeks show abnormal proliferation of granulocytes and hematopoietic dysfunction. Cells isolated from these mice can be effectively transplanted into irradiated homologous mice, indicating that mice transfected with bcr/abl gene Bone marrow cells have a high regenerative capacity and can be used to simulate human CML. The MSC retroviral vector can increase the titer of the virus expressing the ber/abl fusion gene. The CML incubation period is short. All mice receiving the transfected cells can develop CML after the incubation period of 4 to 6 weeks. The main changes include a significant increase in peripheral blood leukocytes, spleen enlargement, and large expression of bcr/abl fusion protein.

　　(2) Model characteristics It was found that no matter what the form of the abl gene, 90% of mice developed tumors. About 50% of these mice showed abnormal bone marrow proliferation, which has many similarities with the chronic phase of human chronic myeloid leukemia, while the remaining mice showed abnormal proliferation of B lymphocytes.

　　(3) Comparative medicine The CML animal model established by this method and clinically human CML still have some differences. For example, most CML model mice often die quickly in a short period of time, and do not experience the chronic phase similar to human CML, and the lungs of the model animals Partial bleeding is obvious, which is rare in human CML.

　　3.bcr/abl transgenic mouse model

　　"(1) Replication method The promoter sequence (-1948 to +22) of the mouse Tec gene is used as a regulatory sequence to prepare a transgene vector. The promoter sequence of Tec gene regulates the expression of P210 protein to prepare transgenic mice. The expression of bcr/abl fusion protein is lethal to mouse embryos.

　　(2) Characteristics of the model At present, it is still not guaranteed that all the obtained transgenic mice and their offspring will show symptoms of CML, and some individuals may show symptoms of acute leukemia.

　　(3) Comparative Medicine Most transgenic mice first develop abnormal granulocyte proliferation and hyperthrombocytosis, and develop myeloproliferative syndrome (MPD) after a certain period of time, which is very similar to human CML. A few transgenic mice showed acute leukemia after birth, but their offspring showed myelodysplastic syndrome after a period of incubation. It shows that choosing the right promoter is very important for constructing CML transgenic mice.