动物模型 z-bo 2020-08-10 512

　　Clinically, aplastic anemia (AA) is a group of hematopoietic tissue failure syndromes. The patients are mostly young adults, and the prognosis of acute cases is extremely poor. Only chronic mild cases can be relieved or cured. The cause of aplastic anemia has not yet been fully elucidated. The known cause is mainly a series of functional and morphological changes caused by damage to the body's bone marrow pluripotent stem cells and the microenvironment, which further lead to pancytopenia; possible causes include physical and chemical And biological factors. In addition, the body's immune factors play a certain role in the occurrence and development of this disease. The main clinical symptoms of patients with aplastic anemia are anemia, bleeding, and repeated infections; three kinds of blood cells are reduced at the same time, without liver and spleen or lymph node enlargement.

　　1 Physical stimulation

　　(1) Copy method Adult mice can be irradiated with 60Co-γ rays 7.0Gy at one time with a dose of 7.0Gy. The irradiation distance is 2m and the radiation dose rate is 2.275Gy/min. The AA animal model can be successfully copied.

　　(2) Characteristics of the model Physical radiation can cause severe hematopoietic dysfunction of the body, as well as damage to various tissues and organs of the model animal, with greater side effects. When using 60CO-γ ray whole body irradiation to create a model, if the transient sexual organ damage is too severe, the mortality rate of the model mouse is very high; and if the transient damage is too light, the hematopoietic function of the mouse can recover by itself, which affects the modeling effect. Therefore, although the model preparation method is simple and easy to operate, the correct choice of radiation dose is a key factor for the success of the model preparation.

　　(3) Comparative medicine After being irradiated with sublethal or lethal X or C-rays, the mouse hematopoietic function can be severely impaired, and the peripheral blood cells are reduced. Its disease characteristics are similar to clinically human acute aplastic anemia. can

　　Used as an aplastic anemia animal model for experimental research.

　　2 Chemical induction method

　　(1) Method of replication: The Malilan was made into a 0.05% suspension with distilled water, and the mice were intragastrically administered at a dose of 4ml/kg body weight for 45 days. After 45 days of administration to model mice, the peripheral blood white blood cells, platelets and hemoglobin of most mice can be reduced by more than 1/3 compared with normal mice, and the number of pluripotent hematopoietic stem cells in the femur of mice is only about 40% of that of normal mice. .

　　(2) Model characteristics The model preparation method is simple and reliable, and the effects of marilan and other drugs are obvious. However, compared with the physical stimulation method, the modeling time period is longer, which is likely to cause permanent bone marrow damage in the model animal.

　　(3) Comparative medicine Malilan can damage hematopoietic stem cells in model animals, significantly reduce the number of hematopoietic stem cells, and slow natural recovery. This model is suitable for research on drug treatment and drug screening for aplastic anemia.

　　3 immune-mediated method

　　(1) Copy method BALB/c mice weighing 16-20g and 8-12 weeks old are used as recipients; DBA/2 mice aged 8-10 weeks are used as donors; both mice Male and female are not restricted. Take DBA/2 mice and kill them by cervical dislocation. After soaking and disinfecting with 95% ethanol for 5 minutes, remove the lymph nodes from their thymus, underarms, and groin aseptically, and add RPMI-1640 culture medium to remove surface blood stains and attached connectives. After cleaning the tissue again, use a scalpel to repeatedly cut the tissue until it becomes a paste, then gently grind and filter with a 200-mesh nylon mesh to make it a single cell suspension. After counting, the concentration is 1×1000000000/L, and the ratio of thymocyte: lymphocyte is 1:2. Take 1 drop of trypan blue and drop it on the glass slide to identify that the cell viability should be above 95%. The BALB/c mice were irradiated whole body with γ-ray 6.0Gy for 3min, and within 1～4h after sublethal dose irradiation, 0.2ml/mouse was used to immediately inject the above cell suspension through the mouse tail vein with a syringe. Make an animal model of aplastic anemia.

　　(2) Model characteristics The AA model replicated by this method has good stability and reproducibility, but the production method is more complicated, requires high preparation conditions, and the mortality of the model animals is relatively high, usually all of them die within 12 to 14 days. Therefore, it is suitable for the experimental study of short-term acute aplastic anemia. If it is used in the study of pharmacodynamics, especially the study of the pharmacodynamics of traditional Chinese medicine, it will be limited by the time of research.

　　(3) Comparative medicine The mouse model of aplastic anemia established by immune-mediated methods, the clinical symptoms, peripheral blood and bone marrow changes in model mice are basically the same as those of clinical human aplastic anemia patients. Among the many negative regulatory factors of hematopoiesis in the body, the abnormally increased levels of IL-2 and TNF can show a significant inhibitory effect on the production of CFU-GM, so it has always been considered to be related to human immune-mediated aplastic anemia. The formation of hematopoietic inhibitory activity is closely related. Therefore, this model is suitable for experimental research of acute aplastic anemia related to immune factors.

　　4 Compound method induction method

　　1. Animal model of aplastic anemia induced by cyclophosphamide plus chloramphenicol

　　(1) Replication method Male KM mice weighing about 20g and aged 6-7 weeks are irradiated with 60Co-γ rays 3.0Gy and given cyclophosphamide (CY) 50.0mg/kg on the 4th day Body weight and chloramphenicol (CH) dose of 62.5 mg/kg body weight were administered continuously for 3 days. On the 8, 12, and 16 days after administration, the blood of the mice was taken for routine blood test after anticoagulation, and the mice were sacrificed on the 8th day, and the left femur was taken, fixed with Bouin solution, and decalcified with nitric acid for routine routine Tissue section, observe under light microscope.

　　(2) Model characteristics: The peripheral blood leukocytes, platelets, and hemoglobin of model mice were significantly lower than those of normal mice on the 8, 12, and 16 days. Histopathological observations under the microscope showed that the bone marrow hyperplasia of the model mice was extremely low, and hematopoietic cells were reduced (the volume of hematopoietic cells<20%); there were more fat cells in the bone marrow, interstitial edema, sinusoid expansion, no megakaryocytes and other changes. The characteristics of aplastic anemia. The model is simple in production method, short in replication period, high in success rate, and low in animal mortality. The disadvantage is that the hematopoietic function of the model mice can be restored to normal on the 25th day. If it is used as an AA model for research, the animal should be sacrificed at about 20 days, and testing of various indicators is ideal.

　　(3) Comparative medicine CY is an alkylating agent in this model making method. It has a cytotoxic effect, can inhibit DNA synthesis of the body's cells, and is sensitive to the hematopoietic system. It can slowly but persistently inhibit the bone marrow of model animals. And CH can inhibit the body's bone marrow hematopoiesis, leading to the body's AA. γ-ray irradiation can affect the body's DNA replication, inhibit cell mitosis, reduce hematopoietic stem cells, and affect the body's immune function.

　　2. Animal model of aplastic anemia induced by cyclophosphamide and toluene

　　(1) Reproduction method Use KM mice weighing about 20 g and aged 6 to 8 weeks. One day before modeling, after disinfection of the local skin of the model animals, the blood picture was checked by tail-cutting blood sampling method, and blood was taken from the mouse orbit to push the blood film, stained with yellow tar blue, reticulocyte count, and animal hemoglobin (Hb) recorded , Platelet (PLT), red blood cell (WBC) and reticulocyte (RET) cell count. The next day, the mice were injected with cyclophosphamide solution subcutaneously at a dose of 50mg/kg body weight, once every other day, 4 times in total; at the same time, the mice were put into the cylinder and given toluene inhalation. The toluene concentration was 30mg/L. Once a day, 2 hours each time, a total of 8 times, on the 10th and 35th day of exposure, the blood of the model animals was taken to observe the blood picture and reticulocytes. On the 11th day of modeling, some of the mice were killed by cervical dislocation. The bone marrow (femur and sternum) was fixed with Bouin solution, and the spleen, liver and other organ tissues were fixed with 10% formaldehyde solution, embedded in paraffin, and made conventional tissue sections. HE staining, observe under light microscope. The blood picture of the remaining model mice was measured on the 35th day of modeling, and the auricle microcirculation was observed (with 30mg/kg body weight sodium pentobarbital anesthesia, the animal’s auricle arterioles and microcirculation were observed under a microcirculation microscope. Vein caliber), and then sacrifice the animal to take bone marrow and spleen for histopathological examination. Peripheral blood tri-line cells decreased, reticulocytes decreased, bone marrow biopsy showed low bone marrow hyperplasia, increased non-hematopoietic cells, and spleen atrophy, which can be judged to be successful.

　　(2) The characteristics of the model The animal model of aplastic anemia has been established and used for experimental research. The modeling methods used mainly include chemical methods (such as cyclophosphamide, marilan, benzene, etc.) and physical methods (such as repeated irradiation, injection Radioisotopes 89Sr, 32P, etc.) and biological (immune-mediated) methods. Cyclophosphamide is an alkylating agent, which can inhibit the DNA synthesis of cells in the body and has a cytotoxic effect that reduces the number of whole blood cells. There are many people who are frequently exposed to toluene (or benzene derivatives) in clinical practice, such as paint sprayers, shoemakers, printing and dyeing workers, etc., who are prone to such diseases. The use of cyclophosphamide and toluene compound method to make aplastic anemia animal model is in line with the common cause of the disease. The model making method is simple, the copy cycle is short, and the model success rate is high. If only cyclophosphamide is used to make the model, it can be found that the number of white blood cells in the peripheral blood of the model animal has decreased significantly, while the number of red blood cells has decreased slowly but not significantly, and the model cycle is shorter. If benzene is used alone, the animal's whole blood cells are reduced and the cycle is short. For example, radiation modeling is better than cyclophosphamide, the decline is more obvious in each series, and the maintenance period is longer. But the dosage is not easy to control. There are reports in the literature that several methods are used to jointly create models, such as 60CO-cyclophosphamide-acetophenazine, which can quickly reduce the number of peripheral red blood cells, with a long and stable cycle, but the experimental requirements are strict, cumbersome, and not easy to be widely used.

　　(3) Comparative medicine. Clinically, aplastic animer (AA) is a disease characterized by hematopoietic tissue failure due to the proliferation of hematopoietic stem/progenitor cells, differentiation disorder or abnormal hematopoietic microenvironment. The cause of aplastic anemia is complex, and patients are often associated with frequent exposure to chemicals, radiation, or autoimmune diseases. Model animals replicated by this method showed pancytopenia, reduced reticulocytes, reduced bone marrow hematopoietic stem cells, increased non-hematopoietic components, spleen atrophy, bleeding, infection, etc., which are similar to clinical symptoms of human aplastic anemia It can be used for research on the pathogenesis, drug treatment and drug screening of human aplastic anemia.

　　3. The animal model of aplastic anemia induced by cyclophosphamide plus 60Co-γ rays plus acetophenazine

　　(1) Reproduction method BA1B/c mice weighing 18-22 g and 6-8 weeks old, 60 BA1B/c mice. On the first day of modeling, mice were injected subcutaneously with acetylphenylhydrazine (APH) at a dose of 80 mg/kg body weight. On the second day, the model mice were treated with 2cGy of 60Co-γ radiation; on the third day after irradiation, Cyclophosphamide (CY) was injected intraperitoneally to mice at a dose of 50 mg/kg body weight. Respectively within a predetermined time, collect mouse blood for peripheral blood test; and sacrifice the mouse by cervical dislocation, take the mouse femur under aseptic condition, wash out the bone marrow cells with 2ml IMDM medium, prepare single cell suspension, draw 100μl for bone marrow Nucleated cell (BMC) count. Use mouse bone marrow cells on day 7 for cell culture to prepare a culture system. The cells were inoculated and cultured in a CO2 incubator. After 7 days, the number of CFU-GM, BFU-E, and CFU-Meg colonies were counted.

　　(2) Model characteristics: The peripheral blood test results on the 7th day after modeling showed that the three-line peripheral blood cells were significantly reduced, and they were still in the bone marrow suppression period at 15 days. The number of BMC decreased after modeling, but the counts of bone marrow nucleated cells (BMC) in mice showed a gradual upward trend on the 7, 11, and 15 days, suggesting that bone marrow suppression can gradually recover over time. On the 7th day after modeling, hematopoietic progenitor cells were cultured, and the colony yield was observed to decrease. The counts of CFU-GM, BFU-E, and CFU-Meg were significantly lower than those of normal control animals. The aplastic anemia model replicated by this method has good characteristics in terms of the level of synergistic decline in peripheral blood picture and the duration of bone marrow suppression. The model replication method is simple and easy to implement, and the modeling cycle is short. The result is stable and reliable and can be used as a better AA model.

　　(3) Comparative medicine The causes of AA are complicated, and patients are often associated with chemical drugs, radiation, and autoimmune diseases. Therefore, the establishment of a good AA animal model is of great significance to further study the pathogenesis and drug treatment of aplastic anemia. At present, there are many kinds of modeling methods for AA animal models, but they all have the disadvantages of unstable aplastic anemia, inconsistent bloodline reduction, cumbersome modeling methods, and long cycle. However, this model adopts a compound modeling method. The model animal has the characteristics of rapidly reducing the number of peripheral red blood cells, long and stable cycle, etc. Cyclophosphamide, as the most commonly used cell cycle non-specific drug to replicate AA animal models, can cross-link with cell DNA , Inhibit the synthesis of DNA, its cytotoxic effect is strong, the bone marrow suppression caused by it is rapid and basically reversible. 60Co-γ rays can cause cell DNA damage, interfere with DNA replication, block mitosis, and inhibit cell proliferation. As a slow-oxidizing drug, acetophenazine can cause red blood cell membrane damage and reduce the number of peripheral red blood cells. Using cyclophosphamide alone to make the model can find that the number of white blood cells in the peripheral blood of the model animal has decreased significantly, but the number of red blood cells has decreased slowly and not significantly. Considering that the mature red blood cells of the animal’s peripheral are not affected by cyclophosphamide, the survival cycle is long, which temporarily conceals the model animal’s Bone marrow suppression; and the model has a short replication cycle, which limits further observation of red blood cells. The results of modeling by radiation alone are better than those of the cyclophosphamide model, the decline in each series is more obvious, and the model maintenance cycle is longer. However, the radiation dose is not easy to control, and it is easy to cause the death of mice or affect the model replication effect. Although this model has the characteristics of rapidly reducing the number of peripheral red blood cells, long and stable cycle, but it still has the disadvantages of requiring special equipment, professionals, and cumbersome process when copying. Compared with other commonly used methods of copying AA animal models, it is still simple , Effective and stable characteristics.