动物造模,肌炎动物模型 z-bio 2022-02-06 706

　　Polymyositis is an autoimmune disease commonly seen in neuromuscular diseases. Abnormal cell-mediated immunity and humoral immunity play an important role in muscle tissue injury.

　　1 Rat myositis model

　　(1) Reproduction method Collect the skeletal muscle of guinea pigs, homogenize it with a knife-type homogenizer to prepare myosin, and place it on the soles of the feet, the sides of the back and the ridges of adult Lewis rats. At 5 o'clock, complete Freund's adjuvant (CFA) and an equal amount of myosin suspension were injected subcutaneously. Immunity is 1 ml/kg body weight per point, and immunization is carried out once a week for 4 consecutive weeks. At the same time, 0.5ml of Bordetella pertussis undiluted solution was injected into the abdominal cavity, and the number of bacteria was 4.0×10 to the power of 10.

　　(2) Model characteristics Most model rats exhibit symptoms such as slow activity, swinging, malaise, weakness, shortness of breath, shortness of breath, lifting back, drooping head, reduced diet, and slow growth. 4 weeks after immunization. Under the optical microscope, the muscle fibers showed varying degrees of degeneration, the cytoplasm became turbid and swollen, the horizontal stripes of the muscle fibers were blurred or disappeared, scattered, and there were local lymphocytes and monocytes in the muscle tissue. Was atrophied in the stroma. Enzymatic histochemical staining of tissue sections showed abnormal mitochondrial metabolism in cardiomyocytes, while ATPase showed that both type I and type II muscle fibers were involved. The content of specific antibodies against myosin in the serum of rats increased significantly. The proliferation of T lymphocytes induced by concanavalin A is obvious.

　　(3) Past comparative medical studies reported that different scholars used rabbits, guinea pigs, humans and chickens to immunize rats with homogenized skeletal muscle. The results of the comparison showed that the skeletal muscles of guinea pigs and humans were immunized. The symptoms of myositis in animals are more severe. Using this model establishment method, guinea pig skeletal muscle myosin was used to immunize rats (Lewis rats, inbred strain, sensitive to autoimmune response), and Freund's complete adjuvant was used to generate specific immune responses in the body. This produces muscle antibodies. At the same time, guinea pig skeletal muscle and rat skeletal muscle have antigen exchange, and finally formed a model of experimental myositis. From the animal's clinical symptoms and pathological biopsy, it is similar to human polymyositis. Experiments have shown that intraperitoneal injection of Bordetella pertussis stock solution is more emulsified than simple subcutaneous injection of myosin and Freund's adjuvant. The substance is more serious and is consistent with the experimental results of intraperitoneal injection of Bordetella pertussis toxin in several previous studies, but the specific mechanism of action is still unclear. In the etiology of polymyositis, abnormalities in cell-mediated immunity and humoral immunity have different effects on muscles, and cell-mediated immunity is the main one. The myositis model generated by this method shows that the muscle tissue of the model animal has inflammatory cell infiltration, muscle fiber degeneration and necrosis, and the modeling process involves a cell-mediated immune response. This result is attributed to myosin which is formed by immunity. Inauguration. Concanavalin A is a T lymphocyte stimulator, which can enhance the body's overall T cell immunity by non-specific activation of polyclonal T cells. Therefore, the proliferation of T lymphocytes induced by concanavalin A represents the overall immune function of human T cells.

　　2 Guinea pig myositis model

　　(1) Replication method First, use fresh rabbit skeletal muscle to prepare a muscle homogenate. Cut the muscle, add pH7.2ψ (H3PO4) = 20% buffer, and then cut it with a knife in an ice water bath. The homogenizer homogenizes. Run 3 times at a speed of 1700r/min, 5 seconds each time, with an interval of 30 seconds. Next, filter with 4 layers of gauze, and homogenize the filtrate 3 times at a speed of 3000 r/min with a refiner homogenizer at an interval of 30 seconds, each time for 5 seconds. Add detergent Triton-X-10, the ratio of the two is 100:1. Centrifuge the mixture at 25009 x 10 minutes (4°C), and make the supernatant into a muscle homogenate in the refrigerator at -20°C Before the experiment, the muscle homogenate was mixed with complete Freund’s adjuvant 2:1 and injected into the back of the guinea pig’s neck every week (0.2 ml of muscle homogenate and 0.1 ml of complete Freund’s adjuvant) for 8 week.

　　(2) Model features 1-2 days after injection, there are 0.5-1 cm hard nodules, redness and swelling in the injection area. After 1 week, the nodules become soft, undulate, ulcers, one body temperature rises and becomes the highest Striking in 2 weeks. The wound began to heal within 4 weeks, the mass gradually became smaller, and became a hard mass within 8 weeks. After 4-6 weeks, mobility, weight loss, and darkening of the fur all decreased to varying degrees. Blood muscle enzymes (CK, LDH, AST) increased at 4 weeks after injection, and reached a peak at 6-8 weeks. The electromyogram shows that the time limit of the motor is significantly reduced, the amplitude is reduced, and the polyphase wave is increased. Tissue tissues at 4, 6 and 8 weeks after immunization. Physical examination found pathological changes characterized by multiple inflammation of skeletal muscle: under the microscope, the striatal muscle was locally degenerated and necrotic, and inflammatory cells were distributed in the infiltrated and thickened muscle cells. Infiltration of small interstitial blood vessels and surrounding inflammatory cells. Under an electron microscope, the bright and dark bands of myofibrils disappeared, myofibrils swelled, ruptured, vacuoles, mitochondria condense, vacuoles, and the gaps between muscle bundles widened, suggesting muscle atrophy.

　　(3) Comparative medicine. When humans develop polymyositis, the level of serum muscle enzymes increases, electromyography shows myogenic damage, and the histopathological features examined under the microscope are mainly muscle, fibrosis, necrosis, and inflammation. Cell infiltration. Rabbit skeletal muscle and guinea pig skeletal muscle have antigenic crosstalk. The experimental myositis model established by repeatedly immunizing guinea pigs with rabbit skeletal muscle homogenate and complete Freund’s adjuvant has the advantages of muscle enzymes, electromyography and histopathology. For human myositis.