动物模型 z-bo 2020-08-10 507

　　Polymyositis is a common autoimmune disease in neuromuscular diseases. Abnormal cellular immunity and humoral immunity play a key role in the damage of muscle tissue.

　　1 Rat myositis model

　　(1) Reproduction method Take guinea pig skeletal muscle, homogenize with a knife homogenizer to prepare myosin, and inject complete Freund's zol subcutaneously on the soles of both feet, both sides of the back and the root of the tail of adult Lewis rats. (CFA) plus the same amount of myosin suspension; the immunization dose is 1ml/kg body weight per point, immunization once a week for 4 consecutive weeks. At the same time, the stock solution of B. pertussis was injected into the abdominal cavity, 0.5ml each, and the number of bacteria was 4.0×10 to the power of 10.

　　(2) Model characteristics Most model rats showed symptoms such as slow activity, walking swaying, fatigue and weakness, shortness of breath, raised back, drooping head, reduced food intake, and slow growth. After 4 weeks of immunization, light microscope The muscle fibers showed varying degrees of degeneration, the cytoplasm was cloudy and swollen, the horizontal stripes of the muscle fibers were blurred or disappeared, scattered and focal lymphocytes and monocytes were seen in the interstitium of the muscle tissue, and some muscle fibers were atrophy. Enzyme histochemical staining of tissue slices showed abnormal metabolism of mitochondria in muscle cells, and ATPase showed that both type Ⅰ and type Ⅱ muscle fibers were involved. The content of myosin-specific antibodies in rat serum was significantly increased. The proliferation of T lymphocytes induced by Concanavalin A was obvious.

　　(3) Comparative medical research reported in the past that different scholars have used rabbit, guinea pig, human, and chicken skeletal muscle homogenate to immunize rats. The comparison of results found that animal muscles immunized with guinea pig and human skeletal muscle homogenate Symptoms of inflammation are more serious. The method of making this model uses guinea pig skeletal muscle myosin plus complete Freund’s adjuvant to immunize rats (Lewis rats, inbred strains, more sensitive to autoimmune reactions), so that specific immune responses can occur in their bodies Thus, muscle antibodies are produced, and guinea pig skeletal muscle and rat skeletal muscle have antigenic crossover, and finally a model of experimental myositis is made.

　　According to the animal’s clinical manifestations and pathological biopsy, it is similar to human polymyositis. Experiments have shown that the inflammatory response of rats by intraperitoneal injection of B. pertussis stock solution at the same time is greater than that of simple subcutaneous injection of myosin and Freund’s adjuvant emulsion. Seriously, this is consistent with some previous studies that reported the application of pertussis toxin intraperitoneal injection, but the specific mechanism of action is unknown. In the pathogenesis of polymyositis, abnormalities of cellular immunity and humoral immunity have different effects on muscles, but cellular immunity is dominant among them. The myositis model produced by this method has inflammatory cell infiltration, muscle fiber degeneration and necrosis in the muscle tissue of the model animal, indicating that the cellular immune response is involved in the model making process, and this result is due to myosin The formation of immune induction. Concanavalin A is a T lymphocyte stimulator, which enhances 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 immune function of the body's overall T cells.

　　2 Guinea pig myositis model

　　(1) Copy method First prepare muscle homogenate with fresh pure skeletal muscle of rabbit: chop the muscle and add pH 7.2ψ(H3PO4)=20% buffer, and homogenize with knife homogenizer at 1700r/min in ice water bath Pulp 3 times, once for 5s, with an interval of 30s. Then filter with 4 layers of gauze, and then homogenize the filtrate with a mill homogenizer at 3000r/min for 3 times, 5S each time, with an interval of 30s. Add detergent Triton-X-10, the ratio of the two is 100:1, and centrifuge at 25009×10min (4℃) after mixing, and the supernatant is the muscle homogenate device in the refrigerator at -20℃. Before the experiment, the muscle homogenate was mixed with complete Freund's adjuvant 2:1, and subcutaneously injected into the back of the guinea pig's nape once a week (0.2ml of muscle homogenate plus 0.1ml of complete Freund's adjuvant) for 8 weeks.

　　(2) Model characteristics 1 to 2 days after injection, there are hard nodules of 0.5 to 1 cm in the injection area, redness and swelling. After 1 week, the nodules become soft, fluctuating, ulcerated, and part of the body temperature rises, most obviously at 2 weeks, 4 The wound healed at the beginning of the week, and the mass gradually became smaller, resulting in a hard lump at 8 weeks. After 4 to 6 weeks, there will be varying degrees of activity reduction, weight loss, and dull coat color. Blood muscle enzymes (CK, LDH, AST) increased at 4 weeks after injection, and reached the peak at 6-8 weeks; EMG showed that the motor unit time limit was significantly shortened, the amplitude was reduced, and the polyphasic wave increased; the tissues were organized at 4, 6, and 8 weeks of immunity The pathological changes characterized by multiple inflammation of skeletal muscle were found in the physical examination: under the microscope, the striated muscle showed focally distributed muscle cell degeneration, necrosis, and inflammatory cell infiltration, thickened interstitial small blood vessel wall, and inflammatory cell infiltration around it. Under the electron microscope, the light and dark bands of myofibrils disappeared, myofibrils were swollen, broken, and vacuolated, mitochondria condensed, vacuolated, and the gap between muscle bundles widened, suggesting muscle atrophy.

　　(3) Comparative medicine When humans develop polymyositis, their serum muscle enzyme levels increase, electromyography shows myogenic damage, and the histopathological features under microscope are mainly muscle fiber degeneration, necrosis, and inflammatory cell infiltration. Rabbit skeletal muscle and guinea pig skeletal muscle have antigenic intersectionality. The experimental myositis model made by immunizing guinea pigs with rabbit skeletal muscle homogenate and complete Freund’s adjuvant for multiple times has the advantages of muscle enzymes, electromyography and histopathology. The changes are similar to human myositis.