动物造模 z-bo 2022-02-04 415

　　[Modeling mechanism] Bleomycin (bleomycin) is a polypeptide anti-tumor drug. The mechanism that causes pulmonary fibrosis is mainly through the action of reactive oxygen species. In the early stage of lung injury caused by bleomycin, that is, the alveolitis stage, a large amount of oxygen free radicals are produced, causing damage to the lung.

　　【Modeling method】

　　Rats: Sprague Dawley rats weighing 170-200g are selected. Bleomycin (30mg/branch), diluted with 0.9% sodium chloride to 4g/L, and 0.25～0.3ml (5mg/kg) of bleomycin solution was dripped into the trachea to establish a pulmonary interstitial fibrosis model. The rats were intraperitoneally anesthetized with 2% sodium pentobarbital (40mg/200g, ie 0.25ml/mouse). After about 3 to 5 minutes, the rats entered a slow state of anesthesia (if the anesthesia is not deep, they must be strengthened with ether). After anesthesia, fix it supine on the rat board, fix the limbs and head, and cut off the neck hair. Disinfect the skin with povidone-iodine (iodine), cut a 1cm long neck incision under aseptic operation, and separate and expose the trachea layer by layer (if the room temperature is low, insulation measures are required). Use curved-point ophthalmic forceps to pass under the trachea, slightly lift the trachea, try to connect to the trachea bifurcation, and raise the head of the rat board to make it 30°～35° with the desktop. Choose a No. 7 needle for injection, and grind the needle into an arc with a sand sheet to make it round and blunt. Before inserting the needle, try to pass the injection needle with a syringe to avoid blockage. When the needle is inserted, it is at 150° from the horizontal plane and inserted between the two cricoid cartilage. The direction of the needle hole faces the surgeon, and there is a sense of failure to prove that the needle has penetrated 1 to 1.5 cm, and the needle is injected at the bifurcation of the trachea and the bleomycin is injected. Inject 0.2ml of bleomycin (approximately 4mg/kg), and then inject 0.2ml of air into the trachea 2 to 3 times to make the medicine evenly distributed in the lungs. With the long axis of the rat's body as the center, rotate the rat board forward and backward for 1 to 2 minutes. The skin is sutured, and local povidone iodine disinfection (or disinfection with penicillin) prevents infection, and the room temperature is kept at 24～25℃. After the animal is naturally awake, it is placed in a cage for regular breeding.

　　Mice: Male Kunming mice weighing 18-20g are selected. Ketamine (100mg/2ml) was injected intraperitoneally (10mg/100g). The experimental animal was anesthetized and lay on its back. The skin was cut longitudinally, the trachea was exposed bluntly, and the trachea was pierced with a 4 gauge needle as close as possible to the tracheal bifurcation. Add ml of bleomycin slowly (8mg of bleomycin per tube, and prepare 0.2% medicinal solution with saline before use), immediately rotate the animal upright to make the medicinal solution evenly distributed in the lungs, and then suture the skin, local Ethanol disinfection prevents infection. Similarly, female BLAB/C mice can also be used. The mice are anesthetized by intraperitoneal injection of chloral hydrate and then fixed on the operating table. Using animal laryngoscope to depress the base of the tongue of the mouse to expose the glottis, the atomizer is inserted into the trachea from the glottis and atomized Inject 100μl (5mg/kg) of bleomycin solution.

　　Goats: Select goats weighing 12-15kg, under 3% sodium pentobarbital anesthesia, slowly instill bleomycin 1.5-3.0 U/kg into the bronchus of the lower lobe of the left lung through a balloon catheter, and instill the total amount of liquid Give it at 2～3ml/kg. After the instillation, the goat is placed on the left side for 30 minutes to facilitate the absorption and uniform distribution of the drug, and replicate it into a pulmonary fibrosis model.

　　[Model Features] The histopathological and pathophysiological changes of the rat model are similar to human pulmonary interstitial fibrosis. The early stage of the lesion is manifested as exudative alveolitis, and inflammatory cells accumulate in the lesion. The late stage is pulmonary interstitial fibrosis, interstitial cell proliferation, and matrix collagen aggregation to replace the normal lung tissue structure. After bleomycin was injected for 2 weeks, the lung coefficient (lung weight/body weight×100%) and the content of hydroxyproline increased significantly. Extensive inflammatory cell infiltration can be seen under the microscope, mainly lymphocytes and mononuclear phagocytes, and there are symptoms of grade II alveolitis such as alveolar wall thickening and fibroblast proliferation. In the 4th week, there were a large number of scattered green-stained collagen fibers in the lung interstitium, the alveolar structure was destroyed, and there were many fibroblasts and other grade III pulmonary fibrosis lesions.

　　mice received bleomycin 15 days later, the lesions were diffuse, but prominent around alveolar septum, blood vessels and small trachea. The alveolar wall thickens at the lesion, the capillaries expand, and the alveolar cavity becomes smaller, which is full of inflammatory cells such as neutrophils, monocytes, and lymphocytes. In the trachea, blood vessels, and near the pleura, inflammatory cell infiltration, mainly neutrophils and monocytes, appeared, showing grade 3 alveolar inflammatory changes. After 30 days, the lesion was diffuse, the alveolar wall thickened and the alveolar interval widened. In the lung tissues around the trachea and blood vessels and near the pleura, inflammatory cell infiltration, dominated by monocytes and lymphocytes, can be seen, the pleura thickens, and the proliferation and aggregation of fibroblasts and collagen fibers can be seen. Mainly showed grade 1 fibrosis changes.

　　The classic method of replicating a mouse model of pulmonary fibrosis is to instill a bleomycin solution into the trachea. Intratracheal drip or nebulization of bleomycin can cause obvious lung tissue damage and pulmonary fibrosis. A single instillation of bleomycin into the trachea can cause obvious pulmonary fibrosis, but the distribution of the lesions is not uniform, and it cannot accurately simulate the diffuse distribution of human primary pulmonary fibrosis. The intratracheal atomization administration method can make bleomycin more evenly distributed in the lungs, and the fibrosis changes are more diffuse, which is closer to the human pulmonary fibrosis changes. The nebulized bleomycin solution in the trachea does not need to puncture the trachea, reduces the injury and pain of the animal, and can be used as a preferred solution for replicating a mouse model of pulmonary fibrosis.

　　Goats were treated with bleomycin on the 3rd to 4th day, patchy shadows and thick lines appeared in the central lobules. At the end of the first week, the above-mentioned areas became larger, fused, and reached the subpleural area. Exudates can be seen in the alveoli and interstitium. From 2 to 3 weeks, the main manifestations are ground glass lesions and fine radioactive lines, located around the central lobules and tracheal vascular bundles, and there is no sign of thickening of the interlobular septa. In the 4th week, abnormality of the lung interstitium was shown. The gross observation of the lung specimens showed a gray color, and the histological examination of the abnormal areas shown on CT showed that the main lesions were around the trachea, and the lesions around the lung lobules below the lung segment were scattered. Within 1 week, lung cell metaplasia was mainly caused by alveolar wall destruction and inflammatory cell infiltration. After the second week, fibroblasts mainly accumulated in the interstitium, accompanied by increased collagen fibers. After the third week, the alveolar septum thickened.

　　[Model Evaluation and Application] There are many inducers used in the production of this model, for example, bleomycin is the most commonly used. One-time injection of bleomycin in the trachea can replicate an animal model similar to the pathological process of human pulmonary interstitial fibrosis. It can be administered systemically or locally, and it can be administered at one time or in multiple doses. A miniature drug pump can be used for continuous administration. The specific methods are as follows: ①Intratracheal administration: It is the most commonly used route of administration at present. It is a local administration method. The inducer is directly injected into the trachea of the animal to cause lung disease. It can be one-time or one-time Repeated many times. For the aqueous solution of bleomycin, transtracheal instillation is mostly adopted. Although this method is simple to operate and low in cost, it has a limited range of lesions and is different from the diffuse distribution of human lesions. Therefore, the use of aerosol inhalation for administration overcomes the disadvantages of the former and makes the lesion distribution evenly diffuse. ②Intraperitoneal administration: It is a method of systemic administration in which an inducer (such as bleomycin) is injected into the abdominal cavity of the animal to exert a fibrotic effect. Generally, it is administered multiple times, with continuous administration every day, and administration at intervals of several days. When the drug accumulates in the body to a certain amount, an animal model can be established. However, compared with intratracheal administration, the lesions caused by the pleura and bronchi are heavier, while the latter causes the lesions to be obvious around the bronchus and the pleura is not obvious. This is different from the human pulmonary fibrosis lesions (the lesions are caused by The beginning of the pleura) is not very consistent, so the method is considered superior to the latter. However, compared with the latter, due to the excessive amount of medicine required, its cost is also high, which limits its application in the country.

　　This model provides a new experimental research method for studying the etiology, pathogenesis and prevention of pulmonary interstitial fibrosis. It can observe the occurrence, development and disease outcome of pulmonary interstitial fibrosis under strict control of various conditions. Different changes in pathomorphology, molecular biology and imaging performance and other laws, improve the level of understanding of interstitial lung diseases.