动物造模 z-bo 2022-02-03 501

　　[Modeling mechanism]: Bleomycin is a peptide antitumor drug. The mechanism of pulmonary fibrosis is mainly due to the action of reactive oxygen species. In the early stage of bleomycin lung injury, that is, the alveolitis stage, large amounts of oxygen free radicals are produced, which damage the lungs.

　　[Modeling method]: Sprague Dawley rats weighing 170-200g were selected. Bleomycin (30 mg/tube), diluted with 0.9% sodium chloride to 4 g/L, 0.25-0.3 ml (5 mg/kg) bleomycin solution was instilled into the trachea to establish lung interstitium Fibrosis model. Rats were anesthetized intraperitoneally with 2% sodium pentobarbital (40 mg/200 g, or 0.25 ml/mouse). After about 3-5 minutes, slowly anesthetize the rat (if the anesthesia is not deep, use ether to strengthen the anesthesia). After anesthesia, use a mouse pad to fix it on the back, fix the limbs and head, and cut off the hair on the neck. Disinfect the skin with povidone iodine, and make a 1 cm long neck incision aseptically to expose the tracheal layer in separate layers (if the room temperature is low, insulation measures are required). Using curved eye drops, pass under the trachea, lift the trachea slightly, connect it to the carina, and then raise the ratchet head of the desktop computer by 30° to 35°. Choose a 7 gauge needle for injection, and polish the needle into an arc to make it round and blunt. Before inserting the needle, make sure to thread the needle with a syringe to avoid clogging. When inserting the needle, the needle makes a 150-degree angle with the horizontal plane and is inserted between the two circular cartilage. The direction of the needle hole faces the surgeon, and it is impossible to prove that the needle has penetrated 1 to 1.5 cm. Therefore, he is injecting and injecting bleomycin into the trachea bifurcation. Inject 0.2 ml of bleomycin (approximately 4 mg/kg), and then inject 0.2 ml of air into the trachea 2-3 times to distribute the drug evenly in the lungs. Rotate the mouse pad back and forth around the long axis of the mouse body for 1-2 minutes. Use local povidone-iodine disinfection (or penicillin disinfection) to suture the skin to prevent infection. Keep the room temperature at 24 to 25°C. After waking up, place it in a cage and store it regularly.

　　Mouse: Choose a male Kunming mouse weighing 18-20 grams. Ketamine (100mg/2ml) (10mg/100g) was injected intraperitoneally. Anesthetize the experimental animal and lie down. Cut the skin longitudinally, expose the trachea straightforwardly, and pierce the trachea as far as possible into the carina with a #4 needle. Slowly add bleomycin (8 mg bleomycin, made into a 0.2% solution in saline before use), and immediately erect the animal to distribute the drug evenly in the lungs and suture the skin locally. Ethanol disinfection can prevent infection. Likewise, female BLAB/C mice can be used. The mice were anesthetized by intraperitoneal injection of chloral hydrate and fixed on the operating table. Use an animal laryngoscope to depress the root of the rat’s tongue to expose the glottis, insert the nebulizer into the trachea through the glottis and spray, and inject 100μl (5 mg/kg) of bleomycin solution.

　　Goats: Select 12-15 kg goats, under 3% sodium pentobarbital anesthesia, slowly inject bleomycin 1.5-3.0 U/kg into the left lower lobe bronchus through a balloon catheter. The total volume is 2-3 ml/kg. After instillation, the goat is placed on the left side for 30 minutes to promote the absorption and distribution of the drug and reproduce the pulmonary fibrosis model. [Model Features] The histopathological and pathophysiological changes of the rat model are similar to human lung interstitial fibrosis. The early stage of the lesion is exudative alveolitis, in which inflammatory cells accumulate in the lesion. The late stage is pulmonary interstitial fibrosis, interstitial cell proliferation and matrix collagen accumulation, replacing normal lung histology. Two weeks after bleomycin injection, lung coefficient (lung weight/body weight x 100%) and hydroxyproline content increased significantly. Extensive inflammatory cell infiltration can be seen under the microscope, mainly lymphocytes and mononuclear phagocytes, with symptoms of grade II alveolar inflammation, including alveolar wall thickening and fibroblast proliferation. In the 4th week, many green stained collagen fibers were scattered in the lung interstitium, which destroyed the alveolar structure, and there were many fibroblasts and other grade III lung fibrotic lesions. After 15 days of administration of bleomycin to mice, the lesions spread, accompanied by obvious alveolar septa, blood vessels and small trachea. The disease causes the alveolar wall to thicken, the capillaries expand, the alveolar cavity shrinks, and is filled with inflammatory cells such as neutrophils, monocytes and lymphocytes. Inflammatory cell infiltration, mainly neutrophils and monocytes, appeared near the trachea, blood vessels, and pleura, indicating grade 3 alveolar inflammatory changes. After 30 days, the disease spread, the alveolar walls became thicker, and the alveolar space became wider. In the lung tissue around the trachea and blood vessels and near the pleura, inflammatory cell infiltration, mainly monocytes and lymphocytes, thickening of the pleura, proliferation and aggregation of fibroblasts and collagen fibers can be seen. .. Mainly manifested as a grade 1 fibrosis change. The classic way to replicate a mouse model of pulmonary fibrosis is to inject a bleomycin solution into the trachea. Intratracheal drip or atomization of bleomycin can cause significant lung tissue damage and pulmonary fibrosis. Although a single instillation of bleomycin into the trachea can cause obvious pulmonary fibrosis, the distribution of the lesions is not uniform and cannot accurately simulate the diffuse distribution of human primary pulmonary fibrosis. Intratracheal nebulization can make bleomycin more evenly distributed in the lungs, and the changes in fibrosis are more diffuse, more similar to those in human lung fibrosis. The nebulized bleomycin solution in the trachea can reduce the injury and pain of the animal without puncturing the trachea, and can be used as the solution of choice for replicating mouse pulmonary fibrosis models. From day 3 to day 4 of goat bleomycin, patchy shadows and thick lines appeared on the central lobules. At the end of the first week, the aforementioned area becomes larger, fuse and reach the subpleural area. Exudates are found in the alveoli and interstitium. The main symptoms for several weeks are frosted glass lesions and fine radiation around the central lobules and tracheal vascular bundles, with no signs of thickening of the interlobular septa. Abnormalities of the lung interstitium were observed in the 4th week. The macroscopic observation of the lung specimen is gray, and the histological examination of the abnormal area shown by CT shows that the main lesion is located around the trachea, and there are scattered lesions around the lung lobules below the lung segment. I proved it. Pulmonary metaplasia occurs within 1 week, mainly due to the destruction of alveolar walls and inflammatory cell infiltration. After 2 weeks, fibroblasts mainly gather in the stroma and collagen fibers. After 3 weeks, the alveolar septum thickened.

　　[Evaluation and Application of Model] Currently, many inducers are used to create this model. For example, bleomycin is the most commonly used. One-time injection of bleomycin into the trachea can reproduce the pathological process of animal models and human pulmonary fibrosis. It can be administered systemically or locally, one or more times. The mini drug pump can be used for continuous administration. The specific method is as follows. (1) Intratracheal administration: This is currently the most commonly used route of administration. This is a partial management method. The inducer is injected directly into the trachea of the animal to cause lung disease. Repeat one or more times. Bleomycin solution is mainly infused through the trachea. This method is easy to operate and low in cost, but the range of lesions is limited and is different from the scattered distribution of lesions in the human body. Therefore, aerosol inhalation is used to overcome the previous shortcomings and evenly distribute the lesion distribution. (2) Intraperitoneal administration: a systemic administration method in which an inducer such as bleomycin is injected into the abdominal cavity of an animal to exert a fibrotic effect. Usually, it is continuously administered multiple times a day or at intervals of several consecutive days. When a certain amount of drugs accumulate in the body, an animal model is established. However, compared with intratracheal administration, the lesions caused by the pleura and bronchi are heavier, while the latter shows that the bronchial and peripleural lesions are not obvious. It is believed that this method is superior to the latter because it is different from and inconsistent with human pulmonary fibrotic lesions (the lesion is the beginning of the pleura). However, compared with the latter, its cost is also high due to excessive amounts of the required drugs, which limits its application in the country. This model provides a new experimental method for studying the etiology, etiology and prevention of pulmonary fibrosis. Under strict control of various conditions and these different changes in pathological morphology, molecular biology, imaging and other laws, it can observe the development, development and disease results of pulmonary fibrosis, and improve the resistance to interstitial lung disease. Understand the level.