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动物模型,胰腺炎动物模型
z-bo
2020-07-17
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Since Bernard injected bile and olive oil in 1856 and made the first animal model of acute pancreatitis in canine pancreas control, after more than a century of development, scholars from various countries have designed many AP animal models and conducted a large number of experiments on them Research, the research overview of the AP animal model is now introduced as follows.
Animals used for modeling mainly include Wister, Spraque-Dawley rats, mice, possums, guinea pigs, dogs, cats, rabbits, pigs, monkeys, etc. Generally, rats and dogs are commonly used.
1. Living pancreatitis model
1.1 Intrusive method
1.1.1 Pancreatic duct injection method: It is the oldest and most widely used method.
1.1.1.1 Methods and characteristics: In 1978, Widdison perfused the cat's main pancreatic duct with a pressure of less than 1.96kPa. The method is to separate the main pancreatic duct at the tail of the pancreas 2 ~ 3mm, insert a 0.6mm diameter polyethylene catheter, 3mm deep, ligate the distal pancreatic duct after fixation, infuse glycerodeoxycholic acid or sodium deoxycholate through the catheter, and then insert Inject 0.5ml of activated pancreatic juice into the tube to induce edema pancreatitis. On this basis, intravenous injection of 16,16 dimethyl prostaglandin E2 can turn edema pancreatitis into ANP. Aho et al. used the first retrograde injection of 5% sodium taurocholate (NaTc) into the pancreatic duct of rats in 1980 and successfully induced an acute hemorrhagic necrotizing pancreatitis (AHNP) model [1]. However, Foitzik et al. [2] believe that many factors affect the severity of AP induced by the model, such as the concentration of perfusate, volume, pressure of perfusion, time of perfusion, etc. can all affect the mortality of the disease, so the model does not Not suitable as a therapeutic study for AP. And the AP produced by this model is uncertain, complex, expensive, and has a low mortality rate. Death cannot be used as the end point of the experiment. Domestic [3] scholars use a micro-medicine injection pump to inject 5% sodium taurocholate 1.0ml/kg, the injection rate is 0.2ml/min, and the injection lasts 10min. It is believed that the pressure in the pancreatic duct can be prevented from rising too fast, and it remains constant The injection speed makes the experimental conditions more standardized and the experimental results more accurate. Therefore, the model is considered to have the following characteristics: (1) The causative factors are similar to clinical ones, which can mimic the etiology of pancreatic duct obstruction, bile reflux, etc.; (2) It can better control the degree of pancreatic lesions, change the injection speed, injection duration, Drug concentration can produce pancreatitis of different severity; (3) suitable for evaluating the efficacy of drugs.
1.1.1.2 Mechanism: The drug damages the pancreatic duct epithelium or duct, the pressure increases, and the permeability of the pancreatic duct increases. Trypsinogen and other large-molecule secreted pancreatic enzymes return to the pancreatic parenchyma, and the pancreas is injured. AP occurs. 16,16 Dimethylprostaglandin E2 increases the permeability of the microvascular bed, which converts AP to ANP [1].
1.1.2 Pancreatic duct ligation
1.1.2.1 Methods and characteristics: In 1975, Nevalainen et al. used 5-0 silk thread to ligature the duodenum with 1 cm each of the opening of the common bile duct. Pancreatic edema appeared 24 hours later, pancreatic head hemorrhage and peripancreatic fat necrosis . This model is similar to AP of human bile reflux. But its shortcoming is that it can be complicated by high gastric retention, resulting in a significant reduction in effective circulating blood volume and blood in a hypercoagulable state, which will inevitably affect the reliability and stability of the experimental AP. Seidel and Pfeffert and others have improved the method of making the model, namely the duodenal closed loop model (CDL), free the first segment of the duodenum 10cm, at the distal end of the pylorus and the second and third segments of the duodenum The common bile duct was cut and ligated, and the gastrointestinal tract was reconstructed with anastomosis of the stomach and duodenum. Pancreatic edema appeared after 4 hours, and hemorrhagic pancreatitis appeared after 9-12 hours. This model requires no drugs and avoids systemic reactions. However, 24 hours after the operation, anastomotic edema and gastric retention were still present [4]. [[2] Foitzik et al. believe that due to the secretion of bile, pancreas and other secretions of duodenal closure loops, the animals are often contaminated with bacteria, and the animals develop venous congestion or severe intestinal ischemia within 2-3 days and die from refractory In addition, the ligated distal bowel segment often affects its peristalsis and internal environment due to lack of secretions such as bile and pancreas. In 1996, Azima et al. [5] circumvented the free common bile duct through a polypropylene rope and loosened it after 24 hours. As a result, it was found that serum amylase and bilirubin were significantly increased on the 1st and 2d. Histological examination It indicates that pancreatic edema, zymogen degranulation, inflammation and infiltration, vacuole formation in acinar cells, local fat saponification and parenchymal necrosis, pancreatic tissue structure is almost completely destroyed at the first week, and the zymogen granules are dissolved, but at the third week , Its histology has shown almost normal pancreatic tissue, zymogen granules recovered, suggesting that acute pancreatitis has completely recovered, so it is believed that this reversible acute pancreatitis model can be used for the study of pathogenesis and therapeutic intervention.
1.1.2.2 Mechanism: The mechanism is that the pressure in the duodenal cavity increases, causing bile to flow back into the pancreatic duct. The bile destroys the mucosal barrier of the pancreatic duct epithelium, and the bile lecithin that flows back into the pancreatic duct is phospholipid of pancreatic juice. Enzyme A2 decomposes into lysophosphatidylcholine which can damage cell membranes and ducts. The activation of protease in the pancreas leads to the self-digestion of pancreatic tissue, causing pancreatic edema, hemorrhage and necrosis.
1.1.3 Even injection under the pancreas:
1.1.3.1 Method: Wang Dan-song, [6] and others improved the method of preparing AHNP animal model by evenly injecting 3% sodium taurocholate under the pancreas capsule. Light anesthesia with ether, strict aseptic operation, through the midline abdominal incision, lift the spleen and pancreatic tail, and evenly inject 1ml of 3% sodium taurocholate under the pancreatic capsule.
1.1.3.2 Features: Pancreatic hemorrhage and necrosis occurred within half an hour after operation, serum amylase and pancreatic tissue TNFα levels reached a peak within 24h, and continued to 48h, while pathological damage of liver and lung tissue and TNFα The level rises. The 2d mortality rate of this model was 0, and it gradually increased in the future, and the mortality rate reached 90% in one week. It is believed that this method can establish ANHP combined with MODS rat model, which overcomes the adverse factors of mild pancreatitis induced by hyaline injection and cholangiopancreatic duct ligation, rapid development of retrograde cholangiopancreatic duct injection, and rapid death , Is an ideal animal model for experimental research in the treatment of ANHP.
1.1.3.3 Mechanism: After injection of the pancreas under the capsule, direct chemical stimulation of sodium taurocholate in the early stage caused damage to the pancreatic tissue under the capsule. Later, bile salts activate pancreatic enzymes to produce self-digestion, which further damages the pancreas.
1.1.4 Model of microvascular pancreatitis (also known as arterial injection microsphere method)
1.1.4.1 Method: In 1990, Redha designed a partial occlusion pancreatic artery method to make an AP model based on the principle of early AP microcirculation change. The method is to perform laparotomy on the rat, remove the spleen, free the distal branch of the splenic artery adjacent to the splenic hilum, insert a fine tube, inject 20μm diameter polystyrene microspheres, and contain 200,000 strains in 0.5ml liquid, and remove the cannula Ligation of the arterial branch, as early as 30min, immunohistochemical techniques can be used to detect changes in the pancreas, and acute hemorrhagic pancreatitis occurs within 24h, with a mortality rate of less than 10%, and progresses to chronic active pancreatitis after 3 weeks. The old model that directly blocked the pancreatic artery or vein in the past has rarely been adopted. Pancreatitis caused by pancreatic microcirculation disorder is rare in clinic, but it can be seen in polyarteritis and similar conditions, and it is seen in AP caused by coagulopathy and microvascular thrombosis. This model is suitable for more chronic studies. It is useful for observing recurrent AP. The endocrine disorder is mild, the islets are still preserved, and the catheter pressure is not affected [4].
1.1.4.2 Mechanism: After the microspheres are injected into the artery, the local blood supply of the pancreas is impaired, the pancreas is hypoxic, the polymorphonuclear cells are infiltrated, and the pancreas is edema. Edema in turn reduces the blood supply further, resulting in an increase in pancreatic interstitial osmotic pressure and vessel wall permeability, increased tissue pressure, further compressing blood vessels, reducing perfusion, and increasing pancreatic hypoxia [1].
1.1.5 Electroacupuncture stimulation method:
1.1.5.1 Methods and features: In 1998, Qin Renyi et al. [7] used electroacupuncture with a voltage of 3 volts and a frequency of 20 Hz to stimulate rats to walk at the end of the bile duct in the duodenum. An animal model similar to clinical acute biliary pancreatitis. The method is to fix the SD rat in a horizontal position, take a 2 cm midline incision into the abdomen, lift the duodenum, expose the end of the bile pancreatic duct, bend the end of the electroacupuncture into a hook shape, and walk under the surgical microscope under twelve The end of the biliary and pancreatic duct in the digit is hooked with an electric needle, and the electric needles on both sides are about 2mm apart.The electric needles are connected to the two electrodes on the DM-A quantitative acupuncture anesthesia treatment instrument, and the voltage and frequency are adjusted to 3 respectively. At 20 Hz and 20 Hz, electroacupuncture continuously stimulates the end of the bile duct. After 1 hour, the pancreas was slightly swollen and congested. After 24 hours of feeding, the blood amylase and urine amylase were significantly increased, and the pancreas was swollen and congested. The pathophysiology of this model is more consistent with the occurrence of clinical acute biliary pancreatitis, and its establishment is conducive to the study of the pathogenesis of acute biliary pancreatitis.
1.1.5.2 Pathogenesis: It may be due to the electroacupuncture stimulation of the end of the bile pancreatic duct, which induces the violent contraction and relaxation of the smooth muscles surrounding it, which is very similar to the clinical biliary stone stimulation of the violent contraction and relaxation of the Oddi sphincter. All of them can lead to changes in neurohumoral fluid and imbalance in the expression balance of gastrointestinal peptide hormones and receptors; it can also increase the pressure in the common bile duct and cause bile or intestinal pancreatic juice to flow back into the pancreatic duct.
1.2 Non-invasive method
1.2.1 Ethionine diet method:
1.2.1.1 Establishment of animal model: In 1950, Farber and Popper gave mice ethionine to induce AP. In 1975, Lombardi applied choline-deficient ethionine (CDE) diet to induce acute hemorrhagic pancreatitis and fat necrosis in female mice. Feed formula: sucrose 55.8%, lard 20%, soy protein 10%, with various vitamins and inorganic salts, plus 3% ethionine. The experimental results showed that the young rats fed with CDE died 100% after 4 days. Necrotizing pancreatitis and fat necrosis of abdominal cavity occurred in autopsy. According to different experimental purposes, different feeding times can be used to control the severity of the disease and the mortality rate between 0 and 100%. Both experimental models and clinical patients have ascites, acidosis, hypoxia and hypovolemia, which can fully evaluate the duration and pathophysiology of model animals [4].
1.2.1.2 The characteristics of animal models: This method is simple, rapid replication, stable model, non-invasive method adopted, less impact on the environment in the body. Therefore, this model is suitable for exploring the pathogenesis of diseases and studying the effects of oxygen free radical scavengers and oral protease inhibitors on acute necrotizing pancreatitis (ANP). The disadvantage is that the degree of damage it produces depends on the sex, age and weight of the experimental rats. Small animals are not easy to use to evaluate the value of new diagnostic methods and surgical techniques.
1.2.1.3 Mechanism: So far it is not completely clear. It is speculated that ethionine may interfere with the metabolism of methionine in the cell, thereby interfering with the synthesis of phospholipids in the cell membrane. This is caused by the lack of choline in the diet. The effect is strengthened, resulting in the pancreas cells exuding being blocked. The sex difference may be due to the harmful effect of estrogen or the ability to neutralize active pancreatic enzymes.
1.2.2 AP animal model induced by hylain
1.2.2.1 Establishment of an animal model of AP induced by hylain
In 1977, Lampel first reported the use of ultra-large doses of hyarine (5μg/kg/h×5h) to continue intravenous drip in rats, causing acute edematous pancreatitis (AEP) characterized by marked edema of the pancreas within a few hours. ). In 1984, Niederau et al. replicated the model of acute necrotizing pancreatitis (ANP) in mice by using 7 consecutive intraperitoneal injections (amount of hyarine 50μg/kg/h, once per hour). In 1985, Niederau et al. used 7 consecutive subcutaneous injections (the dose was the same as before) and developed necrotizing pancreatitis within 8 to 12 hours after administration. The authors compared the two methods of administration and found that intraperitoneal administration caused more severe and long-lasting damage. Some animals developed ascites, but if the amount of hyaluronan continued to increase, the damage to the pancreas would not increase. In 1987, Tani et al. used super-large doses of hyacinin to take two different methods of medication, namely four consecutive subcutaneous injections (20μg/kg body weight, once per hour) and four consecutive intramuscular injections (50μg/kg, once per hour). ), successfully replicated the rat AEP model. In 1989, Robert et al. successfully replicated the rat AEP model with the method of subcutaneous infusion at a constant rate (5μg/kg/h×5h, 1ml/h). In 1992, Schmidt et al., on the basis of continuous intravenous infusion of hyacinin (5μg/kg/h×6h), combined with retrograde infusion of small doses of gly-codeoxycholicacid (GDOC) into the cholangiopancreatic duct, and through adjustment GDOC concentration, dosage, and the length of bolus injection induced different severity of AP in rats. At the same time of intravenous infusion of hyacinin, if 10 mmol/L of GODC was injected into the pancreatic duct at 0.2 ml/2 min, moderate AP was produced; if GODC was changed to 0.5 ml/10 min, severe AP was produced. The advantage of this model is that not only can the severity of the disease be artificially controlled (the animal's 24-hour mortality can be controlled between 6% and 4%), but the pathological changes of the pancreas are very similar to human AP, causing pancreatic acinar cells, The uniform injury of severe necrosis overcomes the shortcomings of AP caused by simple pancreatic duct injection of GDOC. This model is particularly suitable for the study of the treatment of progressive necrosis of AP. In 1994, Castillo et al., based on the intravenous application of serotonin, combined with intestinal kinase injection into the pancreatic duct to replicate the ANP model (serotonin 5μg/kg/h×5h) continuous intravenous infusion; enterokinase 25 units/ml, 10min, 30mmHg Pressure, dosage 0.5～0.55ml. The characteristic of this model is that it causes severe local injury and necrosis of the pancreas, accompanied by multiple hemorrhage in the peritoneum, lungs, soft tissues, etc., and causes early animal death. In 1995, Muttillo et al. reported that subcutaneous injection of super high-dose hyacinthine (20μg/kg/h×5h) caused ANP in mice, and it was observed that all animals died within 72h, and cytoplasmic vacuoles and interstitial edema were relatively mild. The main pathological changes are the degeneration and necrosis of acinar cells accompanied by a large number of granulocyte infiltration. In the same year, Kaiser replicated the mouse ANP model by subcutaneous injection of super high-dose tree frog (50μg/kg/h) for 12 hours. [8]
1.2.2.2 Mechanism of AP caused by serotonin: serotonin is a decapeptide consisting of seven identical amino acids with human CCK octapeptide. The detailed mechanism of the hyperdose of serotonin induced AP in experimental animals has not been fully elucidated, some studies