动物造模,肝衰竭动物模型 z-bio 2021-11-16 280

　　(1) Model method Adult rats were anesthetized by intraperitoneal injection of 100 mg/kg of ether or ketamine, the back was fixed, and the abdomen was opened along the lateral incision in the upper abdomen. Use a coagulator to cauterize the abdominal vessel wall to stop bleeding. Expose the liver, cut the falciform ligament, coronary artery ligament and interlobular ligament, expose the hilar, first ligate and cut the abdominal bile duct of the portal vein scalp branch, portal vein scalp branch and hepatic artery, and ligate the scalp branch and portal vein with 5-0 silk thread . Accompanied by hepatic artery. Then carefully divide the right portal vein branch into the right upper and lower lung lobes, and the related hepatic artery, and ligate the right or right upper lung branch of the portal vein with 5-0 silk thread. At this time, the ligated liver lobe is in an ischemic state. Pierce the portal vein with a 5.5 gauge needle above the ligation, and inject 10 ml/kg body weight of 5% glucose and sodium chloride solution or sodium lactate Ringer's solution until the liver leaves turn white, and then use 5-0 silk thread to remove the liver leaves quickly ligation. In total, 85%-95% of liver tissue was removed. After ensuring that there is no bleeding, suture the abdominal wall incision. After the operation, observe the rat's mental state, activity state, liver function changes and 12-hour survival rate. After the operation, the rats were awake and turned over, which is a sign of successful operation, and rats that stood up and died within 12 hours were considered to have failed the operation. Alternatively, anesthetize healthy piglets by intraperitoneal injection of 30 mg/kg body weight of barbital sodium. Anesthetize the right upper limb, establish the right upper limb, use a nasal catheter to supply oxygen, fix the supine position, and make an upper abdominal midline incision. It stratifies in the abdomen and releases the portal vein and right renal vein. Inject 0.25 mg of heparin into the portal vein, and take a silicone plastic tube (15 cm long and 0.8 cm in diameter) filled with 2% heparin saline, 2-3 cm away from the distal end of the free right renal vein. Fix the inferior vena cava, insert about 1.5 cm from the proximal end to the distal end of the portal vein to fix it, open the bypass tube, check for obvious bleeding, and then open the abdomen. Two days later, I entered the second stage of surgery. After entering the abdomen, cut all the ligaments around the liver, that is, the liver branches of the hepatic artery (including the left and right deltoid ligaments, coronary ligaments, falciform ligaments and hepatogastric ligaments). Suture, and finally ligate the hepatic artery and stomach, the hepatic artery completely blocked the blood flow of the human liver, and the blood pressure was monitored through femoral artery catheterization. One hour, four hours and seven hours after the blood flow to the liver was completely blocked, small pieces of liver tissue were taken out and fixed with 10% formaldehyde and 0.25% glutaraldehyde, respectively. Then close the abdomen. During the operation, 500 ml of balanced salt and 800,000 U of penicillin were intravenously injected into the experimental pigs. After death, an autopsy was performed, and liver samples were collected and examined. During the modeling process, the general condition and death time of the model animals were dynamically observed, and the whole blood was collected to prepare the serum for biochemical examination. After modeling, the animals were sacrificed and livers were collected for histological morphological examination.

　　(2) Model features: Rats can wake up immediately after surgery, stand up and walk around. However, liver failure will appear after 1-2 hours. After 12 hours, hepatic coma appeared continuously, showing indifference and rounding of the body. The mice have increased hair, decreased activity, refused to drink water or food, and their urine turned yellow. The 12-hour survival rate of the model animals that underwent 85% liver tissue resection was 93%, and they all died within 48 hours; the model animals that had undergone 95% liver tissue resection survived for 2, 4, 6 and 12 hours, and the tissue resection rate was 92 , Are %, 75%, 58% and 50 respectively. %, all of them died within 24 hours. Model piglets die within 12 to 16 hours after the blood flow in the human liver is completely blocked. Twelve hours after the operation, alanine aminotransferase (ALT), blood ammonia (NH3) and total bilirubin (TBIL) increased, and blood sugar (GLU) decreased. After the piglet’s liver blood flow is blocked, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), Amm, urea nitrogen (BUN), creatinine (CRE), coagulation The zymogen time (PT) gradually increased. , Albumin (ALB) and Fibrinogen (FIB) gradually decreased. When the rat was sacrificed 12 hours after the operation, the remaining liver tissue was dark red with cysts and hyperemia on the surface. Under the microscope, the structure of the liver lobules is destroyed, a large number of liver cells are degenerated and necrotic, the cytoplasm is loose, the necrotic area is infiltrated by inflammatory cells, and the liver sinusoids are expanded and meshed. , And saw congestion and bleeding. One hour after the model piglet completely blocked the blood flow to the liver, the structure of the liver lobules and the arrangement of liver cells were still normal, the disc space was slightly widened, and the liver cells were turbid and swollen. The nuclear membrane and nucleolus are still transparent; after 4 hours of observation under an optical microscope, the structure of the liver lobules still exists, the position of the liver cells is not disturbed, and the liver plate. The space between the livers is obviously enlarged, the central vein collapses, the intrahepatic vacuole degeneration, the nucleus shrinks, and the nucleus disappears; under the electron microscope of glycogen granules, homogenization of cytoplasm, degranulation of thick vesicles, mitochondrial edema, blurred crystals, and dissolution can be seen In some mitochondria, the microvilli of the bile ducts between cells are significantly reduced, nuclear chromatin accumulates, nuclear deformities and inclusions of pseudonuclei are formed. After 7 hours, the rear microscope showed that the structure of the liver lobules was disordered, the arrangement of liver cells was disordered, the liver plate was dissociated, the liver plate dissociation was more obvious, the liver fluid was cloudy, swollen, and vacuolar. The liver cytoplasm is more prominent. , Nuclear chromatin is marginalized, and some liver cell membranes are not marginalized. It is intact and shows signs of beginning to dissolve necrosis. An immediate liver histological microscopic examination revealed that the structure of the liver lobules of the model pigs was unrecognizable, the liver cells were reduced, the arrangement was disturbed, the liver plate was dissected, and the intervertebral disc space was enlarged when liver failure and death occurred. , And there is an extensive, localized, spotted liver.

　　(3) Comparative Medicine Acute liver failure is a syndrome of sudden severe liver injury caused by diffuse hepatocyte necrosis and/or multiple factors. The patient's mortality rate can be as high as 75%. The cause is mainly caused by the hepatitis virus. It may also be caused by one or more traumas, drugs or toxic overdose. Currently, there are two main types of animal models of acute liver failure: surgery and drug-toxic liver injury. The most typical and commonly used one is total liver or partial liver resection, which is a modeling method. Total hepatectomy will undoubtedly lead to liver failure, but this model has the characteristics of clinical acute liver failure, including irreversible course, transient hepatic coma, lack of circulatory damage or liver cell death, which is far from the pathological process. As a result, toxic substances are continuously released and liver function abnormalities only appear 2-4 hours before the death of the model animal. In view of these shortcomings, people have studied and established an animal model of acute liver failure that replaces partial hepatectomy. Under normal circumstances, animals can tolerate up to 70% to 75% of hepatectomy under normal liver function. When liver resection exceeds 75%, fatal liver failure occurs. If liver resection exceeds 85%, other treatments should be given. The door opened into a hole, and almost 100% of the animals died within 48 hours. This model undergoes partial liver resection to remove 85% to 95% of liver tissue. After 6-12 hours, the animal's liver and kidney functions were abnormal and gradually deteriorated, and hepatic coma appeared. Histological morphological examination showed liver lobule disease, liver cell exhaustion, liver plate dissection, obvious expansion of Disse space, local and speckled extensive hepatocyte necrosis, and all animals died within 24-48 hours after surgery. Compared with the total hepatectomy model, partial hepatectomy is closer to the clinic and has physiological and pathological characteristics that can be reversed. For example, in 90% of hepatectomy rats, intrasplenic transplantation of hepatocytes is performed 3 consecutive days before surgery, while 40% of the models can survive for more than 28 days. So far, people have successfully replicated and established partial hepatectomy models for various animals such as rats, rabbits, dogs and pigs. Among them, small animal models are more suitable for studying the pathophysiological processes of acute liver failure and the effects of drug interference on these processes, while large animal models are used to evaluate the effectiveness and safety of artificial liver technology, so they are more commonly used. The animal model of liver failure of total hepatectomy has unique advantages. The replication cycle is short, can quantify the hepatectomy, and there are no specific pathological changes in the remaining liver tissue. In the clinical symptom model of liver failure, the changes of animal symptoms and related blood indicators are reproducible and reliable, with good stability and high success rate.