动物造模,肾功能衰竭模型 z-bio 2021-11-01 757

　　1. Adriamycin induction model

　　(1) Reproduction method Adult rats weighing about 250g are anesthetized by injecting pentobarbital sodium at a dose of 30 mg/kg body weight through the abdominal cavity. After anesthesia, the rats are fixed on the operating board supine, and the abdominal operating area is routinely disinfected and depilated, along the right The skin was cut through a para-abdominal incision to expose the right kidney. After the renal pedicle was ligated, the right kidney was removed. The incision was sutured and the abdominal cavity was closed by routine surgery. After 15 days, the rats were injected with adriamycin (adriamycin, ADR) at a dose of 5 mg/kg body weight through the tail vein of the rat.

　　CRF model; for long-term model building, 28 to 48 weeks of observation are required. Before and after modeling, the model rats were placed in a metabolic cage to collect 24h urine, and the 24h urine volume and urine protein volume were measured; venous blood samples were taken for the blood biochemical index determination at the predetermined time of the experiment; the animals were put to death after anesthesia, and the left kidney tissues of the animals were taken The specimens were fixed in a fixative solution, embedded in paraffin, and used for regular tissue sectioning, stained with HE, and observed under a microscope.

　　(2) Model characteristics: 4-8 weeks after injection of ADR, model animals can experience increased urine output, decreased activity, and slower weight gain. Proteinuria can appear in model rats 6 to 14 days after injection, and proteinuria is the highest at about 4 weeks. Proteinuria usually lasts for 6 to 9 months, and is accompanied by hypoproteinemia, hyperlipidemia, edema, thoracic cavity and Abdominal effusion; animal systolic blood pressure (SBP) increased, inulin clearance rate (Cin) decreased. Histopathological observations under the microscope showed that the glomeruli of the model animals were obviously hypertrophy in the early stage of modeling, and there were early renal tubular epithelial damage and glomerular sclerosis pathological changes; the glomerular sclerosis was more serious in the late stage of modeling, and the focal kidney was small. The scope of bulbar sclerosis was significantly enlarged and more extensive. The acute phase of this model (3-6 weeks after injection of ADR) is similar to human minimal change nephropathy, that is, renal tubular epithelial cell damage is the main cause. The chronic phase (6-9 months after administration) is similar to human focal segmental glomerulosclerosis.

　　(3) Comparative medicine Doxorubicin is an anthracycline anti-tumor drug commonly used in clinical practice. It can have direct toxic effects on glomerulus and renal tubular epithelium, and make the membrane barrier and absorption function of renal proximal tubule epithelial cells. Damaged, the cell nucleus disintegrates; the shed tubular epithelial cells and the protein and other cellular components in the lumen form a cast, which blocks the lumen, leading to an increase in the internal pressure of the renal tubules, aggravating kidney damage, and ultimately forming CRF. ADR is used to prepare CRF models, and there are various methods, including simple intravenous injection, or injection plus nephrectomy model, and the resection method can also be selected. Therefore, the disease characteristics of different models are not completely consistent. This model has good practical value for the observation of drug efficacy and drug screening in areas such as dilating renal tubules, reducing renal tubular pressure, and improving proteinuria.

　　2. Adenine induction model

　　(1) Reproduction method Adult rats weighing about 300g are fed with casein feed containing 0.5% to 0.75% adenine; or a suspension is prepared with adenine, and the daily dose is 200 mg/kg body weight. Gavage the rat. The model animals can drink and eat freely. The modeling process takes 3 to 4 weeks to replicate the chronic CRF model of metabolic disorders, electrolyte abnormalities, endocrine abnormalities and anemia similar to human GRF. Blood samples were collected for blood biochemical determinations at the predetermined time in the experiment, and kidney tissue samples were taken, fixed in the fixative, and routinely sliced and examined under a light microscope.

　　(2) Model characteristics: Model animals developed polyuria, polydipsia, lethargy, reduced activity, less food intake, weight loss, pale ears, pale red eyes, squinted eyes and eyelids on the 12th to 15th day of administration. Puffy, clammy tail, dry and loose body hair. Animal blood creatinine, blood urea nitrogen, middle molecular substances are significantly increased, red blood cells, hemoglobin, hematocrit, platelets are all decreased, electrolyte metabolism disorders (high phosphorus, high potassium, low calcium, low sodium, etc.), amino acid metabolism disorders (essential Amino acid reduction) and anemia. Observed by naked eyes, the volume of the kidney has increased significantly. Under histopathological observation under the microscope, most of the renal tubules in the renal tissue have been destroyed, and the tubule epithelial cells have edema and necrosis. A large number of needle-like or rectangular purine metabolite crystal deposits can be seen in the lumen and interstitium; There is lymphocyte infiltration, and local fibrous tissue proliferation can be seen. The destruction of renal tubules is the main feature of this model.

　　(3) Comparative medicine So far, the mechanism of adenine-induced chronic renal failure in rats has not been elucidated. It may be that high concentrations of adenine become 2,8-dihydroxyl which is extremely difficult to dissolve in water through the action of xanthine oxidase. Purine, which is blocked after being deposited in the renal tubules, inhibits the excretion of nitrogen compounds in the body, causing azotemia, toxin accumulation and electrolyte and amino acid metabolism disorders in the body, causing animal pathological characteristics similar to human minimal change nephropathy CRF model. This model has a simple manufacturing method and a short model period. With the length of adenine feed feeding time, mild, moderate and severe CRF models can be prepared respectively. Intraperitoneal injection of adenine into animals can also replicate the CRF model, but it is different from the method of adenine gavage in terms of adenine dosage and modeling time. This model is suitable for the observation and evaluation of the efficacy of drugs that can restore renal tubular function during CRF.

　　3. Cadmium chloride induction model

　　(1) Reproduction method Male KM mice weigh about 20 g. It was fed with mixed feed containing 1g/kg body weight of cadmium chloride (63.1g protein, 52.5g fat and 270.2g sugar per kilogram of feed). The medicinal powder was added to the feed to make a mixed feed. The amount of the medicine used was 5% of the lethal intake per day, and each mouse was estimated by eating 3g of feed per day. All mice were free to eat and water. After 45 days, they were changed to normal diet for a total of 125 days of observation. Blood samples were collected for blood biochemical determinations at the predetermined time in the experiment, and kidney tissue samples were taken, fixed in the fixative, and routinely sliced and examined under a light microscope.

　　(2) Model characteristics 15 days after administration, the blood urea nitrogen and blood creatinine concentrations of the model animals were significantly increased; the renal proximal tubule epithelial cell degeneration was observed under a light microscope, the renal interstitium showed inflammatory infiltration of monocytes, and the glomeruli had nuclei The number of cells increased; 125 days after the medication, the renal tubular lesions disappeared, the glomeruli were hypertrophy, the number of nucleated cells increased significantly, and some glomeruli were fibrotic; there was still monocyte inflammatory infiltration in the renal interstitium. This model is inexpensive, simple to make, and has few interference factors. It is very similar to CRF caused by toxic nephropathy in clinical practice.

　　(3) Comparative medicine Long-term exposure to low concentrations of cadmium in life has a toxic effect on human renal tubules, and can cause CRF with renal tubule damage as the main pathological feature. The CRF model established by feeding mice mixed with cadmium chloride feed showed obvious renal function and renal tissue damage, and showed progressive aggravation characteristics, similar to the clinical CRF caused by cadmium poisoning. In chronic cadmium poisoning, the damage to the renal tubular epithelial cells of the body is the main direct effect, and it can be recovered after disengagement; while the damage of cadmium to the glomerulus and renal interstitium is involved in secondary factors, when the damage reaches a certain level At a certain degree, even if the contact is disengaged, secondary factors can continue to exist, making the glomerular and renal interstitial lesions progressively aggravated and eventually forming CRF. This model can be used for clinical research on CRF drug treatment and screening caused by cadmium poisoning.

　　4. Xiaozhiling induction model

　　(1) Replication method Male rats weighing about 200g and tested for negative urine protein were injected with sodium pentobarbital at a dose of 30 mg/kg body weight through the abdominal cavity. After anesthesia, the rats were fixed on the operating board on their back and injected into the abdomen. The area is routinely disinfected, the kidney is fixed with a "kidney locator", and 0.6ml refined Xiaozhiling and the same amount of normal saline are used to prepare 1.2ml Xiaozhiling injection, which is directly injected into both kidneys from both sides of the abdomen. During injection, insert the needle from the lower pole (4 gauge needle) to the upper part of the middle pole, as far as possible to the cortex, with the needle tip facing the cortex, inject slowly (at least 1 min each time), and press the needle eye with an iodine tincture cotton ball after the needle 3min or more. Before and after modeling, the urinary protein, urea nitrogen and creatinine of the model rats were measured. At the same time, the animals were put to death after anesthesia. The left kidney tissue specimens of the animals were taken, fixed in fixative, embedded in paraffin, and made conventional tissue sections, stained with HE and under light microscope Observed. And take the kidney tissue as the electron microscope specimen, observe under the transmission electron microscope.

　　(2) Characteristics of the model The model animals have reduced diet and weight within 3 days after injection, and the body weight has recovered after 1 week, but the weight gain has slowed down; the model rats have loose body hair and hair loss during the modeling period. After the animal was dissected, it was observed with naked eyes that the kidneys were atrophy and were yellowish-brown, with hard texture, uneven surface, and diffuse yellowish-brown spots and plaques. Microscopic histopathological observations showed that within 2 to 4 weeks after modeling, coagulation necrosis could be seen in the kidney tissue of the model animal, the remaining glomeruli were compensated for hypertrophy, the renal tubules were proliferated and expanded, and the tubular epithelial cells were swollen, degenerated, necrotic, and shedding , The protein cast can be seen in the lumen. From 6 weeks after modeling, mesangial cells and matrix have mild to moderate proliferation, some residual glomeruli can be seen with focal hyalinosis, some interstitial and glomerular fibrosis; by 8 weeks, the kidney is small The glomerulus is obviously fibrotic; by 16 weeks, the whole glomerulus is fibrotic, and the renal interstitium also has obvious fibrosis. Ultrastructural observation under the electron microscope showed that 8 weeks after modeling, endothelial and epithelial cells were seen to form vacuoles, the foot processes were fused and disappeared, the basement membrane was thickened and loosened, and collagen fibers and renal tubular epithelium were seen between the basement membrane matrix and the foot processes. The cells are swollen, the microvilli become shorter, the arrangement is disordered, the lumen is occluded, and there are protein casts inside; the rough endoplasmic reticulum expands and increases, and the lysosomes increase. By 16 weeks, the ultrastructural pathological changes were further aggravated, glomerular fibrosis, renal capsule disappeared, capillary basement membrane thickened and shrunk, and electron dense deposits were visible. Late stage: metabolic disorders, severely damaged glomerular and tubular functions, both light and electron microscopy showed high consistency with the pathological changes of human CRF pyknotic kidney.

　　(3) Comparative medicine Xiaozhiling is mainly composed of Chinese medicine gall and alum and other effective ingredients. Tannic acid, the main component of Galla Chinensis, has strong astringency to body tissues, which can coagulate protein and constrict blood vessels. Potassium aluminum sulfate, the main component of alum, has a strong inflammatory effect on the body part and can cause tissue fibrosis. Xiaozhiling preparations can cause vasculitis, arteriovenous thrombosis and proliferative endarteritis in the body, and can promote vascular stenosis and reduce or block local blood flow. The method of injecting an appropriate amount of Xiaozhiling into the rat kidney can destroy most of the nephrons and eventually lead to the pathological manifestations of CRF in the rat. The model making method is simple and easy to implement, with little trauma and no infection, the model is stable and reliable, and the success rate is high. The dose can be strictly controlled to make CRF models with different severity of lesions of mild, moderate and severe. The characteristics of the model are similar to those of human CRF pyknosis in clinical practice. The kidney is similar, and it is suitable for research on drug treatment and screening of similar diseases.

　　5. Daunorubicin induction model

　　(1) Reproduction method After examination, the urine protein was negative, and adult rats weighing about 200g were injected with daunorubicin at a dose of 10mg/kg body weight through the tail vein. After the injection, the animals were kept and observed routinely, and they were free to drink and eat. It takes 4 to 6 weeks to copy the CRF model. In the process of model preparation and observation, urine and blood were collected as indicators, and the animals were anesthetized at the predetermined time in the experiment. Kidney tissue specimens were taken and placed in the fixative for routine tissue sectioning, HE staining, and observation under a light microscope. At the same time, the kidney tissue was taken as an electron microscope specimen and observed under a transmission electron microscope.

　　(2) Model characteristics 1 to 3 weeks after injection of daunorubicin, model animals can develop a large amount of proteinuria (greater than 300mg/24h) and maintain a high level, often accompanied by hyperlipidemia and hypoproteinemia Symptoms and edema of the whole body. Under light microscope, the glomerulus in the kidney tissue of the model rat was slightly swollen, the capillary cavity of a small amount of vascular loops was slightly narrowed, and the renal tubules were mildly affected. There were many casts in the cavity, and there were no blood vessels in the renal interstitium. Obvious pathological changes. Electron microscopy observed the fusion or flattening of foot processes of most glomerular visceral epithelial cells.

　　(3) Comparative medicine The principle of making this model is similar to that of ADR. Daunorubicin can be metabolized and reduced to semiquinone free radicals in a certain part of the kidney. Free radicals react with oxygen to produce reactive oxygen species. These substances can cause lipid peroxidation in glomerular epithelial cells and change glomerular epithelium. Cellular glycoprotein metabolism destroys the structure and function of the glomerular filtration membrane, and finally causes selective changes in the membrane filtration barrier to trigger proteinuria and cause CRF in model animals. Compared with the CRF model established by the adenine method, this model has a heavier degree of urine protein and a longer duration, and its application is the same as that of the adriamycin CRF model.