动物造模,肾小管间质纤维化模型 z-bio 2021-03-26 267

　　(1) Reproductive method Gentamicin (GM) was injected subcutaneously into adult rats weighing 200 to 260 g twice a day for 2 consecutive days, with a body weight of 400 mg/kg. Rat urine was collected on days 2, 5, 8, 11, 15, 15, 22, and 29 after the first injection of GM to collect 24-hour urine output (UV), urinary creatinine (UCr) and natriuresis (UNa). (CCr) and sodium excretion rate (FENa), blood creatinine (SCr), blood urea nitrogen (BUN) and blood sodium level (SNa) are collected in it. Anesthetize the animal and collect the kidneys. , Part of the kidney tissue was fixed with 10% formaldehyde. It is embedded in conventional paraffin, serial tissue sections, conventional HE, PAS staining, and observed with an optical microscope. Another part of the renal cortex tissue was photographed with a transmission electron microscope, fixed with 2.5 glutaraldehyde-1.0 acid, sectioned with a transmission electron microscope, and observed with an electron microscope.

　　(2) Model characteristics Two days after the first injection of GM, the ultraviolet rays of the model animals began to increase after 24 hours, reached a peak on the 8th day, then decreased, and returned to normal on the 22nd day. day. The urine protein of model animals gradually increased, reached a peak on the 5th day, continued to decline after 8 days, and returned to normal on the 22nd day. Serum creatinine (SGr) gradually increased and reached a peak on the 11th day. After that, it dropped sharply. On the 15th day, on the 2nd day of GM injection, blood urea nitrogen (BUN) increased slightly, and significantly increased on the 5th day. After reaching the peak on the 8th and 11th days, blood urea nitrogen (BUN) dropped rapidly and returned to normal on the 22nd day. The creatinine clearance rate (CCr) began to decline on the 5th day, rose slightly on the 8th day, and then continued to decline. On the 22nd day (basically the 29th day) and the sodium excretion rate (FENa) returned to normal. ) Reached its peak on the 15th day, began to decline on the 22nd day, and basically dropped to a normal level on the 29th day. day. Microhistopathological observations showed that 6 hours after injection of GM, the brush border of the proximal tubular epithelial cells was partially shed; on the second day, the renal cortex and extramedullary area were located, and inflammatory cell infiltration was visible, and the proximal tubular epithelial cells swelled . .. Collect vacuum denatured and abnormal PAS-positive inclusion bodies, and some of the brush margins disappeared. On day 5, the proximal tubule epithelial cells of the renal cortex undergo extensive necrosis, and the lumen is filled with red granular necrosis. Type, the tube wall does not seem to be broken or crushed, the PAS-stained basement membrane remains intact, and a small number of proximal tubules begin to cover the basement membrane with a flat epithelial cell. On the 8th and 11th days, the necrotic material in the lumen gradually decreased. The regeneration of the rotating tubules is obvious, the epithelial cells gradually increase and increase, and some papillary proliferations that extend into the lumen show mitosis. PAS staining gradually increased the brush border; on the 15th day, the shape of the EGF-treated group was close to the control group, and only a small amount of inflammatory cell infiltration was seen in the matrix; on the 22nd day, the necrotic material in the renal tubules disappeared and the epithelium The cells proliferate in the form of cubes, and a small amount of inflammatory cell infiltration can still be seen in the kidney matrix; on the 29th day, it is the morphological structure of the kidney tissue of the model animal. Has returned to normal. During the modeling process, the epithelial cells of the distal tubule and the collecting duct of the kidney showed vacuolar degeneration, but no necrotic changes, and a column was found in the distal tubule. Ultrastructural observations by transmission electron microscopy showed that 2 days after injection of GM, a large number of microvilli of the epithelial cells of the renal tubules of model animals were lost; the secondary phagosomes in the cytoplasm increased significantly, and many body types were found in the medulla. Layered or banded high electron density, mitochondria expand, decrease in number, and lose matrix particles. The internal folds of the plasma membrane on the cell base surface are flattened and reduced in number. On day 5, the proximal tubule epithelial cells of the kidney were necrotic, and the lumen was a complete membrane fragment of the medulla, nucleus and organelles; a small amount was in multiple medulla oblongata and a single remaining proximal tubule epithelium; residual luminal microvilli cells; In the initial epithelial cells, covering the surface of the proximal tubules on the 8th and 11th days, thick and short microvilli gradually appeared on the progenitor cell membranes of the lumen and basal surface, and the internal folds of the folds gradually increased; the internal ribosomes, rough Within 15 days, the Gordi of epithelial cells gradually increased, the medulla decreased, and the number of mitochondria increased; swallowing follicles began to appear in the lumen of the proximal tubule epithelial cells; at 22 days, there was still a small amount of bone marrow kidney Proximal tubule epithelial cells; on day 29, the ultrastructure of the animal returned to normal.

　　(3) Comparative Medicine Acute tubular necrosis (ATN) is the most important clinical cause of ARF, and it is also one of the clinically important diseases. Gentamicin (GM) is widely used as an effective aminoglycoside antibiotic for Gram-negative bacterial infections, but its toxic effect on the kidneys and its potential to cause ATN or ARF cannot be ignored. The pathogenic mechanism of this model is that the GM in the body is eliminated mainly through glomerular filtration, but a small part (≤5%) of the filtered material may be reabsorbed by the proximal tubule. Inclusion bodies containing GM are swallowed by lysosomes, inhibit the activity of various phosphonate lipases, and form bone marrow and bone marrow bodies. The formation of the body is a sensitive indicator of genetically modified nephrotoxicity. At the same time, GM can inhibit activity. The role of protein kinase C changes the normal metabolism of mitochondria and other pathogens, resulting in the toxic destruction of renal tubules. The degree of necrosis of GM to renal tubular epithelial cells is related to the dose. Low-dose GM caused the formation of a large number of medulla bodies in the proximal tubular epithelial cells, but there was no obvious change in renal tissue function or renal morphology or kidney under the microscope; and high-dose GM may seriously damage the renal tubules and severe damage Renal tubules. In the ATN model induced by this method, renal tubular reabsorption dysfunction occurred during necrosis and recovery, resulting in a decrease in glomerular filtration rate. This model is characterized by polyuria renal failure, because the renal tubule reabsorption of water and sodium is reduced, which offsets the decrease in primary urine caused by the decrease in glomerular filtration rate. The model is easy to manufacture, low cost, and highly reproducible, and can be used in various clinical trials of acute renal failure.