Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the formation of auto-resistance against a series of self-antigens, and 80-100% of which may involve the kidneys, which is called lupus nephritis (LN). The pathogenesis of lupus nephritis is not yet fully understood, and one of the main obstacles is the lack of a typical animal model. The main obstacle is the lack of a typical animal model. Currently, widely used animal models include the murine spontaneous lupus model, such as NZB/NZW .MLR, and so on. In this paper, we introduce the establishment of an animal model of lupus nephritis (c-hronic graft-versus-host diseatse , eGVHD).
Principle
Depending on the experimental method, the corresponding principles differ:
Chronic autoimmune graft-versus-host reactions are similar to spontaneous systemic lupus erythematosus and almost always involve renal damage. The principle is that self-reactive T-lymphocytes from the donor mouse recognize incompatible structures in the H-2 of B-lymphocytes from the recipient mouse, resulting in abnormal interactions between T- and B-lymphocytes of the same species, leading to the formation of a series of autoantibodies and the development of the lupus nephritis syndrome.
Appliance
The common areas of application of the animal model of lupus-like nephritis are as follows: the animal model can be used for research on the pathogenesis of lupus nephritis in humans.
Operation method
Animal model of lupus-like nephritis
Principle
Chronic autoimmune graft-versus-host reactions are similar to spontaneous systemic lupus erythematosus and almost always involve renal damage. It is based on the recognition of an incompatible structure in the H-2 of recipient murine B lymphocytes by donor murine autoreactive T lymphocytes, resulting in an aberrant homophilic T-B lymphocyte interaction that leads to the formation of a series of autoantibodies and the development of the lupus nephritis syndrome.
Materials and Instruments
Subjects: Move The basic process of the animal model of lupus-like nephritis can be divided into the following steps: . The F1 generation of inbred mice were randomly divided into 2 groups: normal control and model group. Under aseptic conditions, the spleen, lymph nodes (mesentery. Lymph nodes (mesenteric, inguinal and cervical) and thymus were removed, minced, ground on slides, washed with PBS and sieved to make cell suspension, and murine lymphocyte isolate was slowly added along the wall of the centrifuge tube at a ratio of 1:1, centrifuged at 1800 r/min for 20 min, and then the layer of lymphocytes was taken out and washed with diluted PRS for three times (the first time was centrifuged at 1800 r/min for 10 min, and the next two times were centrifuged at 1800 r/min for 5 min), and finally washed with PRS for 5 min. The lymphocyte layer was washed three times with PRS dilution (the first time at 1800 r/min for 10 min, the second time at 1800 r/min for 5 min), and finally the appropriate amount of mixed lymphocyte suspension was prepared with PBS. Cell viability was determined by Tepan blue rejection assay and phase microscopy, and at least 90% of the cells were viable. The total number of cells was determined by flow cytometry. On days 0, 3, 7, and 10, the model group was injected into the tail vein of the recipient mice with 0.25 mL of lymphocyte suspension containing 50 x 10G recipient viable cells per injection. Controls were given equal amounts of PBS each time. B. Animals are fed in metabolic cages with ad libitum access to water and food for 18 hours, and urine (1-3 mL/d) is collected from the metabolic cages from 8 am to 8 am the following day. Starting from 2 weeks after the first injection and every 2 weeks thereafter, the 24-hour urinary serum protein excretion of the model rats was measured (using the radioimmunoassay method). 0.5 mL of blood was collected from the orbital plexus with a glass capillary pipette, and the serum was prepared, stored at -20 ℃, and the anti-ds-DNA antibody was determined by ELISA, and the rats were observed to have any subcutaneous oedema, ascites, weight change, and lifespan. C. In order to understand the morphological changes of the renal lesion process, one mouse from the control group and one mouse from the model group were randomly taken at 4, 8 and 12 weeks after the fourth injection, and then the kidneys were removed by short-neck necropsy and paraffin sections and frozen sections were prepared. Paraffin sections were stained with HE, PAS, PASM and Masson staining; frozen sections were analyzed by direct immunofluorescence for IgG deposition in kidney tissue. At 12 weeks of age, one rat was randomly taken from the model group, and the specimen was prepared for electron microscopy, fixed with 2.5% glutaraldehyde, and then embedded, dehydrated, sectioned, stained and analyzed by electron microscopy. Caveat 1. Changes in blood and urine indicators. Most of the rats showed subcutaneous edema, some showed ascites, and hair loss and poor activity were also seen. Urinary albumin began to increase in all model rats at 2 weeks after the 4th injection, and reached a peak at 12 weeks, while urinary albumin was undetectable in control F1 rats. The level of anti-ds-DNA was significantly increased at 2 weeks after the 4th injection and reached the peak at the 4th week. 2. Pathologic changes. Pathological changes in the kidneys of the model rats appeared at 8 weeks after the 4th injection, and the renal histology was mainly membranous nephropathy at the 12th week, accompanied by inflammatory cell infiltration in the paraglomerular and interstitial compartments of the kidneys, followed by mesangial proliferative nephritis. Immunofluorescence revealed significant IgG, with linear or granular deposits along the glomerular basement membrane. Electron microscopy showed membranous glomerulonephritis with thickening of the basement membrane, deposition of electron-dense material within it, and fusion of epithelial peduncles, all of which are characteristic changes very similar to human lupus nephritis. For more product details, please visit Aladdin Scientific website.
① 8~10 weeks old females (C
57
(BL6 x DBA/2) F1 generation 36 mice at 8~10 weeks of age, weighing 15~20 g. ② 150 female DBA/2 at 6~8 weeks of age, weighing 10~15 g;
② 150 female DBA/2 mice aged 6~8 weeks, weighing 10~15g.
Experimental reagents:
① 2.5% glutaraldehyde.
A