Method for establishing peritoneal dialysis injury model of mouse and application of BMSCs exosome
An injury model and exosome technology, applied in the field of biomedicine, can solve the problem of ineffective prevention or delay of peritoneal dialysis-related peritoneal fibrosis, etc.
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Embodiment 1
[0032] Research Objects and Specimen Collection
[0033] 1. BMSCs extraction and identification:
[0034] (1) Primary isolation of mouse BMSCs (bone marrow mesenchymal stem cells):
[0035] Mice were sacrificed by decervical dissection, soaked in 75% ethanol for 10 min, transferred to ultra-clean workbench, and the bilateral tibia and femur were taken out under aseptic conditions and placed in a Petri dish filled with sterile PBS. Use scissors and forceps to remove musculature from the bone, and cut away the ends of the tibial and femoral epiphyses. Use a 1ml sterile empty needle to inhale the culture medium, then insert it into the epiphysis to rinse the bone marrow cavity repeatedly, and blow the cell suspension with a pipette gun. After standing for 5 min, the isolated primary BMSCs were incubated in a sterile culture dish with 5% CO 2 1. Cultivate in an incubator environment with constant temperature (37°C) and constant humidity. After 24 hours, observe the cell state u...
Embodiment 2
[0044] Isolation, purification and identification of exosomes (in cooperation with Shanghai Jikai Gene Medical Technology Co., Ltd.): Isolation and purification of exosomes from the supernatant of mouse BMSCs, using the most widely used separation method, ultracentrifugation, mainly Relying on their morphological characteristics, particle size, and marker proteins (CD9, CD63, TSG101) to identify exosomes, the main identification methods used in this study include: ①Transmission electron microscopy (TEM); ②Western blotting (WB).
[0045] (1) Exosomes were extracted by ultracentrifugation: Thaw 200ml of BMSCs supernatant at 4°C or on ice, centrifuge at 2000g at 4°C for 30min, and take the supernatant; Centrifuge at 120,000g at 4°C for 2 hours; carefully absorb the supernatant (reserve), and use for precipitation Resuspend in 200μL pre-cooled PBS. A total of 1ml of exosomes were extracted from it, and the concentration of exosomes was calculated based on BCA quantification to be...
Embodiment 3
[0050] Establishment of peritoneal dialysis peritoneal injury model induced by human peritonitis peritonitis effluent in mice and exosome treatment of peritoneal dialysis-related peritoneal injury in mice
[0051] Selection of peritoneal dialysis effluent from human peritonitis: collect peritoneal dialysis effluent from patients with peritonitis associated with peritonitis who meet the inclusion criteria, and then perform centrifugation (948g or 3000r / min, 4°C, 15min), bacterial filtration, -80°C Store in aliquots and rewarm in a constant temperature water bath at 37°C before use. The inclusion criteria for the collected peritonitis peritonitis peritoneal dialysis effluent mainly include: ① stay in the abdomen at night for the first admission; ② no antibiotics; ③ culture of Gram-negative bacilli in ascites; Procalcitonin (PCT)>0.3ng / ml, endotoxin>0.1EU / mL, and other biochemical indicators, such as sugar concentration, urea, electrolytes, creatinine, osmotic pressure, etc., but...
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