动物实验,胰岛分离纯化 z-bo 2020-08-13 599

　　Main reagent

　　Collagenase V, 512 kut/mg (Sigma), DNase (Sigma), Fetal Bovine Serum (Gibco), RPMI1640 Medium (Gibco), Dextran (Pharmacia), Hanks Balanced Salt Solution (Gibco) .

　　Major equipment

　　Centrifuge, syringe, microscope, clean bench, etc.

　　Experimental Materials

　　Adult hybrid pigs are 12 months old and weigh about 150 kg. After the pigs are slaughtered and bled, the pancreas is quickly taken out under relatively sterile conditions, stored in Hanks solution at 4℃, and sent to the laboratory. Warm ischemia time is less than 10 min, cold ischemia time is less than 90 min.

　　Experimental steps

　　1. Islet separation

　　After the pancreas is retrieved, it is placed on an ultra-clean workbench to quickly remove the peripancreatic tissue, fat, blood vessels and surgical envelope, while retaining the proper envelope. Transect the pancreas from the junction of the head of the pancreas and the body of the pancreas to find the main pancreatic duct, insert it with a 20 G trocar, ligate with sutures, and ligate and cut the distal end of the tail. About 15-20 g of tissue are taken each time and injected with 4℃ Composite collagenase solution (1.5 g/L, containing DNase 0.3 g/L) prepared with Ca2 Hanks solution, injection volume = pancreatic mass×2, injection speed 7 mL/min, completed within 3-4 minutes, placed in glass In the container, shake digestion in a 38.5±0.1℃ water bath at a shaking speed of 100 r/min. Add 0.1 mmol/L NaOH intermittently during the digestion process to maintain the pH of the digestion solution at about 7.8 as much as possible, starting from 20 minutes of digestion Sampling was taken every 4 minutes, and dithizone staining was performed under microscopy. When the pancreatic tissue was digested and lysed into fine sand, the microscopy showed that most of the structurally intact pancreatic islets were detached from the exocrine tissue. Use 4℃ cold Hanks solution (including 100 mL/L fetal bovine serum) terminate the digestion, after mixing thoroughly, filter with 40 mesh steel mesh, collect the digested tissue, centrifuge at 4℃ for 2 times, remove fat and other foreign cells, take samples for microscopic examination, count and observe after digestion Islets separation.

　　2. Islet purification

　　Use Dextran to prepare discontinuous density gradient solutions with densities of 1.037, 1.054, 1.070, 1.096, and 1.11 kg/L. Then add 10 mL of each discontinuous density gradient solution to a 50 mL centrifuge tube. Finally, the washed digested tissue Mix 0.5 mL with 10 mL of 1.037 kg/L density gradient solution and carefully move to the upper layer of the liquid in the centrifuge tube to form a discontinuous density gradient. Put 2-4 centrifuge tubes in a low-temperature centrifuge, centrifuge at 800 r/min for 5 min, then at 2,500 r/min for 15 min, and collect purified islets between 1.096-1.054 kg/L after centrifugation. 4 Centrifuge and wash 2 times at ℃. Respectively, samples were taken for microscopic examination and counted to estimate the purity, biological activity and histological identification.

　　3. Islet count and purity determination

　　The separated and purified tissue suspension was stained with dithizone (dithizone 10 mg, absolute ethanol 3 mL, 250 g/L ammonia water 50 mL), the pancreatic islet cell mass was stained scarlet or red under light microscope, exocrine tissue It is not colored, but is round, oval or irregular. Count the islets with a diameter of ≥50 mm under a microscope to calculate the equivalent number of islets separated per gram of pancreatic tissue. The purity is estimated by the ratio of the amount of endocrine and exocrine tissue.

　　4. Identification of the biological activity of islets

　　Place the purified islet cell suspension under a microscope, and use a Pasteur pipette to suck the islets into the culture plate, each 10 islet equivalent (each 1 islet equivalent is equivalent to the islet cell cluster with a diameter of 150 mm, IE) of adult pigs Pancreatic islets (APIs) were placed in one culture hole, 6 wells were 1 group, 4 groups in total. Each group was placed in sugar-free medium, containing 5.6 mmoL/L glucose (low sugar), 16.7 mmoL/L glucose (high sugar), 16.7 mmoL/L glucose plus 10 mmoL/L theophylline (high glycophylline) RPMI1640 culture medium, placed in 37 ℃, 50 mL/L CO2 incubator for 4 h, collect the culture medium, and use insulin to immunize The kit (Institute of Atomic Energy, Chinese Academy of Sciences) measures insulin content.

　　5. Histological examination of islets

　　After centrifugation and purification, the islet cell suspension was collected, the islet tissue was collected, fixed with absolute alcohol, paraffin-embedded section, and HE staining to check the structural integrity of the islet tissue.

　　6. Statistical processing

　　The data obtained are expressed as mean±SD, the mean difference between groups is compared by t test, and P<0.05 is considered as a difference.