Experimental reagents Collagenase V, 512 kut / mg (Sigma), DNase (Sigma), fetal bovine serum (Gibco), RPMI1640 culture medium (Gibco), Dextran (Pharmacia), Hanks balanced salt solution (Gibco) the company).
Experimental equipment centrifuge, syringe, microscope, ultra-clean workbench, etc.
Experimental materials Adult hybrid pigs, 12 months old and weighing about 150 kg. After the pigs were slaughtered and bleed, the pancreas was quickly removed under relatively sterile conditions, stored in Hanks solution at 4 ° C and sent to the laboratory with less warm ischemia time At 10 minutes, the cold ischemia time was less than 90 minutes.
Experimental procedure 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 coating, and retain the inherent coating. Transect the pancreas from the junction of the head of the pancreas and the body of the pancreas, find the main pancreatic duct, insert it with a 20 G trocar, ligate the suture, and ligate and cut the distal end of the tail. Take about 15-20 g of tissue each time and inject at 4 ° C Ca2 Hanks solution prepared compound collagenase solution (1.5 g / L, containing DNase 0.3 g / L), injection volume = pancreas mass × 2, injection speed 7 mL / min, completed within 3-4 minutes, placed in glass Inside the container, shake and digest in a water bath at 38.5 ± 0.1 ℃ with a shaking speed of 100 r / min. Add 0.1 mmol / L NaOH in the middle of the digestion process to keep the pH of the digestion solution as close as possible to 7.8, starting from 20 minutes of digestion Sampling was performed every 4 minutes and stained with dithizone for microscopic examination. When the pancreatic tissue was digested and lysed into fine sand, microscopic examination revealed that most of the structurally complete islets were shed from the exocrine tissue. 100 mL / L fetal bovine serum) to terminate the digestion, after thoroughly mixing, filter with a 40 mesh steel mesh, collect the digested tissue, centrifuge and wash twice at 4 ℃, remove fat and other foreign cells, sample the microscopic count and observe after digest Islet separation.
2. Islet purification
Dextran was used to prepare discrete density gradients with densities of 1.037, 1.054, 1.070, 1.096, and 1.11 kg / L. Discontinuous density gradients were sequentially added 10 mL each to 50 mL centrifuge tubes. Finally, the digested tissue after washing 0.5 mL was mixed with 1.037 kg / L density gradient solution 10 mL, and carefully transferred to the upper layer of the liquid in the centrifuge tube to form a discontinuous density gradient. Place 2-4 centrifuge tubes in a cryogenic 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 Wash twice by centrifugation at ℃. Samples were taken for microscopic examination to estimate purity, biological activity and histological identification.
3. Islet counting and purity determination
The isolated and purified tissue suspension was stained with dithizone (dithizone 10 mg, absolute ethanol 3 mL, 250 g / L ammonia 50 mL), and the pancreatic islet cell mass was stained red or red under light microscope, exocrine tissue No coloration, round, oval or irregular shape. Count the islets with a diameter of ≥50 mm under the 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 tissues.
4. Identification of islet biological activity
Place the purified islet cell suspension under a microscope, use a Pasteur pipette to suck the islets into the culture plate, adult pigs per 10 islet equivalents (each islet equivalent is equivalent to a 150 mm diameter islet cell mass, IE) adult pig The islets (APIs) were put into a culture well, 6 wells were 1 group, a total of 4 groups. Each group was placed in a 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 sugar theophylline) in RPMI1640 culture solution, placed in an incubator at 37 ° C and containing 50 mL / L CO2 for 4 h, collect the culture solution and release it with insulin The kit (Institute of Atomic Energy, Chinese Academy of Sciences) measures insulin content.
5. Islet histological examination
After centrifugal purification, the islet cell suspension was collected, the islet tissue was collected, fixed with anhydrous alcohol, paraffin-embedded sections, HE staining to check the structural integrity of the islet tissue.
6. Statistical processing
The data obtained are expressed as mean ± SD, and the mean difference between groups was compared by t test, P <0.05 was considered as a difference.
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