Experimental reagent
Collagenase V, 512 kut/mg (Sigma), DNase (Sigma), fetal bovine serum (Gibco), RPMI 1640 medium (Gibco), Dextran (Pharmacia), Hanks balanced salt solution (Gibco) .
Laboratory equipment
Centrifuges, syringes, microscopes, clean benches, etc.
Experimental Materials
Adult hybrid pigs, 12 months old, with a body weight of about 150 kg. After the pigs are slaughtered and bled, the pancreas is quickly removed under relatively sterile conditions and stored in Hanks at 4 °C. The warm ischemia time is less than 10 Min, cold ischemia time is less than 90 min.
Experimental procedure
Islet isolation
After the pancreas is retrieved, it is placed on a clean bench to quickly remove the peripancreatic tissue, fat, blood vessels, and surgical mask, retaining the intrinsic envelope. The pancreas was transected from the junction of the pancreatic head and the pancreas, and the main pancreatic duct was found. The 20-gauge trocar was inserted, the suture was ligated, and the distal end of the tail was also ligated and cut. Each time, about 15-20 g of tissue was injected and injected at 4 °C. Ca2 Hanks solution of complex collagenase solution (1.5 g / L, containing DNase 0.3 g / L), injection volume = pancreas mass × 2, injection speed of 7 mL / min, completed within 3-4 min, placed in glass In the container, shake and digest in a water bath of 38.5±0.1 °C, the vibration speed is 100 r/min, and add 0.1 mmol/L NaOH in the middle of the digestion process to maintain the pH value of the digestive juice as much as 7.8, starting from 20 min. Sampling every 4 min, dithizone staining microscopy, when the pancreatic tissue was digested and lysed into fine sand, microscopic examination showed that most of the structurally intact islets emerged from the exocrine tissue, and immediately used cold Hanks liquid at 4 ° C (including 100 mL / L fetal bovine serum) to terminate digestion, fully mixed, filtered with 40 mesh steel mesh, collect the digested tissue, centrifuge 2 times at 4 ° C to remove impurities such as fat, sample microscopic count and observe after digestion Islet separation.
2. Islet purification
Dextran was used to prepare a density density gradient of 1.037, 1.054, 1.070, 1.096, 1.11 kg / L, and 10 mL of each of the discontinuous density gradients was added to a 50 mL centrifuge tube, and finally the washed tissue was washed. Mix 0.5 mL with 10 mL of 1.037 kg/L density gradient and carefully transfer to the upper layer of the liquid in the centrifuge tube to form a discontinuous density gradient. Place 2-4 centrifuge tubes into a cryogenic centrifuge, centrifuge at 800 r/min for 5 min, then centrifuge at 2 500 r/min for 15 min, and collect purified islets between 1.096-1.054 kg/L after centrifugation. Wash twice by centrifugation at °C. Samples were taken for microscopic examination, and purity and biological activity and histological identification were estimated.
3. Islet count 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 islet cell mass was stained red or red, exocrine tissue under light microscope. Not colored, round, elliptical or irregular. Counting islets with a diameter ≥ 50 mm under a microscope to calculate the number of islet equivalents per gram of pancreatic tissue, and the purity is estimated by the ratio of the amount of endogenous and exocrine tissue.
4. Identification of islet biological activity
The purified islet cell suspension was placed under a microscope, and the islets were pipetted into a culture plate using a Pasteur pipette, and adult pigs were exchanged for every 10 islet equivalents (each islet equivalent equivalent to a diameter of 150 mm of islet cell mass, IE). Islet (APIs) were placed in a culture well, and 6 wells were grouped into 4 groups. Each group was placed in a sugar-free medium containing 5.6 mmoL/L glucose (low sugar) and 16.7 mmoL/L glucose (high glucose). 16.7 mmoL/L glucose plus 10 mmoL/L theophylline (high-sugar theophylline) in RPMI1640 medium, incubate in an incubator containing 50 mL/L CO2 at 37 °C for 4 h, collect the culture solution, and excrete with insulin. The kit (Institute of Atomic Energy, Chinese Academy of Sciences) measures insulin content.
5. Islet histological examination
The islet cell suspension was centrifuged and purified, and the islet tissue was collected, fixed with absolute alcohol, embedded in paraffin, and examined for structural integrity of islet tissue by HE staining.
6. Statistical processing
The data obtained were expressed as mean±SD, and the difference between the groups was compared by t test, and P <0.05 was considered to be different.
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