Biological characters of islets isolated from rats and purified with Dextran T40 / 解放军医学杂志

ABSTRACT

Objective To establish a method of isolating and purifying islets from Wistar rat, and to evaluate the biological characters of the isolated and purified islets. Methods The pancreases were surgically removed from adult Wistar rats, carefully minced with scissors as small as possible prior to incubation in 0.9 mg/ml collagenase (Sigma, type Ⅴ) for 10~17min at 37℃. The digested tissue was filtered through a 60 apertures/cm~2 metal sieve and washed with 10% fetal bovine sera (FBS) in Hank’s solution. Purification was performed using Dextran discontinuous density gradient centrifugation to separate the contents of the postcollagenase pellets. Islet samples were stained with DTZ staining for identification. The viability of the purified islet was determined by acridine orange/propidium iodide (AO/PI) double staining. The in vitro function was assayed by different densities of glucose incubating islets for 45min, respectively. Insulin secretion in the supernatant was assayed by radioimmunoassay. Islets were transplanted under the kidney capsule into recipient SD rats that had been rendered diabetic by injecting streptozotocin 50mg/kg, so as to evaluate the in vivo function of islets. Results The islets were recovered between the 11% and 22% Dextran layers, and between the 11% and 1640 layers. The average number of isolated islets was 2181?14 per rat pancreas. After a discontinuous Dextran density gradient purification, about 1826?24 islets were obtained from per rat pancreas, with an average purity over 95%. Islets showed good response to glucose and high efficiency to invert hyperglycemia 7-10d after transplantation. Conclusion These results showed that the digestion methods and purification procedure which we have developed could provide islets from rats for transplantation studies. The biological characters of islets harvested by our method were very well.