On the base of the one step, operator independent method which was set up by Christophe A.E., the pancreas was infused with cold University of Wisconsin(UW) solution for the preservation, ...On the base of the one step, operator independent method which was set up by Christophe A.E., the pancreas was infused with cold University of Wisconsin(UW) solution for the preservation, digested by the collagenase P, circuited with HBSS+5%fetal calf serum(FCS)+10mmol/L Hepes solution, and separated with the stainless steel mesh. The number of the collected islets were 400000～1800000 per pancreas, i.e. about 12150/g pancreas. After purification, the recovery was 350000～1700000 per pancreas, i.e. about 10250/g pancreas, the recovery rate was above 80%, and the purity of the final preparation was above 95%. The insulin secretion in the response to the high concentration glucose (22 mmol/L) stimulation was apparently different on the 1,3,5 day of the cultural islets, which the high level of insulin was three times the low level (5.5 mmol/L) on the 5th day, and the insulin level of the double stimulation under perfusion conditions is apparently higher than low glucose. The result demonstrated that the purified islets were functionally alive. Histological studies also show that the shape of islets are complete, and the β cell was specially stained by the dithizone (DTZ). The Trypan Blue staining had shown the living cell was above 90%. In conclusion, the new method was highly practical and yielded higher concentration of active pancreatic islets.展开更多
文摘On the base of the one step, operator independent method which was set up by Christophe A.E., the pancreas was infused with cold University of Wisconsin(UW) solution for the preservation, digested by the collagenase P, circuited with HBSS+5%fetal calf serum(FCS)+10mmol/L Hepes solution, and separated with the stainless steel mesh. The number of the collected islets were 400000～1800000 per pancreas, i.e. about 12150/g pancreas. After purification, the recovery was 350000～1700000 per pancreas, i.e. about 10250/g pancreas, the recovery rate was above 80%, and the purity of the final preparation was above 95%. The insulin secretion in the response to the high concentration glucose (22 mmol/L) stimulation was apparently different on the 1,3,5 day of the cultural islets, which the high level of insulin was three times the low level (5.5 mmol/L) on the 5th day, and the insulin level of the double stimulation under perfusion conditions is apparently higher than low glucose. The result demonstrated that the purified islets were functionally alive. Histological studies also show that the shape of islets are complete, and the β cell was specially stained by the dithizone (DTZ). The Trypan Blue staining had shown the living cell was above 90%. In conclusion, the new method was highly practical and yielded higher concentration of active pancreatic islets.
