Effects Of Concentration And Time On Rat Islet Cell Isolation With Collagenase

BackgroundIslet cell allograft is an effective method for the treatment of diabetes mellitus. After the islet cell transplantation was carried out, the type 1 diabetes mellitus might be cured without insulin administration. In order to achieve the successful transplantation, the isolation, purification and restoration of pancreas musted be prepared carefully. But effectual islet numbers were limited, which severely limited its treatment effects. Therefore, how to improve the effect of islet isolation and purification and how to get more high quality islets had become the key of islet transplantation. This paper would discuss the effects of concentration and time on rat islet cell isolation with collagenase, and identify the optimized techniques for islet isolation.MethodsThe first step was time experiment. 16 experimental rats were divided into four groups in random, 4 rats for each group. To digest the rats pancreas with collagenase V under the different concentration, each group concentration was 0.5g/L, 1.0g/L, 1.5g/L and 2.0g/L, and observed the condition of digest with collagenase every 10 minutes. Result was analyzed and the best time point of high yield of islet was determined. The second step was concentration experiment. The same group as the first step was performed. According to specified time was determined by the time experiment. And a discontinuous Ficoll 400 solution was applied to the purification of islets, and purity of islets were determined by DTZ and AO-PI staining. To performed the islet transplantation under renal capsulesfor Kunming mouse. 18 experimental animals were divided into three groups in random, 6 mouse for each group. Group A and B were injected STZ to construct model of diabetes mellitus, and group B were performed islet transplantation. Group C was controlled group.ResultsThe best period of high yeild of islet according to a definite concentration of collagenase: group T1 was 50 minutes, group T2 was 40 minutes, group T3 and T4 were 30 minutes. The result of islet cell isolation and purification was obviously different under each concentration of collagenase. The least yield of islet was group C1(P<0.01). The lowest survival rate was group C4(P<0.01). The most quantity of yeild of islet is 552.75 ± 69.25 at group C3.7 mouse (58.3%) constructed the model of diabetes mellitus were successful. 4 mouse of group B were performed islet transplantation, and 3 mouse' s blood sugar recovered normal on the second day after transplantation. One mouse' s blood sugar maintained normal for 21 day under no immune suppression.ConclusionWhen pancreatic ductal Hanks injection is fine, the concentration of collagenase V is controlled at 1.5g/L, and water-bath with vibration in 30 minutes could gain the best yeild of islet. To construct model of diabetes mellitus of mouse for inject STZ was affirmation, and islet transplantation under renal capsule could recover the blood sugar of diabetes mouse visibly.