Isolation And Purification Of Mouse Islet And Different Transplantation Site-Related Research

ObjectiveThrough improve the key steps of islet isolation, including perfusion, digestion and gradient centrifugation, to establish a stable program for islet isolation. Compare the effect of different implant site, to establish a stable model of islet transplantation, laying the foundation for islet transplantation related research.MethodsMale Balb/c and C57BL/6 mice are used for islet isolation. Digest the pancreas by 0.5mg/ml, 1.Omg/ml,1.5mg/ml or 2.0mg/ml collagenaseXI, compare the effect of different concentration. Isolation and purification the islets through different gradient centrifugation strategy, stain the islets with DTZ, and observe the result under microscope, to determine the optimal protocol for islet isolation. AO/PI stain was used to detect the activity of isolated islets. DM models are induced by intraperitoneal injection of STZ into male Balb/c, C57BL/6, or C3H mice. DM is defined as blood glucose>18mmol/L for 2 successive days, while that blood glucose is 18-30mmol/L on the transplantation day is used as recipient. Islets are implanted intrahepatic, renal subcapsular or intra-intestinal-loop, through blood glucose monitoring, post-operation IPGTT and graft pathology examination to evaluate islet graft function, determine the optimal implant site at last. Use SPSS software for statistics analysis. Compare the difference among groups use one-factor analysis of variance, P<0.05 is defined as statistics significance.Results76.4±19.5% of the pancreas volume expanded with antegrade perfusion, obviously surpass retroperfusion(51.1±17.7%, P＜0.05). The best condition for pancreas digestion is infusion 2ml/mouse of 0.5mg/ml collagenaseXI into the pancreas, placed into 6ml of 0.5mg/ml collagenaseXI,37℃waterbath for 15min. Balb/c mice use Ficoll-1.108(4ml), 1.096 (2ml),1.069 (2ml), HBSS (2ml), acceleration 1-deceleration 1,400rpm for 4min transfer to 1800rpm for another 8min, yield is 125±39IEQ, purity is 79.6±10.5%, then undergo another gradient centrifugation use the same strategy, purity significantly increased to 96.2±1.4%. While for C57BL/6 mice,1800rpm gradient centrifugation for 15min is a better choice. AO/PI staining confirms the good activity of isolated islets. The best dosage to induce DM of Balb/c, C57BL/6 and C3H mice is 165mg/Kg, 170mg/Kg and 200mg/Kg respectively. Blood glucose lever become stable 7 days after injection. Subcapsular 300-450IEQ islets are sufficient to retrieve mice from diabetes. On post operation day(POD) 30 the islet bearing kidney is removed, HE and IHC staining showed islets well survived. After retrieved, mice become diabetic again. While intrahepatic infusion of 500IEQ islets can't correct diabetes, recipient easily died from hepatic embolism. And intra-intestinal-loop islet transplantation need as more as 800IEQ islets to cure diabetes.ConclusionThe improved islet isolation method is a better choice. Probe guided through PE tube implant of islets under renal capsule is a stable model for islet transplantation, can be widely used for further research.