Study Of Islet Cell Transplantation Via Portal Vein In Rats And The Effects Of Immunosuppressive Agents On Islet Cells

Objectives: l.To study of method for isolation and purification as well as the evalua-tion of islet cell quality. 2.To investigate the effects of immunosuppressive drugs on insulin secretion of rat islet cells and human islet cells in vitro. 3.To investigate the immunosuppressive effects of FTY720 in combination with Rapamycin and low-dose FK506 in combination with MMF on intraportal islet cells transplantation in rats. Methods: 1.Rats' islet cells were isolated by the solution of the collagenase injected through the common bile duct and purified by Ficoll's discontinuous density gradient centrifugation. 2.The identification of islet cells and alive cells were studied with dithizone(DTZ) and with acridine orange(AO)-propidium iodide (PI). 3.Rat and human islet cells were exposed to various concentrations of four immunosuppressive agents for 24 hr respectively. Glucose -stimulated isulin secretion during subsequent static incubation was measured using the ultrasensitive rat insulin ELISA kit. 4.Appro -ximately 1000-1200 islets were transplanted into the liver of streptozatocin -induced diabetic rats via the portal vein. Rat recipients of islet allografts were either left untreated or treated with FTY720 in combination with Rapamycin or immuno -suppressed with low-dose FK506 and MMF.The plasma glucose and serum c-peptide levels were monitored and grafts were studied morphologically. The duration of normoglycemia and the graft survival time were contrasted .Results: 1 .The yield of islet cells was 450-720 islets/pancreas. The mean yield was 562 110 islets/pancreas. Islet purity was over 80%,islet viability was more than 95%. The insulin release test indicated the islet cells were sensitive to glucose stimulation.2. Glucose-stimulated insulin secretion from either rat islet cells or human islet cells were significant reduced after exposed to high concentrations of MMF and FK506. No significant reduction in insulin secretion were observed from either rat islet cells or human islet cells after exposed to FTY720 and Rapamycin.3.The duration of normoglycemia in FTY720+Rapamycin group was 23.3 12.1 days while 5.0 3.5 days in control group. The difference was very significant between the two groups.4.The duration of normoglycemia in low-dose FK506 + MMF group was 16.3 ?.6 days, which was significantly longer than that in the control group. Conclusions: l.DTZ and AO-PI combined staining may exactly identify the purity, amount and viability of islet cells. Isolation by the collagenase and purification by Ficoll's discontinuous density gradient centrifugation can produce a high yield of viable purified islet cells in rats. 2 .The high concentrations of MMF and FK506 have deleterious effects on insulin secretion in both rat islet cells and human islet cells.Islet allografts can be protected from rejection and maintain an adequate function without obvious signs of toxicity in rats immunosuppressed with low-dose FK506+ MMF. These findings support the use of low-dose immunosuppressive drug protocols in islet cell transplantation and low-dose FK506+ MMF is available for clinical use. 3. FTY720 and Rapamycin did not have adverse effects on insulin secretion in both rat islet cells and human islet cells. FTY720 in combination with Rapamycin could inhibit islet allograft rejection and obviously prolong the survival of islet allografts. FTY720 and Rapamycin could become the useful immunosupressants for future clinical islet allotransplantation.