Experimental Study Of Islet Transplantation To Treat Diabetes

Objectives The aim of the present study was to evaluate the immunosuppressive effect of tacrolimus (TAC) and other immunosuppressants or combined immunosuppressants in discordant islet xenotransplantation. Therefore, more effective immunosuppressive regime might be selected based on the study. Methods Fetal porcine islet-like cell clusters (ICCs) were isolated by cutting and then digested with collagenase. ICCs were transplanted undeF the kidney capsule in normoglycemic rats treated with 1. TAC monotherapy; 2.TAC plus other immunosuppressive drugs or 3. cyclosporin A (CsA) monotherapy; 4.nutreated control. Twelve or 24 days after transplantation, the number of remaining intact ICCs and the extent of a cellular infiltration in the xenografts was evaluated using immunohistochemistry. In some animals, the grafts were examined for antibody and complement deposition and the levels of xenoreactive antibodies in serum were determined. Results In untreated rats, the xenografts were completely rejected after 12days and no intact ICCs remained. Massive infiltrating cells, mainlymacrophages and T cells were observed in the graft. TAC monotherapy (at 0.5 and 1.0 mg/kg b.w.) almost completely inhibited rejection for up to 12 days with only few macrophages inside the graft. In animals treated with TAC monotherapy (at 0.5 mg/kg b.w.), rejection was markedly inhibited for up to 24 days. However, the effect after 24 days was not consistent and in some grafts there were signs of rejection. The protective effect of TAC observed in this study is in contrast to the findings in rats given CsA monotherapy in which no or only a marginal effect on islet xenograft rejection was observed. Most of ICCs were destroyed with massive macrophages and T cells infiltrated into the area. Only when CsA was given at 20 mg/kg b.w., an inhibitory effect could be observed. Immunosuppression with TAC at a suboptimal dose (0.3 mg/kg b.w.) plus 15-deoxyspergualin or brequinar also had an inhibitory effect on the rejection. TAC plus Sirolimus (SIR) had no effect on the rejection. Almost all the ICCs were destroyed by infiltrated Cells. In animals given TAC plus mycophenolate mofetil, a protective effect was observed as well; however, this effect was not consistent. Conclusion:TAC monotherapy had markedly inhibited the rejection process in the discordant pig-to-rat islet xenotransplantation model. In contrast to it, CsA at a low or moderate dose had no inhibitory effect on the rejection. A protective effect only observed when CsA was at a very high dose. Immunosuppression with TAC at a suboptimal dose plus 15-deoxyspergualin or brequinar had an inhibitory effect on the rejection. TAC plus mycophenolate mofetil, aprotective effect was observed as well. Although TAC and CsA are the calcineurin inhibitors and both of them have their immunosuppressive effect by inhibiting the activation of T cells, these two agents might have the different mechanisms on preventing xenogrraft rejection in this model.Objectives: to have better understanding of mechanism of the transdifferentiation of pancreatic stem cells into islet cells through obtaining , cultivation and transdifferentiation of adult human pancreatic duct cells. Methods: cadaveric adult human pancreas was harvested with the aseptic technic. A portion of the adult human pancreas was digested with collagenase followed by non-continuous density gradient to separate islets from exocrine tissue. Duct epithelial cells which are potential stem cells were cultivated in CMRL1066 and then in serum-free DMEM/F12 medium with the addition of growth factors for up to 27 days. Samples were taken at different time points(l,3,5,7,9,15,21,27d) for light and electron microscopy observation and for immunocytochemistry with antibodies against transdifferentiation gene PDX-1 and CK-19. Insulin and amylase content in the culture medium were evaluated to confirm that the exocrine and endocrine cells were discarded with medium change by day 5. Results A large number of duct epithelial cells were harvested after the digestion with the collagenase and purification with non-contineous density gradient by using modified Ricordi's method. At day 1, few duct cells in the culture falsk adhere to the bottom. Duct epithelial cells were then proliferated and expanded rapidly within 5 days. Few adherent cellsor small clusters became cobblestone like clusters and then the monolayer which covered the most part of the bottom of the culture falsk. At day 7 cobblestone like cell clusters proliferated and expanded even biger. Few islet-like buds appeared in the central or peripheral part of the monolayer. Thereafter more three dimensional Dithizon positive staining islet-like clusters were observed under the light microscope. A great number of islet-like structures appeard from day 21 to day 27. Under the electrnic microscope, from day 5 of culture, short stubby microvilli and apical junctional complexes were seen in most of cells. Few or moderate number of various density granules were presented in the area close to the membrane in some cells. Many endoplasmic reticulums and Golgi's apparatus were observed . Interfilaments appeared in some of the cells. At day 9, more interfilaments were seen in many cells. At ~ day 15, short stubby microvilli in some cells disappeared while some interfilaments were presented around the granules of differrent densities. Short stubby microvilli vanished and interfilaments reduced in many cells while some cells formed microlumen at day 21. In most of cells, interfilaments disappeared and some cells had high and some had lower density granules afterwords. Many granules containing cells were connected together to become a big cluster. Immunohistochemistry: at day 1, cells in the four-chamber slide showed strong positive CK-19 antibody staining. These cells were mainly in the form of single cell or small cluster. Cells were also showed positive PDX-1 antibody staining. But these cells stained at different level even in a single cluster. There were islet-like buds appeared from day 7 on. Most of cell clusters and...