| Diabetes is a worldwide health problem characterized with high morbidity and mortality. Islet transplantation can restore the glucose homeostasis to the physiological level. However two major obstacles remain before islet transplantation hits the medical market.: the source of islets and the host immune attack on the islets. The purpose of this study is to investigate the Sertoii cells' immune privilege protection effect on cotransplanted islets and the potential application in reverse of the diabetic state by Sertoli-islet cell aggreg ates (SICA).In the first part of the study, adult rat islets were isolated with type V collagenase and purified by Ficoll gradient centrifugation at densities of 11%,20%,23% and 25%. The islets were then cultured on extracelluar matrix-coated surfaces to investigate the supporting effect of different extracellular matrix, including type I and IV collagens, and Matrigel,on the viability and function of cultured islets. Islets were stained with diphenylthiocarbazone (DTZ) and trypan blue. The results showed that the purity and viability were 80% and 90%, respectively. Cell viability and insulin secretion level were the best in islets cultured in Matrigel-coated group.In the second part of the study, testicular Sertoii cells were prepared by serial digestion of rat testis using type V collagenase (2.5mg/ml), trypsin (25μ g/ml) and Dnase (4 μ g/ml). Then, Sertoii cells were identified by histological, immunohistochemical, and electron microscopic analysis. All the results firmly holded that a high purity sertoli cells were got through our established culture method.In the third part of the study, Sertoii cells and islets were cotransplanted under therenal subcapsular space of diabetic rats. Each diabetic recipient received 2,000 IEQ islets together with different dosage of Sertoli cells which were 1 X 105, 1X 106and 1 X 107. Blood glucose was monitored every other day. In the absence of the immunosuppression, the transplantation of a quantity of Sertoli cells (1 X 107) together with 2,000 purified islets, reversed the diabetic state for 41 ?.5 days. Statistical analysis showed significant difference among the five groups and the best efficiency was the Sertoli cells group at a density of 1 X 107 cells (p<0.05).The immunohistochemical study of the Sertoli cells (1 X 107) group demonstrated positive staining of Fas-L and insulin antibody.In the fourth part of the study, 2,000 IEQ islets and 1X107 Sertoli cells were cocultured in RCCS. Three dimensional Sertoli-islet cell aggregates were obtained. The basal insulin release level and glucose-stimulated insulin secretion level of SICA were assessed by radioimmunoassay. Immunohistochemical and electron microscopic analysis were also performed. SICA or a mixture of islets and Sertoli cells was transplanted under the renal subcapsular space of diabetic rats. In vitro insulin secretion test indicated that the best activity islets appeared in the SICA group. During the in vivo transplantation test, SICA group reversed the diabetic state for 56.0 + 1.7 days, which was of statistical significance in comparison with the Sertoli cells and islets cotransplantation group. The results of scanning electron microscopy of SICA showed intact islets surrounded by Sertoli cells. Fas-L and insulin immunostaining were positive.We concluded that cotransplantation of islets in accordance with certain ratio of Sertoli cells can induce local immune privilege. Secondly, SICA can help improve graft survival by inducing immunosuppression, since the structure of SICA makes it easier for the SC to provide immunosuppression and trophic support for the islets.
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