| Islet transplantation has been considered as an efficient way in the treatment of Type I diabetes. However, the procedure of the isolation of islets may affect the survival and function of islets in vitro. Additional, the immune rejection after transplantation, would also affect the results of transplantation. Therefor, how to obtain islets in good condition and decrease its immunogenicity become focus in islet transplantation field.In this study, we isolated and culture the islets and sertoli cells(SC) in vitro. And a series of detection for islet and SC were performed. After that, the SC and islets were co-cultured in Rotary wall vessel (RWV) to form sertoli-islet culture aggregates(SICA). Correlated detection for morphous and function of SICA were also detected. More importantly, we transplanted SICA into diabetic rat to detect its function in vivo of effecting the blood glucose levels in diabetic rat. The resculs suggested that the SICA formed in RWV were in good conditions. When transplanted into the diabetic rats, they improved the survival rate of diabetic rat and survival time of these animals. This study has an important scientific research value for islet transplantation. The detailed contents are as follows:1. Isolation and culture of the rat isletDextran density gradient centrifuge approach for islet isolation was usually employed in islet isolation. During the process of isolation, the pancreas need to make a second digestion of pancreas was needed. But in this studies, we improved the method by digestion The results demonstrated that the islets obtained by this method had improved survival. The results of glucose stimulation also demonstrated that these islets had better insulin secretion ability. In addition,we also studied the effects of BSA on the islets in the process of isolation. The result demonstrated that we could obtain islets with better insulin secretion ability if added BSA into the isolation medium.In order to study the effects of rotary wall vessel(RWV) environment on the survival and function of islet, we cultured islets in RWV. Based on the higher survival of islets isolated with an improved method ,the islets culture in RWV had better insulin secretion ability and survival compared with prtri-dish cultured islets.2. Isolation of sertoli cells and evaluation of its functionsTo obtain sertoli cells, we employed the method of collagenase V digestion. Immunohistology detection show that abou 95% of cells showed were FasL positive.To study its functions, we cocultured lympholeukocyte with the conditioned medium of sertoli cells. The flow cytometry results demonstrated that sertoli could induced lympholeukocyte apoptosis in vitro. We also cocultured islet with the conditioned medium prepared from sertoli cells. The results demonstrated that islet had a better insulin secretion ability after coculture.3. Coculture islet and sertoli cell in RWV to form SICAIn order to form SICA, we cocultured islets and sertoli cells in RWV. The results from transmission electron microscopy and immunohistol chemical staining demonstrated that islets and sertoli cells could form a complex spheroid, in which sertoli cells and islets were random distribution. There were lots of insulin secretory granules in SICA, which demonstrated that theβcells of islet had good function. The glucose stimulation results demonstrated that SICA had a good insulin secretion ability.4. SICA transplantation in vivo.We transplanted SICA into diabetic rat. The blood glucose level demonstrated that SICA could prolonged the survival time of diabetic rats. Most of the diabetic rats receving SICA could had normal blood level beyond 60 days, which was much longer than rats receving fresh islets and islets cultured in RWV. The results of immunohistol chemical detection demonstrated that SICA was survival and could secreted insulin. The results of blood glucose tolerance demonstrated that the blood level could be euglycemia in 2 hours .The situation were similar with the normal rat, which demonstrated that SICA were sensitive to glucose and had better ability in blood glucose level regulation.There were some reports demonstrated that FasL and TGF-β1 were two important cytokines playing an important role in the progess of immunoprotection of SC. In this research, we made a detection of the expression cell of FasL and TGF-β1 in the grafts. The results demonstrated that the expression of FasL cells would decrease gradually, however, TGF-β1 cells could still remain high expression. When added Anti-FasL and anti-TGF-β1, the decrease of FasL could not affect the blood glucose level.whereas the loss of TGF-β1 would affect the function of SICA, which would cause the the diabetes recurrence. This rescults indicated that the function of SICA had a relationship with TGF-β1.In conclusion, we made SICA with good biologic activity in vitro, After transplanted in vivo, we found that SICA would prolong the survival time of diabetic rat. The results provided a cell biology foundation for islet transplantation in the treatment of diabetes... |
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