Research On Construction Of Tissue Engineered Pancreas In Vitro And Transplantation In Vivo To Cure Diabetes In Rats

Islet transplantation is an effective β-cell replacement therapy for the patients of type I diabetes who are lack of endogenous insulin production. It is emerging as the most desirable treatment to treat type I insulin-dependent diabetes mellitus (IDDM) to achieve spontaneous regulation of blood glucose level, and has received a great deal of attention both in animal models of diabetes and on recent clinical studies. But the immunosuppressive durgs uitilized in transplantation could cause severe complication. How to improve the survival of grafts without using immunosuppressive drugs is very important in islet transplantation. To accomplish successful islets transplantation without immunosuppression for cure of diabetes mellitus Sertoli cells were isolated from prepubertal Wistar rats of 7-9 day old by one-time digestion with collagenase V. Contaminating germ and red blood cells in the isolated cells were eliminated by treatment of sterile 0.5 mmol/L, pH 7.4 low osmotic buffer and differential adhesion. Purified islets were isolated from adult Wistar rats by the method of ligation-perfusion digestion and dextran discontinuous density gradient centrifugation. Islets were cultured in the rotary wall vessel bioreactor (BWV) to detect the function of the micrograity to the islets. Sertoli cells were cultured with lymphocytes and islets respectively to study its immunosuppressive function and trophic supporting. Purified islets and Sertoli cells were cocultured in the rotary wall vessel bioreactor (BWV) for 5 days to form Sertoli cell-islets aggregates(SICA). SICA was transplanted under the kidney capsule into the streptozotocin-induced diabetic Sprague-Dawley rat recipients after endocrine function detection in vitro. Recipients blood level were detected every other day. The recipients with normalized blood level for more than 20 d were conducted glucose tolerance and their kidneys were removed, and detected by HE staining and immunostaining with antibodies for Fas-L and insulin .Sertoli cells, fresh islets and islets cultured in bioreactor were transplanted into the diabetic rats as control groups. The major results were as follow:1. By the method of collagenase V one-time digestion and low osmotic buffer treatment, we can get 6.5×10~5 purified cell/g testis. The primarily isolated Sertoli cells had good cell viability; cell survival rate was above 85% and there was no big cell corps; after planted in...