Research On The Key Techniques And Transplantation In Animals Of Islet Transplantation

Islet transplantation refers to the transplantation of active and functional islet cell clusters(autologous,allogeneic)into diabetic patients to enable patients to resume real-time blood glucose response and regulation,and it is currently the only minimally invasive treatment for diabetes.But there are still some problems in islet transplantation,such as the limitation of donors,life-long immunosuppression,instant blood-mediated inflammatory reaction(IBMIR)after intravascular islet transplantation,hypoxia-induced islet cells apoptosis after transplantation,resulting in it difficult to achieve large-scale use for islet transplantation,and even single donor to recipient islet transplantation is difficult to achieve the insulin independant.Microencapsulated and non-microeneapsulated islet transplantation are two important directions in the field of islet transplantation.Microencapsulated islet transplantation i5 microencapsulate the isolated islets into microcapsules,then transplanted them into animals.The microeapsules commonly used are alginate-polylysine-alginate mieroeapsules(APA microcapsules),which can block the recipient's immune system against the transplanted islets for the barrier of the capsule menbrane,and allow nutrients,the insulin,glucagon,C peptide secreted by pancreatic islet pass through the membrane.It is expected to achieve xenotransplantation without long-term usage of immunosuppressants,so as to solve the problems of donor insufficient and life-long immunosuppression.In this paper,APA-Ca and APA-Ba microcapsules were prepared by using a sharp-hole coagulation bath.The thickness,strength,surface structure,stability of the capsule membrane,permeability and intraperitoneal transplantation were studied.The results showed that APA-Ba microeapsules had higher membrane strength,stable blood glucose control after transplantated in mice.In order to improve the supply of nutrition and oxygen to the microcapsules,we also prepared multi-layer microcapsules,modified artificial blood vessels,and put the multi-layer microcapsules into the artificial vascular graft devices,then performed end-to-end anastomosis to the unilateral carotid artery of rabbits by Cuff to make the microcapsules contact with blood fully.But after five days of transplantation,sever thrombosis were appeared in the artificial blood vessels,which suggested that transplanted microcapsules into blood vessels maybe too difficult.It has a high requirements of material technology and graft site for transplantation with microencapsulation islets,and it also hindered the connection and communication of blood vessels form islets to the host,which limited the clinical application of this technique greatly.Non-microencapsulated islet transplantation has been available in clinical research and applications for many years.The islet transplantation in mice mainly included the isolation of mice islets and the transplantion of purified islets under the renal capsule.Clinical islet transplantation currently mainly included the isolation of-human islets and the infusion of islets into portal vein through percutaneous and transhepatic puncture.The transplanted islet colonization in the blood vessel branches of the liver.The insulin and glucagon were secreted according to the changes of blood glucose,to balance the blood glucose with real-time.In this study,we used a modefied tool to infuse 400 IEQ mouse islets under the kidney capsule of diabetic Balb/c mice,then the mice achieved the normal blood glucose.The model is stable and maintains nonnal blood glucose for more than two months,which set up a foundation for the subsequent research on islet transplantation.To investigate the position and the circumstance of islets transplantated in the blood vessels of the liver,we made decellularized liver bioscaffolds of C57BL/6 mice and SD rats,and infused DTZ-stained islets through the portal vein into the bioscaffolds.As a result,the majority of red islets were embolized in the edges of the peripheral microvascular system of the mouse and rat liver bioscaffolds.The mouse liver was small in size,after inftusion of 300±50 IEQ of mouse islets resulted in almost 30%of hepatic vascular be embolized;The size of rat liver was relatively bigger,the infusion of 800±250 IEQ of rat islets led to about 5%of the liver be embolized.However,when the 50%purity of human islets were infused,a large amount of exocrine tissue co-exist around the islets in the hepatic vascular branches,which might accelerate the depletion of nutrition and oxygen supply to the islets,leading them to ischemia,hypoxia and even death.Therefore,transplantation of high-purity islets would be more preferable to improve the effect of islet transplantation.Promotion of microvascular remodeling of the grafted islets is an important direction for islet transplantation research.Islets transplanted will experience ischemia and hypoxia,resulting in islet cells apoptosis.Studies have shown that basic fibroblast growth factor(bFGF)can stimulate vascular growth and chemotaxis of various types of endometrial cells.It is hopeful to be an important cell factor to promote the islets revascularization after transplantation.However,the stability of bFGF is poor,for the half-life time is only 3?10 min in vivo.In this study,islets were cultured in CMRL 1066 culture medium with different concentrations of bFGF in vitro.The repairation of vascular basement membrane in islets was observed by transmission electron microscopy(TEM),and the islet activity was evaluated by PDA-PI fluorescence staining.In vivo,sodium hyaluronate gel was prepared by the culture medium with different concentrations of bFGF.The islets mixed with the sodium hyaluronate gel was transplanted under the kidney capsule of diabetic mice to investigate the effect of bFGF on islet microvascular remodeling after islet transplantation.The results in TEM showed that a large amount of endocrine granules were released when co-cultured with 1 ?g/mL bFGF.And the groups of islets co-cultured with 20 ng/mL,60 ng/mL and 100 ng/mL bFGF did not affect the islet inner structure and could promote the repairation of the islet endothelium effectively.From the results of FDA-PI staining,the activity of pancreatic islets co-cultured with those three bFGF concentrations was also more than 90%.In vivo,it was showed that compared to single 400 IEQ islet transplantation,200 IEQ islet allografts in sodium hyaluronate gel with 60 ng/mL and 100 ng/mL bFGF also had well blood glucose control,which were all kept in a normal blood glucose level.It was significantly different from that of 200 IEQ islet transplantation in sodium hyaluronate gel with 0 ng/mL bFGF.The results of immunohistochemistry and Western Blot showed that bFGF could promote the microvascular reconstruction of islets grafted and improve the survival of islet cells after co-transplanted in sodium hyaluronate gel with bFGF.Compared with pancreas transplantation,islet transplantation is simple in operation,high safety,and has better prospects for the treatment of diabetes.In this paper,we started with the research on the microencapsulated transplantation of mouse islets,then we compared the differences between microencapsulated and non-microencapsulated islet transplantation,investigated the distribution of islets in the decellularized liver bioscaffolds after islet infusion through the portoral vein,and the microvessels reconstruction of islets after transplantation.We established a new method that could promote microvascular reconstruction after islet transplantation and improve the efficiency of islet transplantation.