| Objective : With the introduction and continuous improvement of the Edmonton transplantation program in the 20 th century,islet transplantation for the treatment of type1 diabetes has gradually matured and is expected to be widely used in clinical practice.Pancreatic islet transplantation is to increase the number of islet cells in the recipient by transplanting allogeneic or xenogeneic islet cells into the recipient,thereby increasing the release of insulin.It has the advantages of simple operation,high safety,few complications,and repeatability.In clinical practice,90% of the treatment plans are to infuse islets through the recipient portal system,which has obvious deficiencies: the direct exposure of islets to the blood environment of the portal vein will induce immediate blood-mediated inflammatory response,transplantation lead to portal hypertension,embolism,etc.Therefore,exploring new islet transplantation sites to circumvent the above-mentioned defects has become one of the popular directions of islet transplantation research.Different transplantation sites can lower blood sugar,but in order to make the hypoglycemic effect more direct and rapid,the pancreas is undoubtedly an ideal site for transplantation.Difficult to develop.On the basis of the previous research of the research group,in this experiment,the decellularized amniotic membrane was used to construct a thylakoid bag structure for islet transplantation on the surface of the pancreas to achieve islet transplantation on the surface of the pancreas.The microenvironment provided by the pancreas was used to promote the survival of the transplanted islets and further improve the transplanted islets.Features.At the same time,there is an anatomical structure,molecular biology,function and clinical relationship between the exocrine and exocrine tissues of the pancreas.The effect of internal pancreatic islet transplantation on pancreatic exocrine acinar cells on the secretory function of pancreatic enzymes has not been reported yet.Based on the above,using decellularized human amniotic membrane to construct a thylakoid bag on the surface of the pancreas for islet transplantation,to verify the feasibility and therapeutic effect of islet transplantation in the thylakoid bag of decellularized human amniotic membrane on the surface of the pancreas to treat diabetes in mice,and to provide a new transplantation for islet transplantation.site;and to explore the effect of pancreatic islets transplanted into the decellularized human amniotic membrane pouch on the pancreatic exocrine acinar cell pancreatic enzyme secretion function and its molecular mechanism.Methods:1.Obtain postpartum human amniotic membranes from healthy full-term women who meet the requirements and prepare decellularized human amniotic membranes.2.Use a single intraperitoneal injection of high-dose streptozotocin to construct an islet transplant recipient model.3.A thylakoid pouch structure was constructed on the surface of mouse pancreas with decellularized human amniotic membrane through 9 0 sterile surgical sutures.4.The islets were extracted by Ficoll density gradient method,and the activity of the extracted islets was detected by AO-EB staining kit.5.Measure the changes of non-fasting blood glucose values in mice after islet transplantation and the intraperitoneal glucose tolerance at different time periods after transplantation,and evaluate the therapeutic effect of islet transplantation.6.The blood of the eyeball was used to obtain the mouse serum,and then the insulin secretion level of the mouse serum was detected by the insulin ELISA detection kit.7.The morphological characteristics of islet grafts were observed by HE staining,immunohistochemical staining and immunofluorescence staining of histopathological sections.8.The blood of the eyeball was used to obtain the mouse serum,and then the amylase level of the mouse serum was detected by the amylase detection kit.9.The mouse primary acinar cells were extracted by collagenase I digestion,and the activity of the extracted primary acinar cells was identified by trypan blue staining.10.To detect the amylase secretion function of mouse primary acinar cells stimulated by different concentrations of cholecystokinin(CCK-8).11.Western blot was used to detect the expression of amylase secretion-related protein Rab27 B in mouse primary acinar cells under diabetes and in different transplantation models.12.Through the co-culture of islet cells and acinar cell line AR42 J in vitro,the secretion function of acinar cell amylase under different culture conditions was detected.13.Through the co-culture of islet cells and acinar cell line AR42 J in vitro,the expression of Rab27 B protein after co-culture was detected by Western blot.14.The Rab27 B protein of AR42 J cells was knocked down by si RNA,and the expression of Rab27 B protein and the secretion of amylase in AR42 J cells were detected by Western blot.15.AR42 J cells were co-cultured with islet cells after knockdown of Rab27 B protein by si RNA.Western blot was used to detect the expression of Rab27 B protein and the changes of its amylase secretion.Results:1.The decellularized human amniotic membrane that meets the requirements was successfully obtained and stored in 100% glycerol in a cryogenic refrigerator,which can be used for the subsequent construction of thylakoid bags on the surface of the pancreas.2.The C57BL/6 mouse diabetes model was successfully constructed by streptozotocin,which is stable and has a high success rate.3.A thylakoid bag structure on the pancreatic surface was successfully formed by temporarily implanting a hollow plastic tube between the surface of the pancreas and the decellularized human amniotic membrane.The constructed thylakoid bag structure has enough accommodation space and can be used for the subsequent modeling of pancreatic islet transplantation on the surface of the pancreas.4.The islet cells extracted by gradient centrifugation have sufficient yield,high purity and strong activity,which meet the requirements of islet transplantation;and successfully constructed the pancreatic islet transplantation model on the surface of the C57BL/6 mouse and the islet transplantation model under the renal capsule.5.After transplanting 300 islets of homologous islets on the surface of the mouse pancreas,the blood sugar of the mice returned to normal blood sugar levels within an average of 13.8±3.9 days,and the blood sugar level was maintained for more than 100days;the mice showed good performance after transplantation The area under the peritoneal glucose tolerance curve was 1393±105.0mmol/L/120 min.6.The serum insulin content of mice with orthotopic pancreatic islet transplantation was significantly increased(P<0.0001).7.The histopathological results of the grafts showed that the islets could colonize and survive well in the thylakoid bag on the surface of the pancreas after transplantation,and there was no obvious inflammatory cell infiltration.8.Compared with the renal capsule transplantation group,the level of serum amylase in the pancreatic orthotopic islet transplantation group was higher(P<0.05).9.The primary acinar cells extracted by collagenase digestion method have complete structure,high activity and good amylase secretion function.10.Pancreatic orthotopic islet transplantation has a certain repairing effect on the impaired amylase secretion function of pancreatic acinar cells in STZ-induced diabetic mice.11.Western blot results showed that orthotopic pancreatic islet grafts could promote the expression of Rab27 B,a protein related to amylase secretion in pancreatic orthotopic acinar cells(P<0.001).12.In the co-culture system of islet cells and acinar cells in vitro,the co-cultured islet cells can repair the amylase secretion function of acinar cells damaged by high glucose culture,and increase the expression of Rab27 B,a protein related to amylase secretion.Conclusion:A mouse model of orthotopic pancreatic islet transplantation was successfully constructed using decellularized human amniotic membrane,which can effectively reverse the hyperglycemia state of diabetic mice.The enzyme secretion function has a repairing effect;the mechanism of this repairing effect may be achieved by promoting the expression of the amylase secretion-related protein Rab27 B. |
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