Method For Isolation And Extraction Of Islets From Rat Pancreas And Detection Of Its Activity

The rat pancreas is a glandular organ that resembles a fat texture,has a reticular interior,but is lighter in color and located deep in the abdominal cavity.In the abdominal cavity,the pancreas is located under the liver,connected to the stomach,duodenum and spleen.It has a rich network of blood vessels.The pancreas has an envelope formed by interstitial cells.The capsule divides the pancreas into pancreatic lobules.The pancreatic lobules are mainly composed of acinus,ducts and islets.The acinar cavity in which the cells are present is surrounded by a single layer of acinar cells,forming a cavity of different sizes.At the same time,the cavity is also present between the islet cells and adjacent acinar cells,and the islet intercellular tubules exist.Between islet cells.The islet cells encapsulated by the envelope mainly include four kinds of cells,where in the islet β cells mainly secrete insulin.In addition,fibroblasts and abundant capillaries are also present in pancreatic tissue.The pancreas can be divided into two parts,the exocrine gland and the endocrine gland.The exocrine gland mainly produces pancreatic juice,which contains various digestive enzymes.The secreted pancreatic juice flows from the pancreatic duct into the duodenum;the endocrine gland is mainly composed of several cells.Islet,which secretes two hormones that dynamically regulate blood sugar levels.Studies have confirmed that diabetes is mainly caused by islet endocrine dysfunction,and digestive diseases are caused by dysfunction of pancreatic exocrine function.Therefore,it is particularly important to obtain a large number of biologically active islets through mature and effective islet isolation and extraction methods,and to achieve the purpose of treating diabetic patients through islet transplantation.The use of bile duct perfusion to separate and extract islets in animal models has the advantages of high efficiency and high activity,but its operation is more complicated and complicated than the traditional islet extraction method.In recent years,many research groups have made some improvements in the separation and extraction operations,and attempted to cryopreservation of isolated islets,which laid the experimental foundation for the subsequent study on islet transplantation in diabetic patients.In this paper,we draw on the existing method of isolating islets in bile duct perfusion,on the basis of which the ligation is improved,and the ligation of the duodenum is changed to the duodenum on both ends of the bile duct and pancreatic duct opening.The maximal retention of the pancreatic duct to transport collagenase,isolation and extraction of islet cells from rat pancreatic tissue,and identification of the integrity of the isolated islet cells by islet specific staining.In this experiment,Wistar male rats were used as experimental subjects,0.2% V-type collagenase was selected,and the enzyme concentration was higher than that of the existing reports.After the traditional shearing method and the improved bile duct perfusion method were used to separate and extract islets,The FDA/PI staining method was used to detect and compare the cell viability extracted by the two methods.It was found by experiments that the activity of islet cells extracted by bile duct perfusion was significantly higher than that of islet cells extracted by shearing.In addition,the isolated islet cells were stimulated with low glucose and high glucose respectively,and the secretion of insulin was detected by ELISA.The results showed that compared with the shearing method,the islet cells obtained by the modified bile duct perfusion can produce normal glucose stimulation.Insulin is secreted by stress reaction,indicating that islet cells after separation and extraction still maintain normal islet secretion function.Again,we observed the islet cells isolated and extracted by two different methods under the microscope.It was found that the islets extracted by the modified bile duct perfusion method had less phenomenon of envelope fragmentation.Finally,we used our RT-q PCR method to assess islet activity and function by detecting m RNA levels of islet secreted protein hormones.Taken together,the results of this study demonstrate that the islet cell activity and secretion function extracted by our modified bile duct perfusion method is more effective than the shear method.This study will lay the experimental foundation and theoretical basis for the follow-up clinical study of diabetes treatment.