The Role Of PI3K-AKT Signaling Pathway In Islet Mesenchymal Stem Cell Survival In Islet Survival

Background: Islet transplantation has been proven to normalize blood glucose in patients with type 1 diabetes,and it can effectively delay the occurrence of diabetic complications.It is a possible treatment for curing diabetes and has broad prospects for development.The development of islet transplantation is hindered by the lack of islet donors and the low survival rate of transplanted islets.Studies have shown that co-transplantation of bone marrow mesenchymal stem cells(BMSCs)with islets can greatly improve the survival rate of transplanted islets.The PI3K-AKT signaling pathway,as a signal pathway that is widely present in various cells and has an important influence on cell survival,plays an important role in promoting the survival of transplanted islets by bone marrow mesenchymal stem cells.Research purposes:This study intends to co-culture mouse bone marrow mesenchymal stem cells with mouse islets in vitro,and use the trypan blue staining microscopic counting method to calculate the islet survival rate and verify the promotion of islet survival by bone marrow mesenchymal stem cells.effect.The PI3K-AKT signaling pathway specific inhibitor LY294002 was used to further investigate the role of PI3K-AKT signaling pathway in bone marrow mesenchymal stem cells and islet co-culture.Research methods: 1.The cells extracted from the tibia and femoral medulla of mice were cultured in vitro.After 24 hours,after advancing,the rounded and partially fusiform cells were left to be cultured,and the sacral cavity was observed after 48 hours.Adherent cells grow into elongated spindle-shaped cells.After about 72 hours,most of the bone marrow mesenchymal stem cells can protrude from the pseudopod,gradually appearing as a fusiform shape and aggregated into a regular shape of polygons.The cells are close to each other and merge,and The rate of proliferation is gradually increasing.After repeated digestion and passage,high-purity bone marrow mesenchymal stem cells were obtained.2.Mouse islets were extracted and purified by collagenase perfusion method and Ficoll-400 density gradient centrifugation,and identified by dithizone staining and glucose stimulation experiments.Islet was determined to be functionally intact,and islet survival was identified under a trypan blue staining microscope.3.The extracted bone marrow mesenchymal stem cells were divided into islets: pancreatic islet group(group 1),PI3K-AKT signaling pathway inhibitor LY294002 pretreated islet group(group 2),BMSCs and islet co-culture group(Group 3),PI3K-AKT signaling pathway inhibitor LY294002 pre-treated islet and BMSCs co-culture group(4 groups).The cells were cultured under high glucose conditions,trypan blue staining was performed on the 3rd,5th,9th,and 13 th day islets,the islet survival rate was counted under the microscope,and the data were analyzed to prove that the PI3K-AKT signaling pathway is in the bone marrow.The role of mesenchymal stem cells in islet co-culture.Results: 1.Bone marrow mesenchymal stem cells were extracted from the tibia and femur of mice.The bone marrow mesenchymal stem cells were purified by using liquid exchange and subculture methods.,high purity bone marrow mesenchymal stem cells;2,using collagenase perfusion method and Ficoll-400 density gradient centrifugation method to successfully extract and purify mouse islets,through staining and glucose stimulation experiments,confirmed the extraction of islet structure and functional integrity;Bone marrow mesenchymal stem cells and islets were cultured in groups under high glucose conditions.The islet survival rate of group 3 was significantly higher than that of group 1 on days 3,5,9,and 13,and the difference was statistically significant.Mesenchymal stem cells have a pro-survival effect on islets;on days 3,5,and 9,the survival rates of islets in Groups 2 and 4 were approximately the same,and the difference was not statistically significant.On the 13 th day,there were no survival islets in both groups,reflecting The survival of islet cells after inhibition of PI3K-AKT signaling pathway by bone marrow mesenchymal stem cells disappeared,demonstrating that PI3K-AKT signaling pathway is involved in mesangial survival of bone marrow mesenchymal stem cells Play an important role.Conclusion: Bone marrow mesenchymal stem cells have a pro-survival effect on islets in vitro co-culture.The PI3K-AKT signaling pathway plays an important role in the survival of mesangial mesenchymal stem cells.