Study On Transplantion Of Fetal Rat Pancreatic Stem Cell To Treat Type 1 Diabetes Mellitus

Clinical pancreatic islet transplantation has developed rapidly these several years. This development makes the cure of diabetes mellitus hopeful, but the low availability of donor limits the number of islet transplants that can be performed. stem cells have great potentialities of the continuous proliferation and differentiation,and thus,it is thought to be a valuable source for generation of islet cells. Our reserch sought to observe the differentiation of stem cell in vivo by cell transplantation, and attempt to offer compelling opportunities for the development of new, innovative approaches for treating Diabetes.Part 1 Effects of transplanted site on fetal rat pancreatic stem cells transdifferented into islet-like cell cluster in vivoOBJECTIVE: To observe the possibility of fetal rat pancreatic stem cell transdifferentiating into islet-like cell cluster by transplanting fetal rat pancreatic stem cells into type 1 diabetic SD rat.METHODS: The pancreatic stem cells (PSCs) were harvested from pancreatic rudiments of SD rat embryos on embryonic day 16 and examined SRY DNA by Fluorescence in situ hybridization (FISH). The pancreatic stem cells were identified by nestin and PDX-1 immunostaining and flow cytometry. Adult female SD rats were divided into five groups including 10 pancreatic parenchymal orthotopic transplantation, 10 intrahepatic transplantation,10 renal subcapsular place transplantation, 10 experimental control and 10 normal control.1×106 fetal pancreatic stem cells per rat were injected into diabetic rat's pancreatic parenchyma, portal vein and renal subcapsular space respectively. Glucose and insulin level were monitored in serum periodic. Related tissues were excised for histological and morphometric analysis to assess the transdifferentiation and SRY DNA were examined by FISH.RESULTS:After passaged 3 generations, the PSCs expressed nestin and PDX-1 according to immunostaining while identified by flow cytometry 74.1% of PSCs expressed nestin. The PSCs'orthotopic transplantation led to stable reduction in hyperglycemia and increasing insulin level in serum (3 weeks after transplantation) and culminated (5 weeks post- transplantation) in restoration of normoglycemia which remained steady during the course of experiment without further relapse. Neither intrahepatic transplantion nor renal subcapsular transplantation showed decrease of blood glucose or increase of insulin content in serum. No exogenous islet-like cell clusters immunostained by anti-insulin mAb and anti-nestin mAb were found except the recipient's pancreata 8 weeks post-transplantation. The exogenous islet-like clusters expressed SRY DNA according to FISH.CONCLUSION:After passaging 3 generation we can get pure pancreatic stem cells.When orthotopic transplant into pancreatic parenchyma PSCs can differenciate into islet-like cell cluster and reverse experimental diabetes.Part 2 Study on orthotopic transplantion 0f fetal rat pancreatic stem cell to treat type 1 diabetes mellitusOBJECTIVE: To observe the possibility of fetal rat pancreatic stem cell differentiating into islet-like cell cluster by transplanting fetal rat pancreatic stem cells into diabetic SD rat pancreatic parenchyma.METHODS: The pancreatic stem cells (PSCs) were harvested from pancreatic rudiments of SD rat embryos on embryonic day 16 and examined SRY DNA to discriminate gender by Fluorescence in situ hybridization (FISH). The pancreatic stem cells were identified by nestin and PDX-1 immunostaining and flow cytometry.Adult SD rats were divided into five groups including 1×105 PSCs per rat pancreatic parenchymal orthotopic transplantation(group A, 10 rats), 1×106 PSCs per rat pancreatic parenchymal orthotopic transplantation(group B, 10 rats), 1×107 PSCs per rat pancreatic parenchymal orthotopic transplantation(group C, 10 rats),10 experimental control(group D, 10 rats) and 10 normal control(group E, 10 rats). In orthotopic transplantatation group male fetal pancreatic stem cells were injected into diabetic rat pancreatic parenchyma while in experimental control group equivalent volume PBS solution was injected into diabetic rat pancreatic parenchyma. Glucose and insulin level in serum were monitored periodic.8 weeks after transplantation pancreata were excised for histological and morphometric analysis. SRY DNA was detected by FISH. Nestin, PDX-1 and insulin mRNA expression in pancreata were detected by RT-PCR, insulin and PDX-1 protein contents were assess by western blot.RESULTS:After passaged 3 generations,the PSCs expressed nestin and PDX-1 according to immunostaining while identified by flow cytometry 74.1% of PSCs expressed nestin. When orthotopic transplanted,both 1×106 and 1×107 PSCs could lead to stable reduction in hyperglycemia and increasing insulin level in serum (3wees after transplantation),culminating (5weeks post-transplantation) in restoration of normoglycemia which remained steady during the course of experiment without further relapse while 1×105 PSCs could also lead to reduction in hyperglycemia and increasing insulin level in serum without culminating in restoration of normoglycemia. Exogenous islet-like cell clusters were found and expressed SRY DNA in the orthotopic transplanted recipient's pancreata 8 weeks post-transplantation. The expression level of insulin mRNA and protein in the recipient pancreata of group B were higher than experimental control(P<0.05),and the expression of nestin and PDX-1 mRNA and protein were also higher than normal control(P<0.05).CONCLUSION:When orthotopic transplant into pancreatic parenchyma PSCs from fetal rat can differenciate into islet-like cell cluster, gain comparable function with normal islets and reverse experimental diabetes.