Research On The Injury And Repair Of The Islets Induced By Streptozotocin In Rats

Objectives1. To compare the distinct injury to pancreas induced by different doses of STZ in ratsand select the optimum model for the study of β cell injury and further repair.2. To investigate injury to β cells of STZ and compensation of pancreas.3. To probe whether stem/progenitor cells exist in pancreas by tissue culture in virto.Materials and MethodsPart1: Rats were divided into five groups:40mg/kg,50mg/kg,60mg/kg,25mg/kg forthree consecutive times every other day (25mg/kg×3) and control according to the dosesof STZ. After first injection, we observed dynamic change of blood glucose in the first72hours (0,1,3,7,11,24,48,72nd hour). Then, monitor the blood glucose every threedays and general conditions of rats. On the9th week, we took the fasting blood forbiochemistry detection and pancreas, kidney, liver, eye for pathological detection.Part2: Induce diabetes in rats with STZ in dose of25mg/kg×3and detect blood glucoseonce every three days after successful induction. On the63rd day, extract blood fromabdominal aorta for detection of fasting blood glucose, insulin, C-Peptide and takepancreas for HE staining, IHC staining of insulin and IF double-staining of insulin andki67. Observe the injury of β cells and ratio of double-staining of insulin and ki67.Part3: Cut the pancreas from the neonatal rat of four days into1mm×1mm size andculture them in the medium of LDMEM/HDMEM, respectively. When cells grew to10-20layers, we passaged them to P1, and when P1grew to80%area of the cell culturedish, to P2. Record cell morphology during the culture of every passage withmicroscope. Meanwhile, take the cells of P1for the detection of cell stem/progenitorproperty with marker of stem/progenitor cells-Oct4, Sox2for IF staining, and pancreaticendocrine related mRNA-Ngn3, Pdx1, InsulinI, InsulinII.Results Part1: STZ in40mg/kg,50mg/kg,60mg/kg and25mg/kg×3can induce the model ofdiabetes. And25mg/kg×3was optimum with stable high blood glucose and lowmortality. Dynamic blood glucose in the first72h among single high dose of STZ(50mg/kg,60mg/kg) fluctuated from first slightly increased to decreased and finalstably increased to a high point. The ratio of successful model of diabetes is70%,80%,100%,100%. We observed cataract on the10th week in40mg/kg while on the8th weekin other experimental groups. Biochemistry detection of insulin, C-Peptide, urea, UA,Cr, ALT, AST, TP, ALB on the9th weeks implied different change among experimentalgroups except40mg/kg. So were the pathology of pancreas, kidney and liver.Part2:25mg/kg×3induced serious damage to β cells. Compared to control, the level ofinsulin and C-Peptide decreased significantly and blood glucose increased dramatically.To compensate, replication rate of insulin-positive cells (IPCs) had increased and,surprisingly, in exocrine epithelial duct, we found IPCs.Part3: LDMEM was more proper for culture of pancreatic cells, and according to theresults of cell identification of P1, we found existence of Sox2-positive cells and mRNAexpression of Ngn3as stem/progenitor marker of pancreatic endocrine orientation.ConclusionWith investigation of STZ induced diabetic mode, we proved that hyperglycemia had asystemic influence to organs. To reduce this kind of effect, the pancreas itself presentscompensation in two ways, which give us a hint that in the process of diabetes, there arestill some potential cells to work. This also help to the theoretical evidence to recoverfrom diabetes by the increase of IPCs.