| Pancreatic stem cells are adult stem cells in pancreas,have the unlimited ability for self-renewal and to diffierentiate into many type of cell lineages in the appropriate local environment.According to the position in pancreas,pancreatic stem cells were divided into three classes,which exist in pancreatic islets called islet stem cells,present in pancreatic duct called pancreatic ductal stem cells,exist in the gland bubble called pancreatic acinar cells.Pancreatic stem cells can be used not only for the growth and development of animal and human pancreatic tissue,but also can be used as “seed cells” and to differentiate into functional islet transplantation in the treatment of diabetes,which has important significance.But,There is no report about differentiation potential between the two kinds of stem cells especially the comparative researches on differentiation into functional islet.A islet stem cells line and A pancreatic ductal stem cells line had been estanlished in our lab.The morphological characteristics,biological characteristics,cell prolife-ration cycle and karyotype analysis of the two cell line had been studied What we didn't study was the functional characteristics that diffierentiated into many type of cell lineages in the appropriate local environment.In this study,we used the two stem cells to differentiate into nerve cells,osteoblasts,adipocytes and islet cell and compared the differentiation capacity of pancreatic stem cells.All of this provides the basis for clearing the multi differentiation potential of the two stem cell and finding one kind of pancreatic stem which is easier to produce biological value of islets.The main contents include:(1)Comparative study on rat islet stem cells and pancreatic duct stem cells differentiation into neurocyteThis study controlled induced rat islet stem cells,pancreatic duct stem cells into nerve cells in vitro,and compared the differentiation ability.of the two stem cells After randomly thawed islet stem cells and pancreatic duct stem cells,they were cultured with RPMI-1640+15%NBS medium until 80% confluent,then started inductive experiment The experement was divided into three groups,each group of islet stem cells,pancreatic duct stem cells in one plates,each group of six repeats.Experiment 1 group added beta mercaptoethanol;Experiment 2 groups added all rats brain tissue extract;Control group added H-DMEM + 10% NBS,induced 8d,the next day in liquid.observed cells every 4 h,and according to the change of cell morphology,screened the better group.On this basis,further discussed the inducing fluid effect of different concentrations.After randomly selected islet stem cells and pancreatic duct stem cells,they were cultured until the cells grown to 80% confluence,began inductive experement The experement was divided into four groups,each group of islet stem cell,pancreatic duct stem cells in one plates,each group of six repeats.Experiment 1 added 5 mg/m L rat brain tissue extract;Experiment 2 groups added 10 mg/m L rat brain tissue extract;Experiment 3 groups added 15 mg/m L rat brain tissue extract;Control group added H-DMEM + 10% NBS,continuous induced 8d,the next day in liquid.observed cells every2 d,and according to the change of cell morphology,screened the better group.Finally,discussed the influence on the two kinds of stem cells to form nerve cells with optimization induced liquids.After randomly selected islet stem cells and pancreatic duct stem cells,maked cells of climbing with the 1 x 105 / cm2 density,until the cells were grown to 80% confluence,began induction test.Each group of islet stem cells,pancreatic duct stem cells in each of 1 plates,each group of six repeats.Experiment group added 5 mg/m L rat brain tissue extract.control group added H-DMEM+10%NBS,continuous induction 8 d,the next day in liquid.observed cells every2 d.At the end of the experiment,the two kinds of induced cells were immunofluorescence stained with NSEThe results showed that the effect of experiment group added 5 mg/m L rat brain tissue extract was obvious.The induced cells had clear neural synaptic and the next cells contacted each other;experiment group added beta mercaptoethanol did not see nerve sample changes.Experiment group added 5 mg/m L rat brain tissue extract was the best induced group,islet stem cells induce positive rate was 75%,pancreatic duct cells positive rate was 40%.Optimization group results show that morphological changes of the islet stem cells was more clear and complete,the shape of cells changed from the polygon to irregular conical or star,stem cell matured,cell body contracted,Finally forming complete sample neural structure.Two kinds of stem cells of NSE immunofluorescence reaction were positive.The positive rate of islet stem cells was higner than pancreatic duct stem cells.In conclusion,the islet stem cells are easier to differentiate into nerval cell than pancreatic duct stem cells(2)Comparative study on rat islet stem cells and pancreatic duct stem cells differentiation into osteoblasts.This study controlled induced rat islet stem cells,pancreatic duct stem cells into osteoblasts in vitro,and compared the differentiation ability.of two kinds of stem cells.After randomly selected islet stem cells and pancreatic duct stem cells,they were cultured with RPMI-1640 +15% NBS medium until 80% confluent,then started inductive experiment.The experement was divided into three groups,each group of islet stem cell,pancreatic duct stem cells in one plates,each group of six repeats.Experiment 1 group added 0.1umol/L dexamethasone,10mmol/L beta glycerol phosphate,50ug/m L ascorbic acid salts induced liquid;Experiment 2 groups added 8 mg/m L rat gray matter brain tissue extract induced liquid;Control group added RPMI-1640+ 10% NBS,induced 28 d,the next day in liquid.observed cells every 2d,and according to the cell morphology change,screening the better group.And on this basis,discussed the influence on the two kinds of stem cells to form osteoblasts with optimization induced liquids.Randomly selected,islet stem cells and pancreatic duct stem cells were cultured until the cells were grown to 80% confluence,began inductive experement.Each group of islet stem cells,pancreatic duct stem cells in one plates,each group of six repeats.Experiment group added 0.1umol/L dexamethasone,10mmol/L beta glycerol phosphate,50ug/m L ascorbic acid salts induced liquid;Control group added RPMI-1640+ 10% NBS,induced 28 d,the next day in liquid.observed cells every 2d.At the end of the experiment,the two kinds of induced cells were stained with Alizarin red and Vonkossa.The results showed that the effect of experiment group added dexamethasone,ascorbic acid salt,beta glycerol phosphate was obvious.Induced cells secrete matrix to form distinct mineralization nodules;Induced cells of experiment group added rat brain gray matter tissue extract appear single flake nodules,scattered distribution on the cell surface.Optimization group results show that the islet stem cells had a better effect.Induced cells secrete matrix to form mineralization nodules,and nodules growed gradually formed globular,island.After the induction,alizarin red staining reaction was positive,positive rate of islet stem cells,pancreatic duct cells were 60% and 35% respectively.Vonkossa staining reaction was positive,positive rate of islet stem cells,pancreatic duct cells were 45% and 40% respectively.During the induction,islet stem cells kept active growth,stable proliferation,clear vision and less impurity,.To sum up,the islet stem cells are easier to differentiate into osteoblasts than pancreatic duct stem cells.(3)Comparative study on rat islet stem cells and pancreatic duct stem cells differentiation into adipocytesThis study controlled induced rat islet stem cells,pancreatic duct stem cells into adipocytes in vitro,and compared the differentiation ability.of two kinds of stem cells.After randomly selected islet stem cells and pancreatic duct stem cells,they were cultured used RPMI-1640 +15% NBS medium until 80% confluent,then started inductive experiment.The experement was divided into three groups,each group of islet stem cells,pancreatic duct stem cells in one plates,each group of six repeats.Experiment 1 group added 1umol/L dexamethasone,1ug/L insulin,0.5mmol/L 3-isobutyl methy-l-xanthine(IBMX)induced liquid;Experiment 2 groups added 4 mg/m L rat epididymal fat tissue extract induced liquid;Control group added H-DMEM+ 10% NBS,induced 28 d,the next day in liquid.observed cells every 2d,and according to the cell morphology change,screening the better group.And on this basis,discussed the influence on the two kinds of stem cells to form adipocytes with optimization induced liquids.Randomly selected,islet stem cells and pancreatic duct stem cells were cultured until the cells were grown to 80% confluence,began inductive experement.Each group of islet stem cells,pancreatic duct stem cells in one plates,each group of six repeats.Experiment group added1umol/L dexamethasone,1ug/L insulin,0.5mmol /L IBMX induced liquid;Control group added H-DMEM+10% NBS,induced 28 d,the next day in liquid.observed cells every 2d.At the end of the experiment,the two kinds of induced cells were stained with oil red O.The results showed that the effect of experiment group added dexamethasone,insulin,IBMX was obvious.Induced cells appeared different size of lipid droplets,and uneven distribution;Induced cells of experiment group added rat epididymal fat tissue extract had less lipid droplets intracellular and uniform size;Optimization group results showed that the islet stem cells had a better effect.There were more lipid droplets intracellular.At the day of 28,oil red O reaction was positive,positive rate of islet stem cells,pancreatic duct cells were 85% and 60% respectively.Comparison of oil red staining results of islet stem cells and pancreatic duct stem cell,islet stem cells haf a higher positive rate and induced cell stained obviously.In conclusion,the islet stem cells are easier to differentiate into adipocytes than pancreatic duct stem cells.(4)Comparative study on rat islet stem cells and pancreatic duct stem cells differentiation into islet.This study controlled induced rat islet stem cells,pancreatic duct stem cells into adipocytes in vitro,and compared the differentiation ability of two kinds of stem cells.After randomly selected islet stem cells and pancreatic duct stem cells,they were cultured Randomly thawed,islet stem cells and pancreatic duct stem cells were cultured with RPMI-1640 +15% NBS medium until 80% confluent,then started inductive experiment.The experement was divided into six groups,each group of islet stem cells,pancreatic duct stem cells in one plates,each group of six repeats.Experiment 1 group added 10.6mmol/L glucose induced liquid;Experiment 2 groups added 15.6mmol/L glucose induced liquid;Experiment 3 groups added 20.6mmol/L glucose induced liquid;Experiment4 groups added 25.6mmol/L glucose induced liquid;Experiment 5 groups added 30.6mmol/L glucose induced liquid;Control group added L-DMEM+ 10% NBS,induced 28 d,the next day in liquid.observed cells every 2d,and according to the cell morphology change,screened the suitable concentration of glucose.Randomly selected,islet stem cells and pancreatic duct stem cells were cultured until the cells were grown to 80% confluence,began inductive experement.Each group of islet stem cells,pancreatic duct stem cells in one plates,each group of six repeats.The experement was divided into five groups,each group of islet stem cells,pancreatic duct stem cells in one plates,each group of six repeats.Experiment 1 group added 5mmol/L nicotinamide induced liquid;Experiment 2 groups added 10mmol/L nicotinamide induced liquid;Experiment 3 groups added 15mmol/L nicotinamide induced liquid;Experiment 4 groups added 20mmol/L nicotinamide induced liquid;Control group added L-DMEM+ 10% NBS,induced 28 d,the next day in liquid.observed cells every 2d,and according to the cell morphology change,screened the suitable concentration of nicotinamide.Randomly selected,islet stem cells and pancreatic duct stem cells were cultured until the cells were grown to 80% confluence,began inductive experement.Each group of islet stem cells,pancreatic duct stem cells in one plates,each group of four repeats.The experement was divided into five groups,each group of islet stem cells,pancreatic duct stem cells in one plates,each group of six repeats.Experiment 1 group added 15.6mmol glucose,10mmol/L nicotinamide combination induced liquid;Experiment 2 group added 20.6mmol glucose,10mmol/L nicotinamide combination induced liquid;Experiment 3 group added 25.6mmol glucose,10mmol/L nicotinamide combination induced liquid;Control group added L-DMEM+10% NBS,induced 28 d,the next day in liquid.observed cells every 2d,and according to the cell morphology change,screened the suitable combination induced liquid.And on this basis,discussed the influence on the two kinds of stem cells to form islet cells with optimization induced liquids.Randomly selected,islet stem cells and pancreatic duct stem cells were cultured until the cells were grown to 80% confluence,began inductive experement.Each group of islet stem cells,pancreatic duct stem cells in one plates,each group of six repeats.Experiment group added25.6mmol glucose,10mmol/L nicotinamide combination induced liquid;Control group added H-DMEM+10% NBS,induced 28 d,the next day in liquid.observed cells every 2d.At the end of the experiment,the two kinds of induced cells were stained with DTZ and made glucose-stimulated insulin secretion.The results showed that 10.6 mmol glucose treatment group,30.6mmol treatment induced glucose results had no significant difference with the control group.Induced cells didn' t form islet like cell,but did programmed cell proliferation and death.15.6 mmol glucose treatment group,20.6 mmol glucose treatment group,25.6 mmol glucose treatment group,could induce the formation of islet like cells of islet stem cells,pancreatic duct stem.and islet like cell clusters formed different diameter.Different concentrations of nicotinamide induced results showed that 10 mmol/L nicotinamide treatment group had a faster proliferation rate.The results of joint glucose and nicotinamide showed that 25.6mmol glucose,10mmol/L nicotinamide treatment group obtained more islet cell clusters.Optimization group results showed that the islet stem cells had a better effect.Islet stem cells proliferated firstly into a monolayer like slabstones and the size of islet clusters increased continuously throughout the induced course.Ultrastructural analysis showed a small ratio of caron and cytoplasm,developed endoplasmic reticulum,mitochondria,and a few excretory granule in cytoplasm.On the induced day 28,most islet clusters became crimson with DTZ staining.The induced pancreatic islet derived from islet stem cells and pancreatic duct stem cells secreted0.414±0.07ng/m L?0.483±0.15ng/m L and 0.401±0.05ng/m L?0.469±0.1ng/m L insulin in 5.5m M and 25 m M glucose media during 2 hours.The stimulation index of islet stem cells was bigger than pancreatic duct stem cells.Compared the inductive rate,biological function as well as the stimulation index,the islet stem cells are easier to differentiate into islet than pancreatic duct stem cells. |
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