| BALB/C MICE PANCREAS ISLET AND EXOCRINECELLS SEPARATION,PURIFICATION AND FUNCTIONTESTINGObjective:The separation and purification of islet Balb/c mice.Methods:Using retrograde bile pancreatic duct perfusion v-shaped separation method of collagenase to absorb Balb/C mice islet, single concentration gradient (10771) and lymphocyte separation medium, inverted microscope manual screening of purified islet, DTZ (dithizone) specific dyeing monitor islet yield and purity, placenta blue staining, the actiity of the islet cells by ELISA kit to detect insulin stimulation index calculated islet function.On the function of pancreatic exocrine cells in vitro testing, collecting supernatant ELISA method for determination of content of trypsin.Result:After purification of each Balb/C mice can be received around 150 high-quality islet, activity and purity were greater than 90%, basic normal glucose stimulated insulin releasing test show that islet function.Pancreatic exocrine cell culture 1 h,4 h after that trypsin supernatant fluid content is 5.8±1.2 ng/ml,7.3±1.0 ng/ml.Conclusion:Application physi CAL mature experiment method, can obtain high purity, high quality of pancreas and pancreas exocrine cells to meet the needs of the next step of islet transplantation follow-up experiments.ESTABLISH OF ISLET CELLTRANSPLANTATION ANIMAL MODEL TOGETHER TOGETHER WITH THE PANCREATIC EXOCRINE CELLS UNDERTHE LEFT RENAL CAPSULE INDIABETIC MICEObjective:Master to establish Balb/C mice diabetes left renal capsule under pure islet transplantation and together with the pancreatic exocrine cells transplantation animal models of research methods and the pancreas exocrine cells damage of islet transplantation.Methods:Male Balb/C mice abdominal cavity injection of STZ, chain urea with cephalosporins induced mice with type 1 diabetes. Simple transplantation group (n= 10) by rat renal capsule left in each transplanted islet cells,250, under the joint transplantation group (n= 10) in each the synchronization between up and down the left kidney capsule transplantation rat islet cells, such as 250 and volume of pancreatic exocrine cells, transplantation continuously monitor the mice tail vein blood sugar change, after a month left renal resection, immunohistochemical staining observation resistance to insulin antibodies and antitrypsin expression.Result:(1) After islet transplantation, transplantation group than pure blood sugar back to normal time delay 2-3 days, the two groups after transplantation of diabetic mice blood glucose were gradually returned to normal.(2) Simple and common transplantation group, a large number of insulin resistance in this case in the renal capsule antibody positive color, confirm for islet transplantation.Islet cell transplantation group jointly group of visible antitrypsin antibody positive particles in great quantities, and simple transplant group no obvious positive particles.(3)The pancreas exocrine cells and islet cells after transplantation, confirmed the pancreas exocrine cells can release a lot of trypsin damage of islet grafts function recovery.Conclusion:(1) Selection of induced diabetic model mice weighed more than 26 g,1.75 mg/kg intraperitoneal injection can induce diabetes model.(2) Successfully established of pancreas and pancreatic exocrine cells transplantation animal model, animal model of preparation work for follow-up study.PROTECTIVE EFFECTS OF SOMATOSTATIN ON MICE ISLETS EARLY INJURY AFTER TRANSPLANTATION BY PANCREAS EXOCRINE CELLSObjective:To investigate the the protective effects of smoatostatin on mice islets injury by protease released from pancreas exocrine cells.Methods:(1) In vitro experiment:30 randomly selected male BALB/C mice, pancreas and pancreas exocrine cells, randomized and short-term training together, using flow cytometry instrument to detect the contents of apoptosis, determination of trypsin in the supernatant and detect pancreatic insulin levels and stimulation index, exocrine cells and islet cell apoptosis by TUNEL staining detection after smear and immunohistochemical determination of P53 protein.(2) In vivo:adopt the method of retrograde perfusion collagenase bile total tube separation and purification of pancreatic exocrine cells and islet respectively, respectively, at the same time in the mouse up and down very islet transplantation 250 pancreatic exocrine cells, and its volume 40 islet transplantation model was established successfully, the random number table method is divided into the experimental group and control group, experimental group of postoperative intraperitoneal injection of somatostatin 10 ng/g, the control of postoperative intraperitoneal injection amount of normal saline, continuous determination of blood glucose levels and then observe the changes of vital signs.Five days after removal of mice left kidney graft, USES the frozen section TUNEL staining to detect pancreatic exocrine cells apoptosis, immunohistochemical SP technique for determination of trypsin in graft to release a quantity.Result:(1) Flow cytometry, according to the results of islet cells respectively, the experimental group and the control group, apoptosis rate (6.76 ±0.30)% and (6.21±0.40)%, two groups of more, there was no statistically significant difference (P> 0.05);Somatostatin respectively, the experimental group and the control group, apoptosis rate (21.34±0.34)% and (15.76 ±0.40)%, two groups of relatively, the experimental group apoptosis rate is significantly higher than the control group, the difference was statistically significant (P< 0.05);(2) The original generation training after that ELISA determination of content of trypsin in the supernatant fluid:purified islet group that trypsin in the supernatant fluid concentration (0.43±1.11) ng/ml, the control group (5.03±1.20) ng/ml, which is significantly higher than the former, the difference was statistically significant (P< 0.01);Experimental group that trypsin in the supernatant fluid concentration (2.43±1.30) ng/ml, lower than the control group, but higher than that of purified islet group, the difference was statistically significant (P< 0.05);(3) The original generation of training after insulin stimulation index comparison:purified islet group and insulin stimulation index was 3.85±1.20, the control group was 1.55±1.12, the former is obviously higher than the latter, the difference was statistically significant (P < 0.01);Experimental group insulin release index was 2.53±1.10, below the purified islet group, but higher than the control group, the difference was statistically significant (P< 0.05);(4) Of the pancreas exocrine cells and islet smear TUNEL results:pancreas exocrine cells apoptosis index:TUNEL results show that the experimental group of pancreatic exocrine cells apoptosis index was (25.37±0.56)%, while the control group of pancreatic exocrine cells apoptosis index was (17.68±0.41)%, the control group significantly lower than that of the experimental group, the difference was statistically significant (P< 0.05);Islet cell no statistical difference.(5) Immunohistochemical SP cells smear results showed that apoptosis cells in brown staining, the experimental group P53 positive staining (10.65±0.45)%, positive control group P53 stain (8.32± 0.30)%, comparing the two groups, the difference was statistically significant (P< 0.05).(6) 5 days after operation, the frozen section in the TUNEL apoptotic detection results:fluorescent staining of exocrine cells in experimental mice is less than that of control group.Experimental pancreas exocrine cells apoptosis index was (18.35±0.45)%, while the control group of pancreatic exocrine cells apoptosis index was (10.23±0.23)%, the control group significantly lower than that of the experimental group, the difference was statistically significant (P< 0.05);No statistical difference was found between islet cell apoptosis index.(7) After 5 days trypsin immunohistochemical results:around the islet cells in control group with a large number of dark brown antitrypsin antibody positive particles, but not seen in the experimental group obviously the dark brown color.Positive control group particle number is (80±4), the experimental group (15±3) number of positive particles, comparing the two groups, the difference was statistically significant (P< 0.05).Conclusion:(1) Somatostatin can induce damage in the process of islet transplantation in mice pancreas exocrine cells apoptosis, and has no effect on islet cell apoptosis.(2) Somatostatin can induce pancreatic exocrine cells apoptosis, and apoptosis is closely relative to regulate the expression of P53 gene.(3) Application of somatostatin in islet transplantation can significantly reduce the damage to the pancreas exocrine pancreas cells in mice. |
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