Application Of Pancreatic Islets And Bone Marrow Mononuclear Cells In Treatment For Diabetic Rats

Background:Insulin-dependent diabetes mellitus is a type of autoimmunity disease characterized by the decreased number of insulin-secreting cells, which lead to elevated blood sugar and sever cardiovascular,eye and renal complications in patients with diabetes. Moreover, the patients used to be injected with trypsin everyday to control blood sugar. However, hypoglycemia, syncope, paralysis and even death caused by over dosage usually occur. The first case of islet transplantation was performed by Lacy in 1982, but low success rats and high mortality hindered its application. Islet transplantation hasn't greatly improved until 2000 when the Edmonton protocol was proposed. Nevertheless, islet transplantation is still faced with many challenges and lacking of donors ranked first. Now, Less than 1% patients have chance to receive it and this phenomenon would be aggravated as the indications of islet transplantation broadened.Objective: 1) To compare the success rate for establishing type 1 diabetic mellitus model by intra-peritoneally injection Streptozotocin (50mg/kg) respectively after taking and fasting food in rats and to determine survival rate after two weeks; 2) To compare and observe outcome of islet after the Pancreases were perfused by cold storage (4℃) collagenase V and Hanks; 3) To investigate the therapeutic effect of bone marrow mononuclear cell and pancreas islet transplantation in liver, tail vein and pancreas tissues; 4) To investigate the possible immune-suppressant effect of bone marrow mononuclear cells; 5) To investigate the relationships between cells and transplantation sites.Method:1) Forty eight Wistar rats,6-8 weeks old, body weight is 200～250g. Intraperitoneally injected with streptozotocin solution (50mg/kg) once. Streptozotocin solution was ice bathed and away from light. This process was accomplished in 30 minutes. Rats were head downward for 30 seconds in order to streptozotocin solution afflux to pancreas. Then put diabetic rat into rearing cage to take food and water freely. Random blood sugar was detected with blood sugar meter twice. The successful model for diabetic mellitus in rat was defined as random blood sugar higher than 16.8mmol/L and the onset of typical clinical symptoms of diabetes mellitus including polydipsia, polyuria and body weight loss; 2) Retro-perfuse pancreas and morsel pieces of 1×1×1mm3.Then digest pancreas with 10ml preheating (37℃) collagenase V solution. Use small amounts clear supernatant to observe under inverted microscope. Add 50ml pre-cooling Hanks to remnant to digest interstitial tissue to detach pancreas islet. Filtrate cell suspension and centrifuge for 5 minutes twice (1500r/min,4℃). Clean the pancreas islet with Ficoll400 solution. Add 25%,23%,20%,10% Ficoll400 solution into panc-reatic tissue in turn. Gradient centrifugation for 10 minutes (1000rpm,4℃). Iisolated pancreas islet focus on 23-20% and 20-10% solution surface layer. Remove pancreas islet to a new testing tube and washing with Hanks twice. Pancreas islets were counted with dithizone; 3) Dissect four limbs bones in aseptic codition. Bathe in penicillin and streptomycin solution for 15 minutes, Then bathe in phosphate buffered saline (PBS) for 10 minutes. Snip epiphysis with bone ribbing rongeur.Douching bone marrow with 5ml low glucose dulbecco minimum essential medium (L-DMEM) which contain 10% fetal bovine serum (FBS). Then douch bone marrow cells with 26G syringe two times. Until bone marrow cells were single cell suspension. Add Lydroxypropylmethyl Cellulose (5ml) into centrifuge tube. Then add bone marrow cell suspension above Lydroxypropylmethyl Cellulose. Gradient centrifugation for 20 minutes (1600r/min). Suck bone marrow mono-nuclear cell with drip tube at solution surface layer. Coloretur bone marrow mononuclear cell with trypan blue and count number with microscope. Adjust cell density to 1×107/ml for transplantation; 4) Diabetic rat model were randomly divide into A,B,C,D,E,F groups. Group A:transplant 1000 pancreatic islets beneath the liver capsule. Group B:transplant 1000 pancreatic islets and 1×107 bone marrow mononuclear cells beneath the liver capsule. Group C:transplant 1000 pancreas islets through tail vein. Group D:transplant 1000 pancreas islets and 1×10 bone marrow mononuclear cells through tail vein. Group C and D incise little nick on abdomen after transplantion.Group E:transplant 1000 pancreas islets beneath the pancreas capsule. Group F:transplant 1000 pancreas islets and 1×107 bone marrow mononuclear cells beneath the pancreas capsule. Determine plasma glucose in pre-transplant 1 day,0,1,2,3,5,7,9,11,13,15,17,19,21 day. Resect rat's pancreas, liver, lung and spleen dying with hematoxylin to observe pathological change; 5) Data were analysed by using SPSS 17.0Results:1) Blood sugar Diabetic rat model was higher than 16.8mmol/L. The success rate is 100%. Establishing diabetic mellitus model in rats by intrperitoneally injiceting STZ without fasting is a reliable and stable method. 2) Separated pancreas islets coloretur with dithizone to count number. Pancreas islets were compact scarlet spherical or mass like. But exocrine gland cell could not b dyed by dithizone and refraction is powerful. Pancreas islets recovery coefficient is 60-70%. Purity of purification cell is more than 90%. Acquire (520±53) pancreas islets every rat; 3) Mononuclear cells were dyed with 2% trypan blue. Then observe with microscope. Mononuclear cells refraction is powerful and purity is high. Dead cells become calamine blue; 4) Group A:start to activate after 3 days.Plasma glucose level reach to normal (7.98±2.28) mmol/L and maintain (3.71±0.95) days. In Group B plasma glucose level reach to (7.72±1.75) mmol/L and maintain (4.86±1.06) days. Group C and D start to activate after 4 days. Group C reach to (7.35±1.40) mmol/L and maintain (7.85±1.46) days.Group D reach to (7.00±0.83) mmol/L and maintain (14.1±1.21) days. Group E and F start to activate after 24 hours. Group E reach to (7.06±2.11) mmol/L and maintain (24.90±2.60) days. Group F reach to (8.21±1.22) mmol/L and maintain (39.80±2.57) days. There is statistical significance in time-dependent tendency of plasma glucose level (P<0.05).There is statistical significance in plasma glucose level maintain normal time (P<0.05).There is interaction between time- dependent tendency of plasma glucose level and the way of transplantation; 5) The specimen diabetes rats pancreatic tissue is loss of volume. The color is similar with fat. The number of pancreas islet Is decreasing. The liver specimen showed severe inflammatory reaction.Conclusion:1) Establishing diabetic mellitus model in rats by intrperitoneally injiceting STZ without fasting is a reliable and stable method.; 2) The islets outcome is same through common bile duct injection of Hank's and injection of collagenase. Perfusion pancreas with pre-cooled cooling Hank's could protection pancreas islet integrality.Injection of Hank's through common bile duct is a ideal method of pancreas perfusion; 3) Selection of ideal approach for transplantation is a key influential factor in this procedure. Iislets transplanted beneath the pancreas capsule maintain plasma glucose for the longest time. The pancreas is a ideal transplantation organ; 4) Bone marrow mononuclear cells have immunosuppressant effect in immunologic rejection process; 5) United transplantation seem to be an ideal way to reduce the blood sugar level.; 6) It is easy to generate inflammatory reaction in liver and lead pancreas islet apoptosis.