Effect Of Trypsin Inhibitor On The Isolation, Purification And Cold Storage Of Rat Islets

Background:Diabetes Mellitus (DM) has been a common and frequently occurring disease throughout the world at the moment and its mortality is up to the 3rd after cardiovascular disease and cerebrovascular disease. According to the statistics from WHO, by 2000 there had been 0.177 billion suffering from Diabetes Mellitus all over the world, and the quantity is still increasing. At present type 2 Diabetes Mellitus is fulminant in China, the number of patients has reached 40 million, and it increases at a speed of at least 3000 per day, and exceeds 1.2 million per year. Although the rate of type 1 Diabetes Mellitus patients is less than 10% in our country, the number of population is large; therefore the number of type 1 Diabetes Mellitus patients is a lot.For type 1 Diabetes Mellitus, there are two major treatment methods--external or intensive insulin therapy and the transplantation of pancreatic islets or pancreas. In recent years, with the development of the basic theory and clinic research, the transplantation of pancreatic islets has been the most promising treatment method for type 1 Diabetes Mellitus. The researches of type 1 Diabetes Mellitus bring about an upsurge, and make a great breakthrough, especially after the proclamation of Edmonton Protocol. The effects after the transplantation of pancreatic islets are updated incessantly. However, lots of obstacles of the transplantation of pancreatic islets need to be conquered. One of the most important obstacles is that the amount of islets is not enough. There was no practical breakthrough, though researchers had taken plenty of methods to enhance the islet yield of the single pancreas. Recent evidence suggests that intrinsic pancreatic proteases may inhibit effective collagenase enzymatic activity during islet isolation, thereby impairing the isolation success. One of our objectives is to improve yield and functional viability of islets isolated from rat pancreases by inhibiting the activity of intrinsic pancreatic proteases. Due to the level of the isolation and purification of rat islets, we cannot isolate enough islets to transplant from one single donor; therefore a period of time is needed to wait for the next donor. Between the obtaining and transplantation of the pancreatic islets, there will be necessarily a period of cold-ischemia time for crossmatching between donor and recipient, counting the number of islets, examining the islets function, preoperative preparation and immunogenic preconditioning, therefore a short-term preservation of islets is necessary. Certainly it will bring certain loss of the yield and function of islet whichever method we take, so it is very important to decrease the loss of the yield and function of the short-term preserved islets. To solve above questions, we design this research. During the isolation of rat islets, we use collagenase with trypsin inhibitor, which inhibits the activity of intrinsic pancreatic proteases during the digesting, and make the isolation and digestion of islets going under the exact control to decrease the islets damage of protease and observe whether the yield of islets increase. To further confirm the effect of trypsin inhibitor on the islets function, we preserve the islets gained by short-time cold storage, and make islet allograft after examination the modality and function of islets, then observe the reverse effect of hyperglycemia on rat model of diabetes mellitus after transplantation. At present associated research is little in domestic or abroad,and exists much controversy in conclusion. Methods and Results:1. Effect of STI on the isolation and purification of rat isletsAccording to whether STI was put into collagenase, we divide rats into experiment group and control group. For the experiment group, we put 2.0mg/ml STI into digestive solution containing collagenase, and the control group does not. For both of the two groups insulin collagenase perfusion was used to isolate islets. We use Ficoll-400 discontinuous density gradient centrifugation to purify islets, count the number and examine modality and function. Results indicate yield and purity of rat islets are obviously increased through putting STI into digestive solution containing collagenase, but there is no apparent effect on function of islets. Islets obtained are enough to meet the need of animal transplant experiment.2. Preparation of diabetes mellitus model in ratsDiabetes mellitus model was induced by a single intraperitoneal injection of streptozotocin citrate buffer, and then we monitored the blood glucose change of the rat. Continuous monitoring was carried out in the first five days after injection, and then monitoring once a week for the next four weeks. Among the 30 diabetes mellitus models we prepared, there were 28 rats whose blood glucose in five times continuous detection were above 16.7mmol/L. It is consistent with the standard of diabetes mellitus model and its achievement ratio is 93.3%. The findings indicated that single intraperitoneal injection of streptozotocin citrate buffer was a success method for preparing stable chemical diabetes mellitus rat model, which would be a suitable recipient for islet transplantation.3. Influences of morphology and functions of cold storage rat islets after adding STI to the collagenaseWe took collagenase solution containing with STI to isolate rat pancreas for obtaining islets, and the control group isolated rat pancreas without STI. Then detected the morphology and functions of islets after they were cold stored respectively at -4℃for 72h, and allogeneic transplanted the islets to diabetes mellitus rat model for advanced observation of the influences of functions of islets after adding STI to collagenase. Results: There was not apparent change in experimental islets morphology, which had been in cold storage for 72h, whereas the control islets became cellular edema, islet surface and internal leakage, structure loose. After 72h cold storage, we took the insulin stimulation release trial, the experiment group's low serum glucose mean insulin-release level was 3.3±0.9μmol/L, high serum glucose mean insulin-release level was 9.8±2.1μmol/L,stimulation index was 3.1±0.5. The control group's low serum glucose mean insulin-release level was 2.2±0.6μmol/L, high serum glucose mean insulin-release level was 5.4±0.7μmol/L,stimulation index was 2.1±0.3; To take cold stored islets for allogeneic transplantation inferior rat renal capsule, under the condition of no immunodepressant, the experimental rats blood glucose restored normal 24h after islets transplantation, maintaining 4.0±0.7 days; and for the control group there was only 1 rat's blood glucose restored normal, maintaining 2 days. The examination of pathematology and serum insulin of recipient after allogeneic islet transplantation indicate that the recovery rate and function of islets of experiment group are higher than control group after cold storage. This manifests that collagenase added with STI during the islets isolation can enhance the islet's resistance to cold storage injury, elevate the restore rate in short time cold storage. The morphology and functions changed little after cold storage and the diabetes mellitus rat's blood glucose restored normal in a short time after islets transplantation, maintaining a long time.Conclusion:Trypsin inhibitor can effectively elevate the yield in islet isolation and purification, impart the obtained islet with stronger cold storage resistance. After cold storage the islets have higher restore rate and less function loss, and can reverse the diabetes mellitus rat's high serum glucose status after allogeneic islet transplantation. The findings can improve the low pancreas islets yield in clinical islet transplantation, relieve the pancreas islet donor deficient stress, provide the theoretical basis for one to one pancreas islet transplantation, and would be a new pathway to elevate cold storage restore rate and prolong cold storage time.