| [Background]Diabetes mellitus (DM) is a metabolic diseases group that is characterized by the levels of blood glucose chronic increased. Hyperglycemia is due to the effect of insulin secretion dysfunction and (or) Insulin function defect. In addition to carbohydrates, metabolism of protein and fat is disorders. A long illness can cause multiple system lesions, severe disease or stress can cause Acute metabolic disorder. The illness can reduce living quality and Shorten Lifespan and increase mortality of patient. Data from International Diabetes Federation (IDF) show that the number of diabetics around the world has been increased to366million, in20years future will reach the number will increased nearly600million. At the moment, the treatment of diabetes mainly includes drug therapy and insulin injections. But whether drugs or insulin injections, can cure diabetes, patients need lifelong medication. Because of compliance is not good, most people with diabetes cannot get good treatment, and occur severe complications. Long-term medication or insulin injections, not only increase suffering, but also bring heavy economic burden on families. Therefore, human society is actively seeking a radical cure of diabetes.Islet cell transplantation is considered to be one of effective methods to cure diabetes mellitus. Because of the shortage of islet sources, and islet cell donor shortage, and Immune rejection, etc., Treatment effect is affected.Our country has a huge number of diabetes patients, heterogeneous islet transplantation for the treatment of diabetes research in our country appears more urgent, but compared with abroad, heterogeneous islet transplantation study in our country is relatively backward, So far only a handful of unit conducted a preliminary clinical trials. Domestic lack data information of preclinical animal experiment effect and safety evaluation for islet cell transplantation in the treatment of diabetes, seriously hindered the develop of the clinical application of islet cell transplantation in the treatment of diabetes. Through the project implementation can gain more data information of preclinical animal experiment effect and safety evaluation for islet cell transplantation in the treatment of diabetes. Minipigs as one of the commonly used experimental animals, have the similar in the use of the absorption of sugar. There is only one amino acid is not the same between Minipigs and human. Minipigs is the main source of the treatment for diabetes, Islet cell transplantation has become new method to treatment of diabetes, and hematology and blood biochemical indicators of pig Approximation with humans, As the growth of the age, pigs can form spontaneous atherosclerosis. Convenient for people to research on diabetes and complications. Provide an perfect animal model for the research of diabetes and the complication.[Objective]This study through the method of drug induced Tibet minipigs establish diabetes model, provide basic data for diabetes research;The purpose of this study is to obtain a large number of islet cells are active, to detect islet cell of Tibet Minipigs, to provide reliable data and experience for preclinical studies of Minipigs.[Method] Select10healthy Tibetan Minipig, Single column feed, feeding standard of per day to3%of animal body weight,free drinking water. Miniature pigs were randomly divided into experimental group (group A) and control group (group B), each group5. Observe the status of animals of eating drinking action and spirit everyday, weigh weight once a week. From the ear vein injection3%pentobarbital sodium to anaesthetize the m Tibetan Minipig, Group A inject the drugs of Streptozotocin and Alloxan from the ear vein, continuous injection of3days, Group B injection amount of normal saline. Before injection, two groups of minipigs fasting12hours(From22PM to10PM the next day); After injection, the minipigs breeding in a relatively warm environment, to maintain body temperature. During the experiment fasting blood glucose of minipigs with glucose meter at10o’clock in the morning every day, continuous monitoring for7days. After the success of the building, on the first day of every week measured fasting glucose, monitoring of the minipigs blood glucose changes until the end of the experiment. Detection of glucose tolerance once a week. Before each test fasting and prohibit drinking water.First put75grams of glucose powder soluble in250-350ml of drinking water to feed to minipigs. Monitoring of blood glucose levels at the time of30,60,120and180minutes. Collecting venous blood from precaval vein at the time of the day before the injection and the seventh day after the injection, separation serum, To detect serum total cholesterol(TC) and triglyceride (TG) by enzyme method, The glycosylated hemoglobin of the blood were measured by kites test methods.Choose healthy seven months Tibet Minipigs, After the euthanasia, Gather the Tibet Minipigs pancreas in sterile conditions and infiltration them in the cold of the PBS, Selection of the pancreatic head cutting tissue block, removed by membrane on the surface of the connective tissue and fat, wash by PBS. Tissue block were cut up and divided into four groups, Compare the number of the islet cells were isolated and effects at the different mass concentration of collagenase (1mg/ml,2mg/ml,3mg/ml,4mg/ml) and Comparison the number of the islet cells were isolated and effects at different time points on the same mass concentration (0min,15min,30min,45min,60min,75min,90min);Use4methods purified islet cell suspension, include Ficoll400density gradient centrifugation and lymphocyte separation medium and centrifugation standing four kinds of methods, Compare the islet cells and effect of ultimate collection; The purity of pancreatic islet cells with DTZ staining method; Islet cell survival rate count with AO/PI dyeing method.[Result]In the experiments of Establishment of animal model of diabetes in Tibet minipigs, after inject, Hyperglycaemia occurred in the minipigs of experimental group at the second day and continuous7days stability remain above11mmol/L. Blood glucose levels did not rise in the minipigs of control group, significant difference comparing the two groups(P<0.05). Sugar tolerance and glycosylated hemoglobin of experimental group reduce, indexes of the control group did not change significantly, determine the minipigs has diabetes when blood glucose have more than twice tendency for7.0mmol/L. Fasting blood-glucose and the blood glucose in any points significantly higher than the7.0mmol/L, prove that Tibet minipigs diabetes model is successfully established. Both of two group of blood total cholesterol and triglyceride levels were not significantly changed, show that the drugs mix by STZ and ALX cannot cause the change of blood fat of minipigs, So can’t application of combined ALX and STZ injection method to make hyperlipidemia animal models.To determine the best time of the quantity of islet cells at different mass concentration of collagenase,1mg/ml collagenase group and2mg/ml collagenase group was45min,3mg/ml collagenase group and4mg/ml collagenase group was30 min. The number of the islet cells obtain at the best time were compared and1mg/ml collagenase group obtain Islet cell number maximum, form a complete, good activity; Four kinds of purification methods were compared, the order of the quality of collection of pancreatic islet cells from more to less was standing> Ficoll400> isodensity centrifugation> centrifugation> Lymphatic fluid centrifugal separation, the pancreatic islet cells obtain from four kinds of purification methods have active. Detection of islet cell activity through AO/PI, more activity than95.00%.[Conclusion]The success of the research on optimum condition of separation and purification of islet cell of Tibet minipigs, Tibet minipigs diabetes model was established by the drugs of STZ and ALX, through the islet transplantation for inspection that the effect of separation and purification of islet cells. Accordingly the research can provide a reliable data and experience for the study of the islet cells of Tibet minipigs in preclinical studies. |
PDF Full Text Request