| BackgroundDiabetes (diabetes mellitus, DM) is a high incidence disease that brings serious impact to human health and life. Diabetes is divided into type I and type II. Type I diabetes is insulin-dependent diabetes, and about5-10%of patients are type I diabetes while the90%of patients are type II diabetes which is non-insulin-dependent. More than one-third of patients with terminal type II diabetes will develop into insulin-dependent diabetes patients. Nowadays, there are366million diabetic patients around the world and the number has increased by30%, compared to285million in2010. Each year,4.6million people died from diabetes and the medical expenses on it are up to$465billion. Insulin injection is the effective way to control blood glucose at present. However, Exogenous insulin can not protect patients from complications like nephropathy, neuron system diseases, retinopathy and cardiovascular diseases because of its failure to work as the endogenous one to regulate blood glucose. Consequently, it will greatly reduce the quality of patients’ life and even result in death. The health-care cost of diabetes in America is12.5%of the entire national health-care costs and in China is up to250billion yuan each year. Therefore, the development and application of effective treatments to diabetes is undoubtedly of great social significance and economic benefits.ObjectiveIslet transplantation is considered to be an effective therapy to diabetes. However, the lack of islet cells donor and immune rejection after transplantation are the two inevitable problems to it. The application of xenotransplantation technology has provided a new solution to the problem of lack of donor cells and it will create immeasurable social value. Porcine insulin is similar to human insulin on its physiological function and structure with only one amino acid difference so as to largely decrease the consequence to immunological rejection. Additionally, the porcine islet cells have the similar set point of blood glucose with human. Thus, the application of porcine islet as the xenogeneic islet tissue donor has gradually aroused the concern of public. However, due to the difficulties in isolating porcine islet cells, there is still no effective and stable method to gain large amount of porcine islet cells.This study aims to investigate the pathogenesis and treatment of type I diabetes by conducting a heterogeneous islet cells transplantation through intrahepatic portal vein on SD rats with type I diabetes. This study first isolates and purifies islet cells from swine, and then establishes the type I diabetes animal models and conducts porcine islet cells transplantation on animal models.Methods and ResultsThis study mainly consists of three parts:(1) Establishment of animal model of type I diabetes mellitus on SD rats.(2) Tibet mini pig islet cells isolation and purification and function tests.(3) The experimental study of the Tibetan miniature pig islet cells xenotransplantation treatment of type I diabetes.(1)Establishment of Animal Model of Type I Diabetes Mellitus on SD RatsSelect the180-220g SD rats, male, healthy. Were randomly divided into control group(n=40) and experimental group(n=40). Animal be breeders in the clean environment. Room temperature is18-22℃, indoor ammonia concentrations below20ppm, relative humidity is40%-70%. Standard feed,each cage breed5animal, free feeding and drinking water. Frequently changes the pad material, to keep the environment ventilation and drying.SD rats via adaptive feeding after one week and then be used to establish model. Determine of fasting blood glucose before making model.Abstinence for12h, According to the weight and through intraperitoneal injection manner given65mg/kg of STZ(Solubility in citric acid buffer solution(0.1mol/L, PH4.0), temporary formulated into4mg/mL concentration). The control group was injected with citric acid buffer solution. Through tail vein collect blood sample to test peripheral blood glucose after7days. If the blood glucose levels lower than8.9mmol/L before inject, and glucose is maintained in the16.7mmol/L above a week after injection. It is considered that the model successfully. During the experiment, mesure the weight of rats before the experiment and after experiment of1week,2weeks,4weeks and6weeks, and according to the testing results plotted in growth curve of the two group s rats. Observated pathological changes of pancreas by HE staining.The results show that treptozotocin can well induce rats with type I diabetes. The blood glucose of rats in experimental group was significantly higher than that of control group after the success of model replication, and has been maintained at a high level. Mould forming rate of90%. Rats exhibited appetite is bigger, the amount of drinking water was increased, polyuria, weight loss, reduced activity, slow response, the wound is easy to be infected, emaciation, hair does not shine, hair removal and other symptoms after the success of model replication. After injection of STZ solution,the rats in the experimental groupe show slow growth trend in the experimental early stage, With the increase of blood glucose, body weight show downward trend gradually. While the body weight of the control group rats was growth sustained. With the extension of time, the control group and the experimental group rats weight difference increasing.(2) Tibet mini pig islets isolation and purification and function determinationTibet miniature pigs, male and six months old, No infectious diseases, weight range in20-25kg, fasting12h before operation.,free drinking water. Through intramuscular injections induced anesthesia by Sumianxin (0.1mL/kg). Wait for the animal unresponsive, cannot stand, nociceptive reflex is not obvious, then used the multi-functional animal anesthesia machine through isoflurane to maintain anesthesia. Removal of the pancreas in a relatively sterile conditions from pancreas. The warm ischemia time not more than10minutes and cold ischemia time not more than45minutes. Use of type V collagenase solution(1g/L) retrograde perfusion via pancreatic duct. water bath box (38.5℃)static digestion about30min, Wait for a large number of islets from down, terminate of digestion by Hanks liquid that contained10%fetal bovine serum. Filter collected the product after digestion by600μm steel screen. The pancreas that incomplete digested need digest one more time and then collected. Using Ficoll400density gradient centrifugation centrifugal to purify of islets of Langerhans, including three density (1.090g/cm3,1.074g/cm3,1.054g/cm3), In the conditions of4℃,1000r/min and2000r/min,centrifuge2min and10min respectively, collect the pancreatic islet cells from1.054g/cm3to1.074g/cm3. Purity and activity:Dithizone staining. Observed under inverted microscope,counting yield and purity of the pancreatic islet cells. Insulin release test to detected the function of the pancreatic islet cells.The results show this method can obtain structural integrity, higher purity, and maintain a good activity of pancreatic islet cells. The pancreatic islet digested by collagenaseV, Observated under inverted microscope, pancreatic islet were round, oval or irregular shape, not the same size.Most pancreatic envelop integrited, refraction performance is well, massive pancreatic exocrine acinar cells and tissue fragments around scattered islet cell clusters. Most of pancreatic islet diameter is in50μm between200μm. After purification, average per gram of miniature pig pancreatic tissue can be obtained (2807±124) IEQ (Islet equivalent quantity). The average purity for (81.1+1.3)%. The purified islets cells is sensitive to glucose stimulation test. The amount of insulin release in high glucose and theophylline group was3.60times higher than low glucose group(P<0.01),and high glucose group was1.67times higher than low glucose group(P<0.01), It indicated that the purified islets has better biological function.(3) The experimental study of xenotransplant the islet cell of miniature pig to therapy type I diabetesTested the blood sugar of diabetic SD rats before24hours of transplantation.3%pentobarbital sodium according to1.0ml/kg dose via intraperitoneal injection to narcotize diabetic SD rats. Supine immobilization in a rat fixing plate. Remove the hair on surgical site, spread the operation towel after iodine disinfection. Cut a length approximately3cm incision in upper abdominal median. Into the peritoneal cavity, exposure of main trunk of portal vein. make a not shrink knot with vascular suture in portal vein, Then connect the syringe that was equipped with islet cell suspensions and venous indwelling needle, exiting the needle core after puncture. Tightening the suture line to avoid hemorrhage. The mimi pig islet cell suspension was injected into the portal vein slowly, tight the vascular suture and knot. This study set up three transplant dose (10000IEQ,20000IEQ and40000IEQ).After determine the best transplant dose, based on this transplant dose.We use Two kinds of immunosuppressive agents (KF506and CSA). Divided into the single and combined application of three methods. Discuss the three immunosuppressive regimens for anti rejection effect. The results show that Tibet mini pig islet xenograft to treatment the diabetes mellitus of SD rats can effectively reduce the blood glucose of rats. Among the10000IEQ,20000IEQ and40000IEQ three different transplant dose,20000IEQ and40000IEQ has the most obvious effect in reduce blood glucose. But between this two transplant dose.But the effect of reduce blood glucose was not statistically significant with the graft volume increases, Therefore, we determined the20000IEQ as the best transplantation dose.In the use of three different immunosuppressive regimens.CSA and KF506in xenotransplantation of porcine islets for the treatment of type1diabetes rats have better resistance to immune rejection, two union application showed obvious synergistic effect, can be effective in prolonging graft survival time.ConclusionsIn summary,STZ can be stably induced rat occur of diabetes, With high rate into a mold, a low mortality rate, and other characteristics, diabetic rats can be maintained for a long time and high blood sugar. Use of the digestion method by collagenase V through retrograde infusion can stably obtain a complete structure, biological function good miniature pig islet cells. Mini pig islet xenograft in the treatment of type1diabetes mellitus rat, can effectively reduce the blood glucose of diabetic rats, the use of immunosuppressive agents, can get good anti rejection effect, can effectively prolong the survival time of allograft. |
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