Study On The Induction Of Islet Transplantation Tolerance In Mice By Donor Antigen-specific Treg Cells

PART ONEIsolate and purify mouse islet cellObjectives: Seek a stable and efficient method acquiring murine pancreatic islet cells and set up standardized program isolating mouse islets.Methods: Expand and digest mice pancreas to get murine pancreatic islet with following step: Perfuse the common bile duct of BALB / c mice with different concentrations of collagenase (0.5mg/ml, 1 mg / ml, 1.5 mg / ml) and then deal with different water bath digestion time (10min, 20min, 30min), discontinuous density gradient centrifugation with Ficoll-400 is applied to purify islet. to calculate islet yield and purity ,Islet specific staining with dithizone (Dithizone, DTZ) In vitro is put into use function of islet is evaluated by glucose-stimulated experiments.Results: Treated with different concentrations of collagenase digestion and different time , Islets qantity yield is greatly different also, the maximum volume , (190±18 ) islet cell mass, purity about 90% ,can be obtain by the use of 0.5mg/ml collagenaseⅤ, 38Ⅴwater bath for 20 minutes. Islet display scarlet after DTZ staining, form completly. insulin concentrations increased with glucose in the Glucose stimulation test,insulin quantity released is 0.535±0.175ng/islet with higher amoutt of glucose stimulating while 1.266±0.321ng/islet with lower amout.Conclusions: Collagenase perfusion, enzyme concentration, digestion time, digestion temperature, mouse strains, and other factors will have a great impact on the islet amount get with the mouse islet isolation process . However, the collagenase concentration, digestion time and temperature are the most important factors affect the outcome of murine islet isolation, dealed with collagenase concentration of 0.5mg/ml, digestion time of 20 minutes, a larger number and better purity of islet cells is available .PART TWOCulture and identify mouse bone marrow-derived immature dendritic cellsObjectives: Study on Murine bone marrow-derived immature dendritic cells culture method in vitro to facilitate further experimentsMethods: Take Babl/c mice bone marrow cells from hind legs, break red cell membrane and then cultured in vitro with cytokine GM-CSF and IL-4 with final concentration of rmGM-CSF 20 ng / ml, IL -4 10ng/ml., culture for 48 hours In Incubator Containing 5% CO2 and then remove the whole cell suspension, added with the same concentration of cytokines in complete medium to hold on culture, half of the medium was changed at the fifth day, anchorage-independent cell is collected at the seventh day . Which is measured on FACS after anti-mouse CD11c, MHC-Ⅱ, CD80 and CD86 antibody stainingResults: Cultured for 48 hours, many adherent cells began to grow; all medium was changed to remove suspended cells, the cells quantity decreased significantly. On the fifth day, a large number of cells multiply, and a lot of cells growing in colonies appeared. At Seventh day of harvest, the number of colonies decreased, while cells became loose, and many cells start to suspended growth, the cells in colony are not more than the fifth day; you can see a large number of cells around the burr under high magnification. Cells show high expression of CD11c, moderate expression of MHC-Ⅱ, low expression of CD80 and CD86 with Flow cytometry analysis, which CD11c-positive are more than 80%, MHC-Ⅱpositive are about 40% ~ 50%, CD80 and CD86 positive cells are less than 10% of the total. Which mean that the cell content is mainly dendritic cells with high purity of more than 80%, most of which are immature dendritic cells.Conclusions: Babl/c mouse bone marrow cells cultured for 7 days in vitro with medium in Concentrations of rmGM-CSF 20ng/ml and IL-4 10ng/ml of method can get more than 80% purity of dendritic cells, which are mostly immature dendritic cellsPART THREECulture and identification of donor antigen-specific Treg cellsObjectives: The methods of murine antigen-specific Treg cells in vitro, and identify whether the amplified antigen-specific cells.Methods: First, selected the CD4 + CD25 + T cells from C57 spleen cells using immunomagnetic beads; and then with Babl/c mouse bone marrow-derived immature DC 1:1 mixed culture; and to add high concentrations of mouse IL-2 (2000 U / ml), mixed culture of cells harvested after 7 days, take part of the cell flow analysis to detect CD4~+ CD25~+ Fop3~+ cells in the cells after mixed culture. Part of an ability to suppress T cell proliferation test, test divided into two groups: experimental group than in the C57 mice CFSE staining of CD4 + T cells, MMC-treated Babl / c mice, mature DC of Treg cells and amplified cells in mixed culture; third-party stimulating cells in the control group than in the C57 mice CFSE staining of CD4 + T cells, MMC-treated mice mature DC cells and amplified the Treg cells mixed culture. Judgments out whether the Treg cells have antigen-specific from the different amplification of T cells.Results: Using immunomagnetic beads cell sorting method can be divided purity over 90% of selected Treg cells; use of immature DC were significantly increased cell number, access to more than 60% purity of CD4 + CD25 + Fop3 + Treg cells. T cell proliferation and inhibition in the experimental group was amplified in the Treg cells suppression of T cells was stronger than the control group, indicating that the Treg cells was amplified with some specificity.Conclusions: Source of mouse donor immature DC cells can be greatly amplified in vitro with donor antigen-specific Treg cells in the presence of high concentration of IL-2. PART FOURInduction of islet transplantation tolerance in mice by donor antigen-specific Treg cellsObjectives: Study whether the antigen-specific Treg cells in vitro amplification can induce tolerance to islet transplantation in miceMethods: Produce the 1 type diabetic mice model by the method of one time intraperitoneal injection of STZ200mg/kg to C57 mice. And then create from Balb/c mice to C57 mice model of islet transplantation, blood glucose observed after transplantation the situation to determine the survival time of transplanted islet cells. Experimental groups: divided into three groups, the first group (no treatment control group): not to deal with, only blood glucose changes; the second group (pure islet transplantation group): for islet transplantation, not injected with Treg cells; the third group (experimental group): 1 day before surgery from the intravenous administration of from 1×106 donor-specific Treg cells, and then proceed to islet transplantationResults: Obtained 15 C57 mice by intraperitoneal injection of STZ 200mg/kg press the blood glucose above 16.7mmol / l standard has 12 modeling success into the model was 80%, mortality rate was 0. Islet transplantation, the no treatment control group the blood glucose of mice stability, no significant changes during the observation; pure islet transplantation group the blood glucose of mice full return to normal after islet transplantation 1 to 2 days, to 7 ~ 11 days began to increase gradually, and maintained at preoperative levels; experimental group the blood glucose of mice return to normal after islet transplantation 1 to 2 days, and the 5 mice blood glucose more than 16.7mmol/l in the 8,19,23,23,31 after islet transplantation.Conclusions: Donor antigen-specific Treg cells can significantly prolong the survival time of islet transplantation; and have a positive role in islet transplantation tolerance.