Simultaneous Blockade Of Inducible Co-stimulator And CD28 Pathway On Prevention Of Mice Islet Allografts Rejection

IntroductionIslet transplantation can provide physiological insulin for the patients who get diabetes mellitus. It has more merits than pancreatic transplantation, such as less traumatic occlusion , safety , easy to repeat and less complication. However, compared with pancreatic transplantation, there is a large difference in quantitatively and qualitatively . The key point to impede the development of islet transplantation is immunological rejection after transplantation. We prepare the model of diabetic mice , and transplant the islets of kunMing mice into the renal caps of C57 mice. Then allograft models are constructed successfully. C57 mice are transfected by pcDNA3 - CTLA4Ig through the right thigh before transplantation and injected with ICOSmAb on the day of 1,3 ,5 after islet transplantation. We observed whether CTLA4Ig could be expressed in the mice serum and the survival days of each group could be prolonged . These results provide proof - of -principle that costimulatory blockade CD28 and ICOS by transfecting plasmid of pcDNA3 - CTLA4Ig in conjunction with injection of ICOSmAb obviously prolonged survival days and reduce the allograft rejection .Material and MethodsKunming mice (BW30 ± 2 g) were used as donors of islets and C57 male mice were used as receptors. Diabetic mice were induced by streptozotocin (200mg/kg, STZ) , and diabetes was defined as the nonfasting blood glucose level was greater than 16. 8mmol/L on two consecutive measurement . The blood glucose level was measured by the meter.Islet Isolation: Islet were isolated and purified according to modified Minnedota program . After infusion of 2 - 4ml cold Hank' s balanced solution containing 1.5mg/ml of type V collagenase (C9263, Sigma) through cholecyst, pancreas were surgically procured and digested at 31X, for 10 - 15 min. During the digestion , the pancreas should be observed closely , and stopped digestion bu cold Hank' s solution when emulsion appeared. The islets were purified bu discontinuous Ficoll density gradient (25% ,23% ,20. 5% , 11% ) centrifugation [4X. ,2400rpm x lOmin) ,The distinct islets were collected and washed. The islets free of acinar cells, vessels, lymph nodes and duct were used for transplantation.Experiment one: The diabetic mice were divided into three groups in random ( n = 10) , named as : the control group (islets were transplanted into the capsule of the kidney alone);the blank control group (the receptors were firstly transfected by pcDNA3 plasmid, and then islets were transplanted into its'capsule of the kidney );the transfected group (the receptors were firstly transfected by pcDNA3 -CTLA4Ig plasmid, and then islets were transplanted into its'capsule of the kidney). The plasmid mixed with lipofectamine was injected into the right thigh of receptor mice,, and the level of CTLA4Ig in the serum was detected by the way of Western blot on the day 5 after being transfected. Blood glucose were detected after operation every other day. HE stain for kidney were used to evaluate the histology , and immunohistochemical stain of insulin - Ab were used to determine the expression of insulin . The test of T lymphocyte increment were performed to detect the ability of T lymphocyte increment.Experiment two;The diabetic mice were divided into four groups in random (n = 10) , named as : the control group (islets were transplanted into the capsule of the kidney alone);the first experiment group (receptor were injected with ICOSmAb at the dosage of 50ug/kg on the day 1X3 S5 after transplantation;the second experiment group (receptor were injected with ICOSmAb at the dosage of lOOug/kg on the day 1%3N5 after transplantation;the third experiment group (receptor were injected with ICOSmAb at the dosage of 200ug/kg on the day 1N3 %5 after transplantation. HE stain for kidney were used to evaluate the histology , and immunohistochemical stain of insulin - Ab were used to determinethe expression of insulin . The test of T lymphocyte increment were performed to detect the ability of T lymphocyte increment. The expression of ICOS molecule was detected by flow cytometry on the day 7 after transplantation.Experiment three: The diabetic mice were divided into three groups in random (n = 10) , named as : the transfected group (receptors were firstly trans-fected by pcDNA3 - CTLA4Ig plasmid, and then islets were transplanted into its 'capsule of the kidney);the ICOS group(receptor were injected with ICOSmAb at the dosage of lOOug/kg on the day 1 N3>5 after transplantation);the associated blockade group ( receptors were firstly transfected by pcDNA3 - CTLA4Ig plasmid, then islets were transplanted into its' capsule of the kidney, and were injected with ICOSmAb at the dosage of lOOug/kg on the day 1X3 N5 after transplantation ). The plasmid mixed with lipofectamine was injected into the right thigh of receptor mice, and the level of CTLA4Ig in the serum was detected by the way of Western blot on the day 5 after being transfected. Blood glucose were detected after operation ever)' other day. HE stain for kidney were used to evaluate the histology , and immunohistochemical stain of insulin - Ab were used to determine the expression of insulin . The test of T lymphocyte increment were performed to detect the ability of T lymphocyte increment.Data are expressed as mean and SD or SEM. The significant of difference was evaluated by q test of ANOVA. A p value <0.05 was considered statistical significant.ResultsExperiment one: CTLA4Ig was detected in the serum by the way of Western blot 5 days after being transfected. Eleven of the 40 C57 mice succeeded in being transfected, and the transfected rate was 27.5% . 100 - 150 IE islets were procured from donor mouse. The degree of purify was 85 -90%. Insulin secretion response to glucose challenge in vitro showed the mean value of insulin in the low - glucose medium was 191.4 ±99. l[xIU/m, while that of high - glucose medium was 882. 9 ± 178. 9|xIU/ml, SI = 4. 62 ± 0. 62, that means the grafts functioned well. The blood glucose level of the diabetic recipient restoredto normal 3 days after transplantation, and the median survival time of islet grafts was 19. 0 ± 1. 10d, which had significant different compared with the two other groups( p < 0. 05 ). But there didnl have significant different compared with each other of the latter Itwo groups (P > 0. 05). HE and immunohistochemi-cal stain showed that large amount of islet grafts were seen under the capsule of the kidney, which were positive stained by insulin - Ab. The similar mass were also observed in the two other groups ,but with few positive cells stained by insulin - Ab. The mean value of cpm of the transfected group was 177.219 ±3. 412min~ ,and those of the blank control group and the control group were 249. 664 ± 5. 937min~ and 250. 925 ± 3. 189min~. The mean value of cpm of the transfected group had significant different compared with the two other groups(p <0.05). But there didnt have significant different compared with each other of the later two groups (P > 0. 05).Experiment two: The blood glucose level of the diabetic recipient of the control group restored to normal 3 days after transplantation, then steped up soon . and the median survival time of islet grafts was 6. 0 ±0.16d. The median survival time of islet grafts of the first experiment group was 9. 0 ±1. 08d. Both the second and the third experiment group had longer median survival time than the latter two groups, which had significant different compared with the two oiher groups(p < 0. 05). But there didn't have significant different compared with each other ( P >0. 05). HE and immunohistochemical stain showed that large a-mount of islet grafts were seen under the capsule of the kidney in the two former groups, which were positive stained by insulin - Ab. The similar mass were also observed in the two latter groups , but with few positive cells stained by insulin -Ab. The mean values of cpm of the two former groups were significant different compared with the two other groups (p <0. 05) , but there didnt have significant different compared with each other s( P >0.05). The expression of ICOS molecule of the control group was up - regulation on the day 7 after .transplantation, but both the second experiment and the third experiment group didnt have the same change. That of the first experiment group was up - regulation to appropriate extent.Experiment three: The blood glucose level of the diabetic recipient of theassociated blockade group restored to normal 3 days after transplantation, and the median survival time of islet grafts was 31.0 ±6. 57d, which were significant different compared with the two other groups ( p <0. 05). But there didnt have significant different between the ICOS group and the pcDNA3 - CTAL4Ig trans-fected group (P >0. 05). HE and immunohistochemical stain showed that large amount of islet grafts were seen under the capsule of the kidney, which were positive stained by insulin - Ab. The similar mass were also observed in the two other groups, but with few positive cells stained by insulin - Ab. The mean value of epm of the associated blockade group was 170. 027 ± 1. 547min~ , and those of the ICOS group and the transfected group were 247. 054 ±4. 340min" and 250. 996 ±3. 537min~ respectively. The mean value of cpm of the associated blockade group had significant different compared with the two other groups ( p <0.05 ). But there didnt have significant different compared with each other of the later two groups (P > 0. 05).Conclusion1 The mouse CTLA4 gene could be transferred into mouse muscle and express its products in the serum by cation liposome.2 In situ infusion, collagenase digestion, purify by Ficoll gradient centrifu-gation are the effective methods for islets isolation.3 The rejection of islet grafts could be inhibited significant by blockade with the way of CD28/B7.4 The expression of the molecule of ICOS is up - regulation on T lymphocytes when rejection happens.5 The rejection of islet grafts could be inhibited significant with ICOSmAb by blockade with the way of ICOS/ICOSL.6 The best dosage of ICOSmAb is 100fxg/kg.7 The rejection of islet grafts could be significant inhibited by costimulatory blockade with the way of CD28/B7 and ICOS/ICOSL, which is the better way than that of single blockade.