| IntroductionIslet transplantation offers a physiological approach for precise restoration of glucose homeostasis, thereby reversing the metabolic and neurovascular complication of diabetes. One of the main obstacles to islet transplantation is the rejection that leads to islet allograft injure. To overcome them, novel immunosup-pressive targets and strategies need to be defined. Chemokines are important regulators in the development, differentiation, and anatomic location of leucocytes. CC chemokine receptor 5 ( CCR5 ) and CXC chemokine receptor 3 ( CXCR3 ) are receptors for the proinflammatory chemokines and paly an important role on allograft rejection. Synthetic molecules that can bind with high sequence specificity to a chosen target in a gene sequence are of major interest in medicinal and biotechnological contexts. They show promise for the development of gene therapeutic agents. Peptide nucleic acid (PNA) is a nucleic acid analog in which the sugar phosphate;backbone of natural nucleic acid has been replaced by a synthetic peptide backbone usually formed from N - (2 - amino - ethyl) ,-glycine units, resulting in an achiral and uncharged mimic. Studies indicate that PNA could inhibit both transcription and translation of genes to which it has been targeted, which holds promise for its use for antigene and antisense therapy. In this study we investigated the effects of targeting CCR5 and CXCR3 using antisense PNA on islet allograft survival in murine recipients rendered diabetic using streptozotocin.Materials and MethodsAnimals : Kunming mice as donors and C57 mice as recipients were obtained from Animal Center of Shenyang Pharmacon University. Adult mice of ( 30 ± 2 ) g were used throughout this study. The recipients were rendered diabetic by a single intraperitoneal injection of 150mg/kg streptozotocin ( STZ) and considered diabetic when the tail vein blood glucose concentration was more than 16. 8mmol/L for 3 consecutive days.Islet isolation and transplantation : Mice were anesthetized with ether. After intraductal injection of 2 to 4ml of cold Hank' s balanced salt solution containing 1. 5mg/ml of collagenase V(Sigma, USA) , pancreata were surgically procured and digested at 371 for 12 to 15min. Islets were washed with Hank' s balanced salt solution and then purified by four - layer discontinuous gradient centrifugation using Ficoll (25% , 23% , 20. 5% and 11% ) and hand picked under an inverted microscope. For each transplantation procedure, recipients were anesthetized with introperitoneal butaylone. A lateral flank incision was made above the right kidney, and islets were inserted under the kidney capsule at the anterior surface of the kidney through a polypropylene tube. Three hundreds islets were used in each transplantation.Experiment one: The recipient mice of islet transplantation were divided into four groups in random(n = 10) , named as : the saline control group;PNA CCR5 group;PNA CXCR3 group and PNA mismatch group. The sequences of PNA antisense were as follows: PNA CCR5 antisense, 434(5'-Tyr -CCGTTCT-GACTTTT - Lys - 3 ')42i;PNA CXCR3 antisense, 884( 5 ' - Tyr - ACGTG-GCTTTTTCG - Lys - 3 0?;PNA mismatch sequence, Tyr - TTTCCAGCT-GCTTT - Lys ( randomly sanitized). Mice received 2. 5mg/kg daily of three types of PNA antisense, via i. v. injections for 7 days, with the first injection on the day of the transplantation. Tail vein blood glucose was monitored to assess islet graft function. Rejection was defined when nonfasting blood glucose levels were higher than 11. 2mmol/L on two consecutive days. After transplantation, we harvested islet grafts, spleen and blood of five recipients per group at seventhday or rejection. Extracting the total RNA of islet allograft, mRNA levels of CCR5, CXCR3 and their ligands were analyzed. CCR5 and CXCR3 protein levels were evaluated by Western Blotting. At the same time, mRNA levels of CCR5, CXCR3 of native mice were detected. Spleen cell suspensions from recipient mice were preparecl. We used flow cytometry for detection of CCR5 or CXCR3 on T lymphocytes. In vitro T cells proliferative capability were assessed by lymphocyte proliferative reaction of mitogen stimulation. HE and immunohis-tochemical stain of Insulin for islet allografts were used to evaluate the histology.Experiment two: The recipient mice were divided into three group: the control group: the mice received saline via i. v injection;the PNA CCR5 group: the mice received 2. 5mg/kg daily of PNA CCR5, via i. v. injections for 7 days, with the first injection on the day of the transplantation;the combination treatment group: the mice received PNA CCR5 and rapamycin by oral for 7 consecutive days, starting the day of transplantation at doses of 0. 2mg/kg per day. After transplantation, we harvested islet grafts, spleen and blood of five recipients per group at seventh day or rejection. Extracting the total RNA of islet allograft, mRNA levels of IL -2, IFN - 7 and IL - 10 were analyzed. CCR5 protein levels were evaluated by Western Blotting. In vitro T cells proliferative capability were assessed by lymphocyte proliferative reaction of mitogen stimulation. HE and immunohistochemical stain of Insulin for islet allografts were used to evaluate the histology.Data were analyzed by SPSS 13.0 software. Results are expressed as mean ± SD. Mean survival times were calculated and Kaplan - Meier survival graphs constructed. The log - rank comparisons of groups were used to calculate P values. For multiple comparisons, a One - Way ANOVA was used. In both cases, P<0.05 was considered significant.ResultsExperiment one: The blood glucose level of isolated islet transplantation mice restored to normal 3 days after transplantation. The median survival time (MST) of islet allograft for saline and PNA mismatch - treated was (6. 50 ±0.58) days and (6.50±0.50) days versus (12.00 ±1.75) days(P<0.01) and (9.70 ± 1.00) days( P <0.05) for PNA CCR5 - and PNA CXCR3 - treated recipients, respectively. PNA CCR5 - and PNA CXCR3 - treated recipients demonstrated statistically significant prolongation in functional allograft survival when compared with saline or PNA mismatch - treated recipients. The mR-NA levels of CCR5 and CXCR3 markedly increased at the time of acute rejection compared to native mice. The mRNA expression of CCR5, CXCR3 at day 7 after transplantation were significantly up - regulated in saline - and PNA mismatch - treated islet allografts compared to native mice(P <0.01 and P <0.01, respectively ). Among them, the expression of CCR5 was significantly downregulated by PNA CCR5 treatment compared with saline - and PNA mismatch -treatment allografts ( P < 0. 01 ) , the expression of CXCR3 was significantly downregulated by PNA CXCR3 treatment( P < 0. 01). Wheras their ligands were not significantly suppressed. Densitometric semi - quantitative date of Western Blotting showed that the level of CCR5 protein markedly decreased in PNA CCR5 treatment allografts compared with saline and PNA mismatch treatment allografts (all P <0. 01) , the level of CXCR3 protein decreased in PNA CXCR3 treatment allografts compared with saline and PNA mismatch treatment allografts (all P <0. 01). The PNA CXCR3 significantly blocked the expression of CX-CR3 in spleen CD3 + T cells(0. 39 ±0. 05 ) , wheras there was a considerable number of CXCR3 + fractions in spleen T cells from mice treated with normal physiological saline (0. 66 ±0. 03) and with mismatched PNA(0. 70 ±0. 03). CCR5 was also lessly expressed in CD3 + spleen T cells from mice treated with PNA CCR5 ( 0. 32 ±0.03) , whereas there were 0. 81 ±0.03 and 0. 74 ±0.04 of CCR5 + cell fractions from mice treated with normal physiological saline and with mismatched PNA, respectively. Lymphocytes from PNA CCR5 and PNA CXCR3 treatment mice exhibited similar degree of proliferation comparable to that of saline and PNA mismatch treatment mice( P > 0. 05, P > 0. 05, respectively). The mean cpm of PNA CCR5, PNA CXCR3, saline, and PNA mismatch treatment recipients was 200.12 ± 12.08, 207.66 ± 14.98, 228.6 ± 12. 51 and 206. 28 ± 16. 88, respectively. By day 7 after transplantation, massive cellular infiltration around islet allografts were observed in saline - and PNAmismatch - treated mice. By contrast, less cellular infiltration were identified in PNA CCR5 - and PNA CXCR3 - treated mice.Experiment two: The median survival time(MST) of islet allograft for saline, PNA CCR5, PNA CCR5 and low - dose rapamycin treated was (6. 50 ± 0.58) days, (12.00 ± 1. 75 ) days and (16.20 ± 3.13 ) days respectively. The MST of combination treatment allografts is longer than that of PNA CCR5 treatment allograft(P <0.05). At day 7 after transplantation, the mRNA expression of IFN - -y of combination treatment allografts was significantly down - regulated compared to saline -treated allografts (P <0. 05). Compared with saline controls , allografts from PNA CCR5 treatment mice exhibited a decrease in the mRNA expression of IL-2(9. 73 ±0. 62, P<0.01), which was accentuated by the use of rapamycin (6. 86 ±0. 59, P < 0. 01 ). Similarly, PNA CCR5 increased the mRNA expression of IL - 10 compared with saline controls ( P < 0.05) , and this effect was accentuated by rapamycin(P <0. 01). The mitogen - induced T cell proliferation was significantly down - regulated in PNA CCR5 and low - dose rapamycin treatment recipients compared with saline and PNA CCR5 treatment recipients. Histologically, the combination applying of PNA CCR5 and low - dose rapamycin further decreased the lymphocytes infiltration in islet allografts.Conclusion1. PNA CCR5 antisense and PNA CXCR3 antisense can significantly and specifically down - regulated the mRNA expression and the protein production of CCR5 and CXCR3 of islet allografts.2. PNA CCR5 antisense and PNA CXCR3 antisense can decreased the CCR5 and CXCR3 expression in CD3 + T cells, but not influence the proliferate capability.3. Both PNA CCR5 and PNA CXCR3 can prolong the survival time of islet allografts. The survival time of PNA CCR5 - treated mice is longer than that of PNA CXCR3 - treated mice.4. Rapamycin can not only inhibit activation of T cells, but also decreasethe proliferate capability of T cells.5. Rapamycin can accentuate the role of PNA CCR5. The combination applying of PNA CCR5 and low - dose rapamycin further alleviate acute rejection and prolong the survival time of islet allografts. |
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