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TCS protein and IL-12p40 gene in mouse pancreatic islet allografts rejection and mechanism of action

Author: ShiWei
Tutor: CaiDuan
School: Fudan University
Course: Surgery
Keywords: islets of Langerhans transplantation trichosanthin regulatory T cell IL-12p40
CLC: R657.5
Type: PhD thesis
Year: 2008
Downloads: 390
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Abstract


Diabetes mellitus is one of the common chronic diseases with a lot of complications threatening health and life of patients.Although the application of insulin had saved lives of many diabetes patients,hypoglycemia is one of its adverse effects and complications of diabetes mellitus can not be controlled thoroughly.Islet transplantation is a new hopeful therapeutic measure in instead of insulin injection. Patients who accepted islet transplantation could get rid of insulin in some extent.But islet allograft could not be functional constantly,rejection toward allograft is the main reason.This study was interested in mechanisms of allograft rejection in islet transplantation.In order to prolong allograft survival of islet transplantation,we applied immunosuppressive drugs to recipient to prevent the rejection.We established the model of streptozotocin induced diabetes mouse on the first step and then allogenic islets were transplanted beneath the capsule of the left kidney.We modified the method of pancreas digestion and islet purification based on previous studies.Then we studied the allo-transplantantion of islet from BALB/c mice to C57BL/6 mice.Trichosanthin was injected intraperitoneally to affect the immune response of recipient mice.Influence to function of islet and immunosuppressive effect was assessed and its mechanisms were investigated.In purpose of comprehend the role of IL-12p40 in allograft rejection of islet transplantation,we compared the immune rejections in recipients of IL-12p40 knock-out mice and wild type C57BL/6 mice.PartⅠEstablishment of islet transplantation model in mouseObjective:To obtain a convenient and high performance method of harvesting mouse islet,and establish a stable model of islet transplantation in diabetes mouse.Methods:(1) Mouse pancreas was digested by collagenase P which was perfused by means of a gallbladder puncture.We observed the result of digestion and difficulty of operation. We compared the results of Ficoll density gradient centrifugation and filtration in islet purification(n=6 in each group).(2) We also performed islet transplantation in streptozotocin induced diabetes mice to observe the stability of diabetes model and correction of hyperglycemia after transplantation. Results:(1) The method of collagenase P perfusion by gallbladder puncture in pancreas digestion is easy and high performance.Filtration is better than Ficoll density gradient centrifugation in islet purification for quantity and purity of islets were elevated (P<0.05),operating time was saved significantly(P<0.01).Insulin release test showed that islets achieved by filtration released more insulin than Ficoll density gradient centrifugation(P<0.05).(2) Hyperglycemia(BG>16.7mmol/L) was induced by streptozotocin injection within 48 hours and it was corrected within 24 hours after islet transplantation(n=10) until rejection happened.Conclusions:The method of collagenase P perfusion by gallbladder puncture is convenient and high performance in obtaining mouse islet.The animal model of islet transplantation in streptozotocin induced diabetes mouse is stable and reliable.PartⅡApplieation of trichosanthin in mouse islet allotransplantationObjective:To understand the inhibition effect of trichosanthin(TCS) to allograft rejection in mouse islet transplantation and investigate its possible mechanisms.To initially find the affect of TCS upon the islet function.Methods:(1) Control group(n=6) and TCS injection group(n=6) were set in mice with BALB/c to C57BL/6 islet transplantation.Blood glucose was tested in both groups daily to obtain the survival time of allografts.(2) Control group,TCS injection group, islet transplant group and transplant plus TCS group(n=5 in each group) were set.In all groups,T cell proliferation rate,IFN-γsecreting level and T cell ratio in peripheral lymph organs were detected to investigate mechanisms of trichosanthin in inhibiting rejection.(3) In vivo,we detected C peptide level secreted by islet graft before and after trichosanthin injection.In vitro,insulin release test was applied after islet culture with trichosanthin to comprehend whether trichosanthin could affect islet function.Results:(1) The mean survival time of islet allograft in TCS group was 12.2±1.7 days,and it was 9.7±1.4 days in control group,survival time was prolonged after trichosanthin injection(P<0.05).(2) Donor antigen specific T cell proliferation rate and IFN-γ secreting level were depressed and regulatory T cell ratio in peripheral lymph organs was elevated after trichosanthin injection.(3) In vivo,C peptide levels were similar before and after trichosanthin injection.In vitro,insulin secreting level of islet was depressed after cultivation with trichosanthin(P<0.05).Conclusions:Trichosanthin could inhibit allograft rejection in mouse islet transplantation. Inhibition of antigen specific T cell proliferation and up-regulation of regulatory T cell are possible mechanisms.Thrichosanthin could inhibit function of mouse islet when contact directly.PartⅢStudying the role of IL-12p40 in allograft rejection of islet transplantationObjective:To comprehend the role of IL-12p40 gene in allograft rejection in mouse islet transplantation and investigate whether IL-12p40 gene deficiency induced Thl to Th2 immune deviation could lead to immune tolerance to islet allotransplantation.Methods:The survival time of islet allografts,infiltration of CD4~+ and CD8~+ T cell and expression levels of IL-4 and IFN-γin graft region were observed and compared between IL-12p40 knock-out(n=7) and wild type(n=6) recipients.Results:Mean survival time of islet allograft in recipients of IL-12p40 knock-out mice was 10.0±2.9 days,and it was 9.7±2.1 days in wild type recipients,there’s no difference between them(P>0.05).Infiltration of CD4~+ and CD8~+ T cell and expression levels of IL-4 and IFN-γin recipients of IL- 12p40 knock-out mice were similar to those in wild type recipients.Conclusions:Th1 to Th2 immune deviation existing in IL-12p40 deficient mouse recipient could not inhibit allograft rejection in islet transplantation and could not induce immune tolerance either.We studied mechanisms and prevention methods of allograft rejection in mouse islet transplantation by means of drug intervention and gene knock-out.Initially applied trichosanthin which is component of Chinese medicine to inhibit allograft rejection in mouse islet transplantation and revealed its possible mechanisms.We also make it clear that IL-12p40 gene deficiency is not sufficient to induce tolerance to islet allograft.Our study is helpful for researchers to understand immune rejection and tolerance in islet transplantation,and it’s positive to apply and investigate trichosanthin or other component of Chinese medicine in transplant area.

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