口服RDP58肽诱导的Treg源性exosomes抑制大鼠肾移植排斥的研究

发布时间：2018-01-06 11:46

本文关键词：口服RDP58肽诱导的Treg源性exosomes抑制大鼠肾移植排斥的研究　出处：《第三军医大学》2012年博士论文　论文类型：学位论文

更多相关文章： RDP58 CD4+CD25+Treg exosome 肾移植

【摘要】：背景和目的排斥反应是导致移植后器官功能丧失的最主要原因，是移植物长期存活的主要障碍。探寻安全有效的抗原特异性免疫抑制方法，以实现移植物长期存活是器官移植领域最重要的任务之一，尽管近几年各种免疫抑制剂的合理使用，有效的降低了器官排斥，增加了器官移植的成功率，对器官移植患者的长期存活和移植物保持良好功能都起着重要作用，但免疫抑制剂同时有自身不可克服的缺陷—抑制机体正常免疫功能。口服耐受是机体免疫系统对肠道接触的大量食物抗原和各种微生物形成的不反应状态，是后天条件下机体维持对必须进入机体的外来抗原(食物抗原，肠道共生菌抗原)耐受的后天机制。其中，以肠系膜淋巴结为主的肠道淋巴系统中的调节性T细胞(regulatory T cells, Treg)在上述耐受形成中起着至关重要的作用。体内CD4+CD25+Treg是一群具有重要免疫调节功能的细胞，对效应性T细胞的活化和增殖起负调控作用；同时在器官移植后促使机体内免疫平衡向耐受状态发展并维持耐受状态中起着极其重要的作用。有效利用机体自身CD4+CD25+Treg调节移植术后异体移植物与受者间免疫功能调节能避免免疫抑制剂带来的副作用。我们知道，exosome是多种活细胞晚期内体以胞吐的方式分泌到细胞外形成，且不同细胞来源的exosome具有不同免疫功能，例如DC来源的exosome和B细胞来源的exosome富含MHC II分子和共刺激分子(CD86、CD80)，是T细胞活化的必需共刺激分子；人类鼻咽癌来源的exosome含latent membrane protein gene1(LMP1)，能抑制T细胞活化效应；某些肿瘤来源的exosome能异常地表达Fas ligand (FasL)，诱导活化的细胞死亡(activated induced cell death，，AICD)。现已证实B细胞，T细胞，树突状细胞(DendriticCells，DC细胞，肥大细胞和多种肿瘤细胞等均能分泌exosome。那么CD4+CD25+Treg细胞是否也能分泌exosome，并通过其进行远端调控？为证实这一设想，我们通过口服RDP58肽与霍乱毒素B型亚单位(cholera toxin B subunit，CTB)的交联物诱导肠内CD4+CD25+Treg细胞，并收集到CD4+CD25+Treg源性exosome。材料和方法本实验旨在收集口服RDP58与CTB交联后的产物诱导的特异性CD4+CD25+Treg细胞分泌的exosome，并建立大鼠急性肾移植模型后，过继转移入CD4+CD25+Treg细胞源性exosome，体内观察exosome对移植肾存活时间、肾功能及移植肾病理组织学变化的影响；体外通过混合淋巴细胞培养，观察其对T淋巴细胞增殖的影响。最后通过SDS-Page和质谱系统(High Performance Liquid Chromatography-CHIP-MassSpectrometer, HPLC-CHIP-MS)对CD4+CD25+Treg源性exosome囊泡内所含蛋白的组成进行检测与分析： (1)人工合成RDP58肽，并完成其分子量及纯度检测； (2)用交联剂SPDP交联RDP58衍生肽与CTB，用HPLC纯化后并进行检测； (3)建立大鼠肾移植模型，观察RDP58-CTB交联物口服后，对异体肾移植排斥的抑制效果，从而验证RDP58肽结构被衍生后，其功能学的变化； (4)口服灌胃RDP58-CTB交联物，诱导肠系膜特异性CD4+CD25+Treg细胞； (5)收集肠系膜淋巴结，流式分选收集CD4+CD25+Treg细胞，培养1-2周后，取上清液超速离心和密度梯度离心法收集exosome； (6)建立大鼠肾移植模型，过继转移入CD4+CD25+Treg源性exosome，并观察其在体内及体外的生物学功能； (7)Exosome蛋白质谱分析：SDS-Page法和HPLC-CHIP-MS法来观察exosome囊泡内蛋白数量和种类。结果 1、人工合成RDP58肽和衍生RDP58肽，纯度均达95%以上，并用交联剂SPDP，完成RDP58衍生肽与CTB的交联； 2、用prolene滑线作内支架法建立大鼠肾移植模型，可有效提高手术成功率和减少缺血时间； 3、口服交联产物RDP58-CTB后能有效提高机体HO-1活性和抑制T淋巴细胞增殖，相对单独应用RDP58而言能显著改善移植肾存活时间和病理组织变化，并达到与腹腔注射RDP58相同的治疗效果； 4、超滤法和密度梯度法收集到CD4+CD25+Treg细胞源性exosomes，电镜下可见其为30-100nm均匀圆形小体,密度为1.13to1.21g/ml (漂浮在30%蔗糖/D2O中)； 5、过继转移入CD4+CD25+Treg细胞源性exosomes后，体内实验有效延长移植肾存活时间、改善移植肾肌酐和尿素氮水平和组织学病理变化；体外能抑制T淋巴细胞增殖； 6、用SDS-polyacrylamide gel (SDP-Page)电泳初步观察Exosome中蛋白组成数量，并进一步行蛋白质谱分析，证实有数十种蛋白组成，包括cap adenylylcyclase-associated protein1、ldha L-lactate dehydrogenase A chain等蛋白。结论 1、 RDP58肽与CTB交联后，能有效提高抗原免疫原性，在体内提高HO-1活性，延长移植肾存活时间；其治疗效果与腹腔注射相同，但口服途径诱导耐受有无可比拟的安全性和方便性； 2、 CD4+CD25+Treg细胞能分泌exosomes，且exosomes过继转移入大鼠急性肾排斥模型后，能明显改善移植肾存活时间和病理组织变化，这可能是CD4+CD25+Treg细胞实现远端调控的重要机制之一； 3、 Exosome经SDS-PAGE和蛋白质谱分析，证实是由十余种蛋白组成的的囊泡状结构。
[Abstract]:Background and purpose
Rejection is the main cause of the loss of organ function after transplantation, is a major obstacle to long-term graft survival. To explore the immune antigen specific safe and effective suppression methods, to achieve the long-term graft survival is one of the most important tasks in the field of organ transplantation, although in recent years the rational use of immunosuppressive agents, effectively reduce the the organ rejection, increase the success rate of organ transplantation, plays an important role to maintain good function of long-term survival in organ transplantation and graft, but also have immunosuppressive insurmountable defects and inhibit the normal immune function.
Oral tolerance is the immune system of the intestinal contact to form a large number of food antigens and various microbial reaction state is acquired under the condition of the body to maintain foreign antigens must enter the body (food antigens and commensal antigens) acquired tolerance mechanism. Among them, the mesenteric lymph nodes of the intestinal lymphatic system mainly in the regulation of T cells (regulatory T cells, Treg) in the development of tolerance plays a crucial role.
The body is a group of CD4+CD25+Treg has an important immunoregulatory function of cells, play a role in negative regulation of effector T cell activation and proliferation; at the same time after organ transplantation to body immune tolerance to the balanced development and plays an important role in maintaining tolerance. Effective use of the body after transplantation of allogeneic CD4+CD25+Treg regulation the relationship between plants and regulating immune function can avoid the side effects of immunosuppressive drugs bring. As we know, exosome is a variety of living cells to late endosomes in exocytosis way secreted into the form, and different sources have different exosome cell immune function, such as exosome II DC MHC rich molecular sources of exosome and B cells and costimulatory molecules (CD86, CD80), is required for activation of T cell costimulatory molecule; human nasopharyngeal carcinoma derived exosome membrane protein gene1 LM (containing latent P1), can inhibit T cell activation effect; some sources of tumor exosome aberrant expression of Fas ligand (FasL), induced cell death (activated induced, cell death, AICD). It has been confirmed that B cells, T cells, dendritic cells (DendriticCells, DC cells, mast cells and tumor cells were so whether the CD4+CD25+Treg cells secrete exosome. can secrete exosome, and through the remote control? To confirm this idea, we through the oral administration of RDP58 peptide and cholera toxin B subunit (cholera toxin B subunit, CTB) of CD4+CD25+Treg cells induced by intestinal crosslinking, and the collection of CD4+CD25+Treg source exosome.
Materials and methods
The purpose of this experiment was to induce secretion of oral RDP58 and CTB products collected after crosslinking of CD4+CD25+Treg cells specific for exosome, and the establishment of rat renal transplantation model after adoptive transfer into CD4+CD25+Treg cells derived exosome, exosome observed in vivo survival time of transplanted kidney, renal function and renal pathological changes by mixed lymphocyte in vitro; culture, to observe its effects on T lymphocyte proliferation. Finally, using SDS-Page and mass spectrometry system (High Performance Liquid Chromatography-CHIP-MassSpectrometer, HPLC-CHIP-MS) detection and analysis of CD4+ CD25+Treg derived exosome protein containing vesicles of capsule:
(1) synthetic RDP58 peptide was synthesized and its molecular weight and purity were detected.
(2) cross linker SPDP cross-linked RDP58 derived peptide and CTB, purified and detected by HPLC;
(3) to establish a rat kidney transplantation model, observe the inhibitory effect of RDP58-CTB crosslinking on allograft rejection after oral administration, so as to verify the functional changes of RDP58 peptide structure after being derived.
(4) intragastric RDP58-CTB crosslinking was administered orally to induce mesenteric specific CD4+CD25+Treg cells.
(5) the mesenteric lymph nodes were collected and CD4+CD25+Treg cells were collected by flow sorting. After 1-2 weeks of culture, the supernatant centrifugation and density gradient centrifugation were collected to collect exosome.
(6) a rat model of kidney transplantation was established and transferred into CD4+CD25+Treg derived exosome, and its biological function in vivo and in vitro was observed.
(7) Exosome mass spectrometric analysis: SDS-Page and HPLC-CHIP-MS methods were used to observe the number and type of protein in the exosome vesicles.
Result
1, RDP58 peptide and derived RDP58 peptide were synthesized artificially, with the purity of more than 95%, and the crosslinking agent SPDP was used to complete the cross-linking of RDP58 derived peptide and CTB.
2, the model of rat kidney transplantation was established by using the Prolene skid line as the internal stent, which could effectively improve the success rate and reduce the time of ischemia.
3, oral crosslinking product RDP58-CTB can effectively improve the HO-1 activity and inhibit the proliferation of T lymphocytes. Compared with RDP58 alone, it can significantly improve the survival time and pathological changes of transplanted kidney, and achieve the same therapeutic effect as intraperitoneal injection of RDP58.
4, CD4+CD25+Treg cell-derived exosomes was collected by ultrafiltration and density gradient method. Under electron microscope, it was 30-100nm homogeneous round body with a density of 1.13to1.21g/ml (floating in 30% sucrose /D2O).
5, after adoptive transfer into CD4+CD25+Treg cell-derived exosomes, in vivo experiments effectively prolong the survival time of renal allograft, improve the level of renal creatinine and urea nitrogen and histopathological changes, and inhibit the proliferation of T lymphocytes in vitro.
6, we used SDS-polyacrylamide gel (SDP-Page) electrophoresis to preliminarily observe the number of protein components in Exosome, and further carry out protein mass spectrometry analysis, which confirmed that there are ten protein components, including cap adenylylcyclase-associated protein1, ldhA L-lactate dehydrogenase A, and other proteins.
conclusion
1, the cross-linking of RDP58 peptide and CTB can effectively improve the immunogenicity of antigen, increase HO-1 activity and prolong survival time in vivo. The therapeutic effect is the same as that of intraperitoneal injection, but the tolerance induced by oral route has unparalleled safety and convenience.
2, CD4+CD25+Treg cells can secrete exosomes, and exosomes can significantly improve the survival time and pathological changes of transplanted kidney after the adoptive transfer of exosomes into the rat acute kidney rejection model. This may be one of the important mechanisms for the distal regulation of CD4+CD25+Treg cells.
3, Exosome was analyzed by SDS-PAGE and protein mass spectrometry and proved to be a vesicular structure consisting of more than ten proteins.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

【引证文献】