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  本文關(guān)鍵詞：口服RDP58肽誘導(dǎo)的Treg源性exosomes抑制大鼠腎移植排斥的研究　出處：《第三軍醫(yī)大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： RDP58 CD4+CD25+Treg exosome 腎移植

【摘要】：背景和目的 排斥反應(yīng)是導(dǎo)致移植后器官功能喪失的最主要原因，是移植物長(zhǎng)期存活的主要障礙。探尋安全有效的抗原特異性免疫抑制方法，以實(shí)現(xiàn)移植物長(zhǎng)期存活是器官移植領(lǐng)域最重要的任務(wù)之一，盡管近幾年各種免疫抑制劑的合理使用，有效的降低了器官排斥，增加了器官移植的成功率，對(duì)器官移植患者的長(zhǎng)期存活和移植物保持良好功能都起著重要作用，但免疫抑制劑同時(shí)有自身不可克服的缺陷—抑制機(jī)體正常免疫功能。 口服耐受是機(jī)體免疫系統(tǒng)對(duì)腸道接觸的大量食物抗原和各種微生物形成的不反應(yīng)狀態(tài)，是后天條件下機(jī)體維持對(duì)必須進(jìn)入機(jī)體的外來(lái)抗原(食物抗原，腸道共生菌抗原)耐受的后天機(jī)制。其中，以腸系膜淋巴結(jié)為主的腸道淋巴系統(tǒng)中的調(diào)節(jié)性T細(xì)胞(regulatory T cells, Treg)在上述耐受形成中起著至關(guān)重要的作用。 體內(nèi)CD4+CD25+Treg是一群具有重要免疫調(diào)節(jié)功能的細(xì)胞，對(duì)效應(yīng)性T細(xì)胞的活化和增殖起負(fù)調(diào)控作用；同時(shí)在器官移植后促使機(jī)體內(nèi)免疫平衡向耐受狀態(tài)發(fā)展并維持耐受狀態(tài)中起著極其重要的作用。有效利用機(jī)體自身CD4+CD25+Treg調(diào)節(jié)移植術(shù)后異體移植物與受者間免疫功能調(diào)節(jié)能避免免疫抑制劑帶來(lái)的副作用。我們知道，exosome是多種活細(xì)胞晚期內(nèi)體以胞吐的方式分泌到細(xì)胞外形成，且不同細(xì)胞來(lái)源的exosome具有不同免疫功能，例如DC來(lái)源的exosome和B細(xì)胞來(lái)源的exosome富含MHC II分子和共刺激分子(CD86、CD80)，是T細(xì)胞活化的必需共刺激分子；人類鼻咽癌來(lái)源的exosome含latent membrane protein gene1(LMP1)，能抑制T細(xì)胞活化效應(yīng)；某些腫瘤來(lái)源的exosome能異常地表達(dá)Fas ligand (FasL)，誘導(dǎo)活化的細(xì)胞死亡(activated induced cell death，，AICD)�，F(xiàn)已證實(shí)B細(xì)胞，T細(xì)胞，樹(shù)突狀細(xì)胞(DendriticCells，DC細(xì)胞，肥大細(xì)胞和多種腫瘤細(xì)胞等均能分泌exosome。那么CD4+CD25+Treg細(xì)胞是否也能分泌exosome，并通過(guò)其進(jìn)行遠(yuǎn)端調(diào)控？為證實(shí)這一設(shè)想，我們通過(guò)口服RDP58肽與霍亂毒素B型亞單位(cholera toxin B subunit，CTB)的交聯(lián)物誘導(dǎo)腸內(nèi)CD4+CD25+Treg細(xì)胞，并收集到CD4+CD25+Treg源性exosome。 材料和方法 本實(shí)驗(yàn)旨在收集口服RDP58與CTB交聯(lián)后的產(chǎn)物誘導(dǎo)的特異性CD4+CD25+Treg細(xì)胞分泌的exosome，并建立大鼠急性腎移植模型后，過(guò)繼轉(zhuǎn)移入CD4+CD25+Treg細(xì)胞源性exosome，體內(nèi)觀察exosome對(duì)移植腎存活時(shí)間、腎功能及移植腎病理組織學(xué)變化的影響；體外通過(guò)混合淋巴細(xì)胞培養(yǎng)，觀察其對(duì)T淋巴細(xì)胞增殖的影響。最后通過(guò)SDS-Page和質(zhì)譜系統(tǒng)(High Performance Liquid Chromatography-CHIP-MassSpectrometer, HPLC-CHIP-MS)對(duì)CD4+CD25+Treg源性exosome囊泡內(nèi)所含蛋白的組成進(jìn)行檢測(cè)與分析： (1)人工合成RDP58肽，并完成其分子量及純度檢測(cè)； (2)用交聯(lián)劑SPDP交聯(lián)RDP58衍生肽與CTB，用HPLC純化后并進(jìn)行檢測(cè)； (3)建立大鼠腎移植模型，觀察RDP58-CTB交聯(lián)物口服后，對(duì)異體腎移植排斥的抑制效果，從而驗(yàn)證RDP58肽結(jié)構(gòu)被衍生后，其功能學(xué)的變化； (4)口服灌胃RDP58-CTB交聯(lián)物，誘導(dǎo)腸系膜特異性CD4+CD25+Treg細(xì)胞； (5)收集腸系膜淋巴結(jié)，流式分選收集CD4+CD25+Treg細(xì)胞，培養(yǎng)1-2周后，取上清液超速離心和密度梯度離心法收集exosome； (6)建立大鼠腎移植模型，過(guò)繼轉(zhuǎn)移入CD4+CD25+Treg源性exosome，并觀察其在體內(nèi)及體外的生物學(xué)功能； (7)Exosome蛋白質(zhì)譜分析：SDS-Page法和HPLC-CHIP-MS法來(lái)觀察exosome囊泡內(nèi)蛋白數(shù)量和種類。 結(jié)果 1、人工合成RDP58肽和衍生RDP58肽，純度均達(dá)95%以上，并用交聯(lián)劑SPDP，完成RDP58衍生肽與CTB的交聯(lián)； 2、用prolene滑線作內(nèi)支架法建立大鼠腎移植模型，可有效提高手術(shù)成功率和減少缺血時(shí)間； 3、口服交聯(lián)產(chǎn)物RDP58-CTB后能有效提高機(jī)體HO-1活性和抑制T淋巴細(xì)胞增殖，相對(duì)單獨(dú)應(yīng)用RDP58而言能顯著改善移植腎存活時(shí)間和病理組織變化，并達(dá)到與腹腔注射RDP58相同的治療效果； 4、超濾法和密度梯度法收集到CD4+CD25+Treg細(xì)胞源性exosomes，電鏡下可見(jiàn)其為30-100nm均勻圓形小體,密度為1.13to1.21g/ml (漂浮在30%蔗糖/D2O中)； 5、過(guò)繼轉(zhuǎn)移入CD4+CD25+Treg細(xì)胞源性exosomes后，體內(nèi)實(shí)驗(yàn)有效延長(zhǎng)移植腎存活時(shí)間、改善移植腎肌酐和尿素氮水平和組織學(xué)病理變化；體外能抑制T淋巴細(xì)胞增殖； 6、用SDS-polyacrylamide gel (SDP-Page)電泳初步觀察Exosome中蛋白組成數(shù)量，并進(jìn)一步行蛋白質(zhì)譜分析，證實(shí)有數(shù)十種蛋白組成，包括cap adenylylcyclase-associated protein1、ldha L-lactate dehydrogenase A chain等蛋白。 結(jié)論 1、 RDP58肽與CTB交聯(lián)后，能有效提高抗原免疫原性，在體內(nèi)提高HO-1活性，延長(zhǎng)移植腎存活時(shí)間；其治療效果與腹腔注射相同，但口服途徑誘導(dǎo)耐受有無(wú)可比擬的安全性和方便性； 2、 CD4+CD25+Treg細(xì)胞能分泌exosomes，且exosomes過(guò)繼轉(zhuǎn)移入大鼠急性腎排斥模型后，能明顯改善移植腎存活時(shí)間和病理組織變化，這可能是CD4+CD25+Treg細(xì)胞實(shí)現(xiàn)遠(yuǎn)端調(diào)控的重要機(jī)制之一； 3、 Exosome經(jīng)SDS-PAGE和蛋白質(zhì)譜分析，證實(shí)是由十余種蛋白組成的的囊泡狀結(jié)構(gòu)。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陳春媛;牛鑫;汪泱;;Exosomes與腎臟生理及疾病[J];上海交通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2014年04期

本文編號(hào)：1387747