摘要
器官移植现已成为心、肝、肾等多种脏器终末期疾病的有效治疗手段。随着外科技术的日臻完善、器官保存和组织培养方法的进步以及免疫抑制剂的有效应用,超急性排斥的预防和急性排斥的防治已取得重大进展,慢性排斥反应导致移植器官慢性失功逐渐成为制约器官移植成功的“瓶颈”,因此,诱导针对供者特异性的免疫耐受是当代移植领域亟待解决的重要课题。
受者T细胞对供者抗原的免疫识别、活化和增殖是执行器官移植细胞免疫的基础,在同种异体移植排斥反应中起着核心作用。受者T细胞对同种异体移植抗原的识别包括直接识别和间接识别,目前越来越多的学者认为T细胞间接识别途径在移植物慢性失功的发生机制中占据关键地位。因此,阻断间接识别信号通路并阐明其分子生物学发生机制在移植免疫耐受中更值得关注。
在间接识别途径中受者T细胞的活化同样需要抗原呈递细胞表面共刺激分子与受者T细胞表面的相应受体的结合,这其中CD80/CD86-CD28是最重要的一条共刺激通路。为此,本课题采用目前国际上较先进的RNA干扰技术——慢病毒介导RNA干扰,抑制受者树突状细胞表面CD80和CD86的表达,通过体内、体外实验观察阻断间接识别通路对小鼠心脏移植的影响,并探讨其相关机制。
第一部分小鼠CD80/CD86基因RNA干扰慢病毒载体的构建与鉴定
目的构建小鼠CD80和CD86基因RNA干扰慢病毒表达载体,并在体外观察其对小鼠骨髓源性树突状细胞的作用。方法针对已经筛选确定的CD80基因RNA干扰有效靶序列,合成靶序列的双链DNA,接入pGCL-GFP载体,再与pHelper1.0和pHelper2.0质粒共转染293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平测定病毒滴度；同法构建出CD86基因RNA干扰慢病毒载体。慢病毒感染体外培养的树突状细胞,通过荧光显微镜检测感染效率,Annexin V/PI双染色法检测感染细胞凋亡和坏死情况,流式细胞仪检测CD80和CD86的表达情况。结果PCR和测序证实,LV-GFP-shCD80和LV-RFP-shCD86慢病毒载体构建正确,病毒滴度均达2×107TU/ml,适合感染树突状细胞的MOI值为20,此时慢病毒对树突状细胞具有低毒性,感染效率为85.42%。CD80和CD86表达的抑制率分别为82.05%和77.78%。结论成功构建出小鼠CD80和CD86基因RNA干扰慢病毒载体,其能明显抑制树突状细胞表面CD80和CD86的表达,这为抗移植物排斥提供了新的治疗手段。
第二部分重组慢病毒预处理的负载供者抗原的受者树突状细胞阻断共刺激通路作用的体外研究
目的观察重组慢病毒处理受者树突状细胞吞噬供者抗原的能力以及吞噬后树突状细胞生物免疫学特性的变化。方法在树突状细胞培养过程中加入供者抗原,通过与受者脾脏T细胞进行单向混合淋巴细胞反应,观察受者树突状细胞负载供者抗原的有效性。应用实时定量RT-PCR和流式细胞仪检测CD80/CD86 mRNA和蛋白的表达情况。通过初次混合淋巴细胞反应,了解重组慢病毒处理树突状细胞刺激受者T细胞的增殖能力,并用这种树突状细胞预致敏小鼠行再次混合淋巴细胞反应,观察受者T细胞针对间接识别途径或直接识别途径提呈的供者抗原的免疫活性。应用酶联免疫吸附法检测初次混合淋巴细胞反应培养上清中IL-2、IL-4、IL-10和INF-γ的含量。结果在培养过程中加入供者抗原的受者树突状细胞具有显著刺激受者T细胞增殖的能力。LV-GFP-shCD80和LV-RFP-shCD86同时感染树突状细胞进一步降低CD80/CD86mRNA和蛋白的表达,这种抑制作用不可被脂多糖等刺激所逆转。初次混合淋巴细胞反应显示,重组慢病毒处理受者树突状细胞能够有效减弱对受者T细胞的刺激增殖作用。再次混合淋巴细胞反应结果显示,重组慢病毒处理受者树突状细胞能够诱导产生针对供者抗原的免疫低反应。培养上清中Th1细胞因子(IL-2和INF-γ)的含量明显减少,而Th2细胞因子(IL-10)的含量显著增高。结论受者树突状细胞能够有效的提呈供者抗原。重组慢病毒明显抑制树突状细胞表面CD80和CD86的表达。重组慢病毒处理的树突状细胞可抑制其刺激受者T细胞的增殖反应,诱导局部细胞因子谱系向Th2型偏移；重组慢病毒处理的树突状细胞可诱导抗原特异性的T细胞免疫低反应性。
第三部分重组慢病毒预处理负载供者抗原的受者树突状细胞在小鼠心脏移植模型中抗排斥反应的研究
目的建立同种异体小鼠异位心脏移植模型,探讨重组慢病毒处理的受者树突状细胞在体内诱导移植免疫耐受的可行性及其免疫学机制。方法改进Cuff技术制作小鼠颈部异位心脏移植模型,使用自制套管将供心肺动脉套接于受者右颈外静脉,将供心升主动脉套接于受者右颈总动脉。移植术前7天经尾静脉输注慢病毒处理的受者树突状细胞预处理小鼠或联合腹腔注射小剂量环孢素A。观察小鼠移植心脏的存活时间和组织病理学改变。Annexin V/CD3双染色法检测受者淋巴组织和移植心脏中T细胞凋亡情况。流式细胞仪检测受者脾脏和移植心脏中调节性T细胞的含量。采用半定量RT-PCR方法检测小鼠移植心脏中细胞因子IL-2、INF-γ、TNF-α、IL-4、IL-6和IL-10mRNA的表达水平以及脾脏T细胞中凋亡信号通路相关基因的表达水平。结果小鼠颈部异位心脏移植手术成功率92.5%。手术时间约60分钟,供心冷缺血时间小于30分钟。重组慢病毒处理受者树突状细胞明显延长小鼠移植心脏的存活时间(平均存活时间为21.8天)。联合腹腔注射小剂量环孢素A,平均存活时间延长至72天,其中有3只小鼠移植心脏存活时间超过100天。小鼠移植心脏组织中Th1细胞因子(IL-2、INF-γ和TNF-α)的表达明显低下,而Th2细胞因子(IL-10)的表达显著升高。重组慢病毒处理受者树突状细胞诱导受者脾脏、肠系膜淋巴结和移植心脏中T细胞的凋亡,诱导移植心脏中调节性T细胞的产生。小鼠脾脏T细胞中Bax和GRP78 mRNA的表达有明显增高；小鼠脾脏T细胞中CHOP mRNA表达显著上调、Bcl-xL mRNA表达显著下调。结论改良Cuff技术成功建立小鼠颈部异位心脏移植模型。重组慢病毒处理的DC联合小剂量环孢素A可延长移植物的存活时间,甚至诱导移植免疫耐受。诱导IL-10等抑制性细胞因子、调节性T细胞的局部表达和受者体内T细胞凋亡,可能是移植心脏存活时间延长的主要机制；T细胞的凋亡存在线粒体凋亡途径和内质网应激反应性凋亡途径。
Organ transplantation has now become an effective method to cure end-stage heart or liver or renal or other organs disease. With the gradual perfection of surgical technique, the progress of organ preservation and tissue culture, and the effective application of immunosuppressive agents, hyperacute rejection and acute rejection prevention have achieved great progress. Chronic graft dysfunction caused by chronic rejection gradually become the bottleneck in successful organ transplantation. Therefore, inducing donor specific immune tolerance is an important subject needed to be solved urgently in current transplant field.
Immune recognition, activation, proliferation of recipient T cells aiming at donor antigen is the basis of organ transplant cell immunity, and this also plays an essential role in allograft rejection. Transplantation antigen allorecognition of recipient T cells include direct recognition and indirect recognition. Nowadays more and more scholars consider that indirect recognition pathway may be substantially more important in the occurrence mechanism of chronic graft dysfunction. Therefore, blocking indirect recognition pathway and illustrating its molecular mechanism deserve paying much more attention in transplantation tolerance.
Recipient T cells activation demand the co-stimulatory signal which is delivered through interactions between recipient T cells and antigen presenting cells. CD80/CD86-CD28 is the most important co-stimulatory pathway. So we knocked down CD80 and CD86 expression in recipient dentritic cells using lentiviral mediated RNA interference, and tried to find out what happened in murine heart transplantation after blocking indirect recognition pathway in vitro or in vivo, and tried to explore the possible mechanisms.
PartⅠ
Construction and identification of lentiviral vectors targeting mouse CD80 and CD86 genes by RNA interference
Objective To construct lentiviral vectors targeting mouse CD80 and CD86 genes by RNA interference and study their effects on bone marrow-derived dendritic cells in vitro.
Methods The effective sequence of siRNA targeting CD80 gene was confirmed in our previous experiment. The complementary DNA containing both sense and antisense oligonucleotides of the targeting sequence was designed, synthesized. After annealed, double-stranded DNA was inserted into the pGCL-GFP vector. The resulting lentiviral vector was named pGCL-GFP-CD80shRNA.293T cells were cotransfected with pGCL-GFP-CD80shRNA、pHelper1.0 and pHelper2.0. The titer of virus was tested according to the expression level of GFP. Lentiviral vector targeting mouse CD86 gene by RNA interference was constructed in the same way. The recombinant lentiviruses infected dendritic cells that were separated from femurs and tibias of mice in vitro. The infection efficiency was assessed by fluorescence microscope. The cell viability of infected dendritic cells was determined by annexin V and propidium iodine staining. The expression of CD80 and CD86 was analyzed by flow cytometry. Results PCR and DNA sequencing demonstrated that LV-GFP-shCD80 and LV-RFP-shCD86 were constructed successfully. The titer of the recombinant lentiviruses was both 2x107TU/ml and the best MOI for lentivirus infecting dendritic cells was 20. Lentiviruses demonstrated a high (85.42%) infection efficiency of dendritic cells without affecting cellular viability. The inhibitory rates of CD80 and CD86 expression were 82.05% and 77.78% respectively. Conclusions Lentiviral vectors targeting mouse CD80 and CD86 genes by RNA interference were constructed successfully. The recombinant lentiviruses show significantly inhibiting effects on CD80 and CD86 expression in dendritic cells. This approach is a potential therapeutic option for allograft rejection.
Part II
Effects of blocking co-stimulatory pathway in vitro on alloantigen pulsed recipient dendritic cells pretreated by recombinant lentivirus
Objective To measure the phagocytic capacity and the bio-immunological characteristics of recombinant lentivirus pretreated recipient dendritic cells loaded with alloantigen. Methods In the culture process of dendritic cells, donor antigens were added, the validity of dendritic cells loading antigens was investigated by one-way mixed lymphocyte reaction using recipient splenic T cells and recipient dendritic cells. The transcription of CD80/CD86 gene and the expression of CD80/CD86 protein were determined by real-time quantitative reverse transcription-polymerase chain reaction and flow cytometry, respectively. The stimulating capacity to recipient T-cell proliferative response was testified in primary mixed lymphocyte reaction. To determine immunological capacity of recipient T cells recognizing donor antigens which were presented through indirect or direct recognition pathway, recipient mice were primed with these dendritic cells and secondary mixed lymphocyte reactions were performed. IL-2, IL-4, IL-10 and INF-γlevels in primary mixed lymphocyte reaction culture supernatants were measured using enzyme linked immunosorbent assay.
Results Recipient dendritic cells which were cultured with donor antigens could stimulate the proliferation of recipient T cells significantly. The transcription of CD80/CD86 gene and the expression of CD80/CD86 protein enhanced down-regulated when LV-GFP-shCD80 and LV-RFP-shCD86 infected dendritic cells simultaneously, and this inhibition was not reversed by lipopolysaccharide. Recombinant lentivirus pretreated recipient dendritic cells effectively inhibited the proliferation of recipient T cells in primary mixed lymphocyte reaction and induced specific T-cell hyporesponsiveness to donor antigen in secondary mixed lymphocyte reaction. A reduction of Thl cytokines (IL-2 and INF-γ) and an induction of Th2 cytokines (IL-10) were found in primary mixed lymphocyte reaction culture supernatants. Conclusions Recipient dendritic cells could present donor antigens effectively. Recombinant lentivirus could specifically and effectively knock down CD80 and CD86 gene expression. Recombinant lentivirus pretreated recipient dendritic cells markedly suppressed recipient T-cell proliferative response, induced local cytokines prone to Th2, and induced antigen specific T-cell hyporesponsiveness.
PartⅢ
Anti-rejection effect of alloantigen pulsed recipient dendritic cells pretreated by recombinant lentivirus in murine heart transplantation
Objective To establish allogeneic heterotopic heart transplantation model in mice. To investigate the mechanism of recipient-derived dendritic cells treated with recombinant lentivirus inducing transplant immune tolerance in vivo. Methods Improved Cuff technique was used in making the model of cervical heterotopic heart transplantation in mice. By using self-made cuffs, the donor pulmonary artery was anastomosed to the recipient right external jugular vein, the donor ascending aorta was anastomosed to the recipient right common carotid artery. Recipient mice were given one injection of recipient dendritic cells treated with lentivirus via the lateral tail vein 7 days prior to heart transplantation, or combined with intraperitoneal injection of a subtherapeutic dose Cyclosporin A. Graft survival was assessed by daily palpation. Rejection was defined by the cessation of heartbeat and further confirmed by histological analysis. The apoptosis of T cells in recipient lymphoid tissues and allografts was determined by Annexin V and CD3 staining. Regulatory T-cell in recipient spleens and allografts was analyzed by flow cytometry. The expression of cytokine genes (IL-2, INF-γ, TNF-α, IL-4, IL-6 and IL-10 mRNA) within cardiac allografts were evaluated by semi-quantity RT-PCR. The expression of apoptosis signal pathway associated genes in splenic T cells were also examined by semi-quantity RT-PCR. Results Murine cervical heterotopic heart transplantations have been performed with a successful rate of 92.5%. The operative time was about 60 minutes, the cold ischemic time for donor heart was smaller than 30 minutes. Recipient-derived dendritic cells treated with recombinant lentivirus significantly prolonged heart allograft survival (Median survival time was 21.8 days). Combined with intraperitoneal injection of Cyclosporin A prolonged the median survival time to 72 days, with 3 grafts surviving beyond 100 days. These dendritic cells down-regulated Thl cytokines (IL-2, INF-y and TNF-a) expression, but enhanced the Th2 cytokines (IL-10) expression within allografts. We found a higher percentage of apoptotic T cells in recipient spleens, mesenteric lymph nodes and grafts. A higher percentage of regulatory T cells were also found in grafts. Splenic T cells expressed high expression of GRP78 and Bax. These changes in endoplasmic reticulum and mitochondrial pathway were enhanced by up-regulation of CHOP and suppression of Bcl-xL expression among these cells. Conclusions Improved Cuff technique successfully established stable heterotopic cardiac allograft model in mice. Recipient dendritic cells treated with lentivirus combined with Cyclosporin A prolonged heart allograft survival, and even induced transplant immune tolerance. Cytokine secretion prone to Th2, regulatory T cells and recipient T-cell apoptosis might be crucial for the equipped dendritic cells prolonging graft survival. Mitochondrial apoptosis pathway and endoplasmic reticulum stress-mediated apoptosis pathway may be involved in allogeneic T-cell apoptosis.
引文
[1]Monk NJ, Hargreaves RE, Simpson E, et al. Transplant tolerance:models, concepts and facts[J]. J Mol Med,2006,84(4):295-304.
[2]Mirenda V, Berton I, Read J, et al. Modified dendritic cells coexpressing self and allogeneic major histocompatability complex molecules:an efficient way to induce indirect pathway regulation[J]. J Am Soc Nephrol,2004,15(4):987-997.
[3]Lechler R, Ng WF, Steinman RM. Dendritic cells in transplantation-friend or foe[J]? Immunity,2001,14(4):357-368.
[4]Thomson AW, Robbins PD. Tolerogenic dendritic cells for autoimmune disease and transplantation[J]. Ann Rheum Dis,2008,67(S3):90-96.
[5]O’Rourke RW, Kang SM, Lower JA, et al. A dendritic cell line genetically modified to express CTLA4-IG as a means to prolong islet allograft survival[J]. Transplantation,2000,69(7):1440-1446.
[6]李红卫,孟自力,张卫民,等.阻断B7/CD28途径诱导异种免疫耐受的体外研究[J].同济大学学报(医学版),2004,25(2)：108-111.
[7]吴小剑,兰平,王东平,等.无能T淋巴细胞的建立及其免疫生物学特性的研究[J].中华器官移植杂志,2005,26(1)：40-43.
[8]Lu L, Li W, Fu F, et al. Blockade of the CD40-CD40 ligand pathway potentiates the capacity of donor-derived dendritic cell progenitors to induce long-term cardiac allograft survival[J]. Transplantation,1997,64(12):1808-1815.
[9]Liang X, Lu L, Chen Z, et al. Administration of dendritic cells transduced with antisense oligodeoxyribonucleotides targeting CD80 or CD86 prolongs allograft survival[J]. Transplantation,2003,76(4):721-729.
[10]姚宇锋,项建斌,蔡端.B7反义肽预处理的受体树突状细胞抑制移植动脉的内膜增生[J].复旦学报(医学版),2007,34(3)：355-357.
[11]Kita Y, Li XK, Ohba M, et al. Prolonged cardiac allograft survival in rats systemically injected adeno viral vectors containing CTLA4Ig-gene[J]. Transplantation,1999,68(6):758-766.
[12]Chen Z, Lu L, Li J, et al. Prolonged survival of heart allografts transduced with AAV-CTLA4Ig[J]. Microsurgery,2003,23(5):489-493.
[13]Min WP, Gorczynski R, Huang XY, et al. Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival[J]. J Immunol,2000,164(1):161-167.
[14]Min W, Huang X, Gorczynski R, et al. Fas ligand-transfected dendritic cells induce apoptosis of antigen-specific T cells[J]. Transplant Proc,2001,33(1-2):234.
[15]Buonocore S, Van Meirvenne S, Demoor FX, et al. Dendritic cells transduced with viral interleukin 10 or Fas ligand: no evidence for induction of allotolerance in vivo[J]. Transplantation,2002,73(S1):27-30.
[16]Gorczynski RM, Bransom J, Cattral M, et al. Synergy in induction of increased renal allograft survival after portal vein infusion of dendritic cells transduced to express TGFbeta and IL-10, along with administration of CHO cells expressing the regulatory molecule OX-2[J]. Clin Immunol,2000,95(3):182-189.
[17]Giannoukakis N, Bonham CA, Qian S, et al. Prolongation of cardiac allograft survival using dendritic cells treated with NF-kB decoy oligodeoxyribonucleotides[J]. Mol Ther,2000,1(5):430-437.
[18]Bonham CA, Peng L, Liang X, et al. Marked prolongation of cardiac allograft survival by dendritic cells genetically engineered with NF-kappa B oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA4-Ig[J]. J Immunol,2002,169(6):3382-3391.
[19]Xu DL, Liu Y, Tan JM, et al. Marked prolongation of murine cardiac allograft survival using recipient immature dendritic cells loaded with donor-derived apoptotic cells[J]. Scand J Immunol,2004,59(6):536-544.
[20]Garrovillo M, Ali A, Depaz HA, et al. Induction of transplant tolerance with immunodominant allopeptide-pulsed host lymphoid and myeloid dendritic cells[J]. Am J Transplant,2001,1(2):129-137.
[21]Spriewald BM, Billing JS, Ensminger SM, et al. Linked unresponsiveness:early cytokine gene expression profiles in cardiac allografts following pretreatment of recipients with bone marrow cells expressing donor MHC alloantigen[J]. Cytokine, 2002,19(1):6-13.
[22]Snanoudj R, de Preneuf H, Creput C, et al. Costimulation blockade and its possible future use in clinical transplantation [J]. Transpl Int,2006,19(9):693-704.
[23]Flinn IW, Lazarus HM. Monoclonal antibodies and autologous stem cell transplantation for lymphoma[J]. Bone Marrow Transplant,2001,27(6):565-569.
[24]Dias N, Stein CA. Antisense oligonucleotides:basic concepts and mechanisms[J]. Mol Cancer Ther,2002,1(5):347-355.
[25]Marshall E. Gene therapy death prompts review of adenovirus vector[J]. Science, 1999,286(5448):2244-2245.
[26]Hannon GJ. RNA interference[J]. Nature,2002,418(6894):244-251.
[27]Abbas-Terki T, Blanco-Bose W, Deglon N, et al. Lentiviral-mediated RNA interference[J]. Hum Gene Ther,2002,13(18):2197-2201.
[28]Frauwirth KA, Thompson CB. Activation and inhibition of lymphocytes by costimulation[J]. J Clin Invest,2002,109(3):295-299.
[29]Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells[J]. Science,2002,296(5567):550-553.
[30]Gu X, Xiang J, Yao Y, et al. Effects of RNA interference on CD80 and CD86 expression in bone marrow-derived murine dendritic cells[J]. Scand J Immunol, 2006,64(6):588-594.
[31]Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference[J]. Nat Genet,2003,33(3):401-406.
[32]Elbashir SM, Harborth J, Weber K, et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs[J]. Methods,2002,26(2):199-213.
[33]Lutz MB, Kukutsch N, Ogilvie AL, et al. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow[J]. J Immunol Methods,1999,223(1):77-92.
[34]Ni K, O'Neill HC. Improved FACS analysis confirms generation of immature dendritic cells in long-term stromal-dependent spleen cultures[J]. Immunol Cell Biol, 2000,78(3):196-204.
[35]Ni K, O'Neill HC. Development of dendritic cells from GM-CSF-/- mice in vitro: GM-CSF enhances production and survival of cells[J]. Dev Immunol,2001,8(2): 133-146.
[36]Rocheleau CE, Downs WD, Lin R, et al. Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos[J]. Cell,1997,90(4):707-716.
[37]Guo S, Kemphues KJ. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed[J]. Cell,1995,81(4):611-620.
[38]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature,1998,391(6669): 806-811.
[39]Cerutti H. RNA interference:traveling in the cell and gaining functions[J]? Trends Genet,2003,19(1):39-46.
[40]Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs[J]. Genes Dev,2001,15(2):188-200.
[41]Ichim TE, Zhong R, Min WP. Prevention of allograft rejection by in vitro generated tolerogenic dendritic cells [J]. Transpl Immunol,2003,11(3-4):295-306.
[42]Laderach D, Compagno D, Danos O, et al. RNA interference shows critical requirement for NF-kappa B p50 in the production of IL-12 by human dendritic cells [J]. J Immunol,2003,171(4):1750-1757.
[43]Hill JA, Ichim TE, Kusznieruk KP, et al. Immune modulation by IL-12 production in dendritic cells using intering RNA[J]. J Immunol,2003,171(2):691-696.
[44]Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells[J]. RNA,2003,9(4):493-501.
[45]Perletti G, Osti D, Marras E, et al. Generation of VSV-G pseudotyped lentiviral particles in 293T cells[J]. J Cell Mol Med,2004,8(1):142-143.
[46]Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells[J]. Annu Rev Immunol,2003,21(1):685-711.
[47]Ensminger SM, Spriewald BM, Witzke O, et al. Indirect allorecognition can play an important role in the development of transplant arteriosclerosis[J]. Transplantation, 2002,73(2):279-286.
[48]Onodera K, Chandraker A, Volk HD, et al. Distinct tolerance pathways in sensitized allograft recipients after selective blockade of activation signal 1 or signal 2[J]. Transplantation,1999,68(2):288-293.
[49]Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution[J]. J Exp Med,1973, 137(5):1142-1162.
[50]Hart DN, Hill GR. Dendritic cell immunotherapy for ancer: application to low-grade lymphoma and multiply myeloma[J]. Immunol Cell Biol,1999,77(5):451-459.
[51]Hanada K, Tsunoda R, Hamada H. GM-CSF-induced in vivo expansion of splenic dendritic cells and their strong costimulation activity[J]. J Leukoc Biol,1996,60(2): 181-190.
[52]Ferlazzo G, Klein J, Paliard X, et al. Dendritic cells generated from CD34+ progenitor cells with flt3 ligand, c-kit ligand, GM-CSF, IL-4, and TNF-alpha are functional antigen-presenting cells resembling mature monocyte-derived dendritic cells[J]. J Immunother,2000,23 (1):48-58.
[53]Inaba K, Inaba M, Romani N, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor[J]. J Exp Med,1992,176(6):1693-1702.
[54]许化溪,严俊.实验动物免疫细胞功能功能的测定[A].见：沈关心,周如麟主编.现代免疫学实验技术[M].湖北：湖北科学技术出版社,1998：328-341.
[55]Norbury CC, Chambers BJ, Prescott AR, et al. Constitutive macropinocytosis allows TAP-dependent major histocompatibility complex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells[J]. Eur J Immunol,1997, 27(1):280-288.
[56]Ji J, Wernli M, Klimkait T, et al. Enhanced gene silencing by the application of multiple specific small interfering RNAs[J]. FEBS Lett,2003,552(2-3):247-252.
[57]Kim KS, Denton MD, Chandraker A, et al. CD28-B7-mediated T cell costimulation in chronic cardiac allograft rejection: differential role of B7-1 in initiation versus progression of graft arteriosclerosis[J]. Am J Pathol,2001,158(3):977-986.
[58]Slavik JM, Hutchcroft JE, Bierer BE. CD80 and CD86 are not equivalent in their ability to induce the tyrosine phosphorylation of CD28[J]. J Biol Chem,1999, 274(5):3116-3124.
[59]Lang TJ, Nguyen P, Peach R, et al. In vivo CD86 blockade inhibits CD4+ T cell activation, whereas CD80 blockade potentiates CD8+ T cell activation and CTL effector function[J]. J Immunol,2002,168(8):3786-3792.
[60]Xiang J, Gu X, Qian S, et al. Graded function of CD80 and CD86 in initiation of T-cell immune response and cardiac allograft survival[J]. Transpl Int,2008,21(2): 163-168.
[61]Min WP, Zhou D, Ichim TE, et al. Inhibitory feedback loop between tolerogenic dendritic cells and regulatory T cells in transplant tolerance[J]. J Immunol,2003, 170(3):1304-1312.
[62]Caballero A, Fernandez N, Lavado R, et al. Tolerogenic response:allorecognition pathways[J]. Transpl Immunol,2006,17(1):3-6.
[63]Mandelbrot DA, Kishimoto K, Auchincloss H Jr, et al. Rejection of mouse cardiac allografts by costimulation in trans[J]. J Immunol,2001,167(3):1174-1178.
[64]Morelli AE, Thomson AW. Dendritic cells:regulators of alloimmunity and opportunities for tolerance induction[J]. Immunol Rev,2003,196:125-146.
[65]Vincenti F, Larsen C, Durrbach A, et al. Costimulation blockade with belatacept in renal transplantation[J]. N Engl J Med,2005,353(8):770-781.
[66]Chen ZH. A technique of cervical heterotopic heart transplantation in mice[J]. Transplantation,1991,52(6):1099-1101.
[67]Matsuura A, Abe T, Yasuura K. Simplified mouse cervical heart transplangtation using a cuff technique[J]. Transplantation,1991,51(4):896-898.
[68]Tomita Y, Zhang QW, Yoshikawa M, et al. Improved technique of heterotopic cervical heart transplangtation in mice[J]. Transplantation,1997,64(11):1598-1601.
[69]Wang Q, Liu Y, Li XK. Simplified technique for heterotopic vascularized cervical heart transplantation in mice[J]. Microsurgery,2005,25(1):76-79.
[70]冯剑锷,孙宗全.应用Cuff技术建立小鼠异位心脏移植模型[J].中华实验外科杂志,2005,22(12)：1576-1577.
[71]Valujskikh A, Pantenburg B, Heeger PS. Primed allospecific T cells prevent the effects of costimulatory blockade on prolonged cardiac allograft survival in mice[J]. Am J Transplant,2002,2(6):501-509.
[72]Colvin BL, Thomson AW. Location, location, location: dendritic cell trafficking and transplant tolerance[J]. Curr Opin Organ Tran,2007,12(1):1-4.
[73]Kawai M, Kitade H, Mathieu C, et al. Inhibitory and stimulatory effects of cyclosporine A on the development of regulatory T cells in vivo[J]. Transplantation, 2005,79(9):1073-1077.
[74]宋光民,宋惠民,张振国,等.大鼠心脏移植与心肌细胞坏死[J].中华器官移植杂志,1999,20(4)：227-228.
[75]Wang Z, Larregina AT, Shufesky WJ, et al. Use of the inhibitory effect of apoptotic cells on dendritic cells for graft survival via T-cell deletion and regulatory T cells[J]. Am J Transplant,2006,6(6):1297-1311.
[76]Sanchez-Fueyo A, Domenig CM, Mariat C, et al. Influence of direct and indirect allorecognition pathways on CD4+CD25+ regulatory T-cell function in transplantation[J]. Transpl Int,2007,20(6):534-541.
[77]Coenen JJ, Koenen HJ, van Rijssen E, et al. Tolerizing effects of co-stimulation blockade rest on functional dominance of CD4+CD25+ regulatory T cells[J]. Transplantation,2005,79(2):147-156.
[78]Li W, Kuhr CS, Zheng XX, et al. New insights into mechanisms of spontaneous liver transplant tolerance:the role of Foxp3-expressing CD25+CD4+ regulatory T cells[J]. Am J Transplant,2008,8(8):1639-1651.
[79]Ke B, Coito AJ, Kato H, et al. Fas ligand gene transfer prolongs rat renal allograft survival and down-regulates anti-apoptotic Bag-1 in parallel with enhanced Th2-type cytokine expression[J]. Transplantation,2000,69(8):1690-1694.
[80]Larsen CP, Elwood ET, Alexander DZ, et al. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways[J]. Nature,1996, 381(6581):434-438.
[81]Le Moine A, Flamand V, Demoor FX, et al. Critical roles for IL-4, IL-5, and eosinophils in chronic skin allograft rejection[J]. J Clin Invest,1999,103(12): 1659-1667.
[82]Pirenne J, Kitade H, Kawai M, et al. Regulatory cells, TH1/TH2 unbalance, and antibody-induced chronic rejection in operational tolerance induced by donor-specific blood transfusion[J]. Transplantation,2005,79(S3):25-27.
[83]Newell KA, Larsen CP, Kirk AD. Transplant tolerance:converging on a moving target[J]. Transplantation,2006,81(1):1-6.
[84]Wells AD, Li XC, Strom TB, et al. The role of peripheral T-cell deletion in transplantation tolerance[J]. Philos Trans R Soc Lond B Biol Sci,2001,356(1409): 617-623.
[85]Li XC, Wells AD, Strom TB, et al. The role of T cell apoptosis in transplantation tolerance[J]. Curr Opin Immunol,2000,12(5):522-527.
[86]Janssen O, Sanzenbacher R, Kabelitz D. Regulation of activation-induced cell death of mature T-lymphocyte populations[J]. Cell Tissue Res,2000,301(1):85-99.
[87]Wekerle T, Kurtz J, Sayegh M, et al. Peripheral deletion after bone marrow transplantation with costimulatory blockade has features of both activation-induced cell death and passive cell death[J]. J Immunol,2001,166(4):2311-2316.
[88]Tung TC, Oshima K, Cui G, et al. Dual upregulation of Fas and Bax promotes alloreactive T cell apoptosis in IL-10 gene targeting of cardiac allografts[J]. Am J Physiol Heart Circ Physiol,2003,285(3):964-973.
[89]Furukawa H, Oshima K, Tung T, et al. Overexpressed exogenous IL-4 and IL-10 paradoxically regulate allogenic T-cell and cardiac myocytes apoptosis through FAS/FASL pathway[J]. Transplantation,2008,85(3):437-446.
[90]Li J, Lee AS. Stress induction of GRP78/BiP and its role in cancer[J]. Curr Mol Med, 2006,6(1):45-54.
[91]Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress[J]. Cell Death Differ,2004,11(4):381-389.
[92]Harding HP, Zhang Y, Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase[J]. Nature,1999,397(6716):271-274.
[93]Iwawaki T, Hosoda A, Okuda T, et al. Translational control by the ER transmembrane kinase/ribonuclease IRE1 under ER stress[J]. Nat Cell Biol,2001, 3(2):158-164.
[94]Calfon M, Zeng H, Urano F, et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA[J]. Nature,2002,415(6867): 92-96.
[95]Yoshida H, Okada T, Haze K, et al. ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response[J]. Mol Cell Biol,2000,20(18):6755-6767.
[96]Sayeed A, Ng DT. Search and destroy: ER quality control and ER-associated protein degradation[J]. Crit Rev Biochem Mol Biol,2005,40(2):75-91.
[97]Yoshida H, Matsui T, Hosokawa N, et al. A time-dependent phase shift in the mammalian unfolded protein response[J]. Dev Cell,2003,4(2):265-271.
[98]Mori K. Tripartite management of unfolded proteins in the endoplasmic reticulum[J]. Cell,2000,101(5):451-454.
[99]Rao RV, Hermel E, Castro-Obregon S, et al. Coupling endoplasmic reticulum stress to the cell death program. Mechanism of caspase activation[J]. J Biol Chem,2001, 276(36):33869-33874.
[100]Yoneda T, Imaizumi K, Oono K, et al. Activation of caspase-12, an endoplastic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor 2-dependent mechanism in response to the ER stress[J]. J Biol Chem,2001, 276(17):13935-13940.
[101]Jimno A, Fujita E, Kouroku Y, et al. ER stress induces caspase-8 activation, stimulating cytochrome c release and caspase-9 activation[J]. Exp Cell Res,2003, 283(2):156-166.
[102]Tournier C, Hess P, Yang DD, et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway[J]. Science,2000, 288(5467):870-874.
[103]Ron D. Translational control in the endoplasmic reticulum stress response[J]. J Clin Invest,2002,110(10):1383-1388.
[104]Unterberger U, Hoftberger R, Gelpi E, et al. Endoplasmic reticulum stress features are prominent in Alzheimer disease but not in prion diseases in vivo[J]. J Neuropathol Exp Neurol,2006,65(4):348-357.
[105]Ozcan U, Cao Q, Yilmaz E, et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes[J]. Science,2004,306(5695):457-461.
[106]Azfer A, Niu J, Rogers LM, et al. Activation of endoplasmic reticulum stress response during the development of ischemic heart disease[J]. Am J Physiol Heart Circ Physiol,2006,291(3):1411-1420.
[107]Wootz H, Hansson I, Korhonen L, et al. Caspase-12 cleavage and increased oxidative stress during motoneuron degeneration in transgenic mouse model of ALS[J]. Biochem Biophys Res Commun,2004,322(1):281-286.
[108]Lou LX, Geng B, Yu F, et al. Endoplasmic reticulum stress response is involved in the pathogenesis of stress induced gastric lesions in rats[J]. Life Sci,2006,79(19): 1856-1864.
[109]Fribley A, Wang CY. Proteasome inhibitor induces apoptosis through induction of endoplasmic reticulum stress[J]. Cancer Biol Ther,2006,5(7):745-748.
[110]Carracedo A, Gironella M, Lorente M, et al. Cannabinoids induce apoptosis of pancreatic tumor cells via endoplasmic reticulum stress-related genes[J]. Cancer Res, 2006,66(13):6748-6755.
[111]Zhao L, Ackerman SL. Endoplasmic reticulum stress in health and disease[J]. Curr Opin Cell Biol,2006,18(4):444-452.
[112]Emadali A, Nguyen DT, Rochon C, et al. Distinct endoplasmic reticulum stress responses are triggered during human liver transplantation[J]. J Pathol,2005,207(1): 111-118.
[113]Pino SC, O'Sullivan-Murphy B, Lidstone EA, et al. Protein kinase C signaling during T cell activation induces the endoplasmic reticulum stress response[J]. Cell Stress Chaperones,2008,13(4):421-434.
[1]Selenko-Gebauer N, Majdic 0, Szekeres A, et al. B7-H1 (programmed death-1 ligand) on dendritic cells is involved in the induction and maintenance of T cell anergy[J]. J Immunol,2003,170(7):3637-3644.
[2]Emmer PM, van der Vlag J, Adema GJ, et al. Dendritic cells activated by lipopolysaccharide after dexamethasone treatment induce donor-specific allograft hyporesponsiveness[J]. Transplantation,2006,81(10):1451-1459.
[3]Hackstein H, Taner T, Zahorchak AF, et al. Rapamycin inhibits IL-4-induced dendritic cell maturation in vitro and dendritic cell mobilization and function in vivo[J]. Blood,2003,101(11):4457-4463.
[4]Bohana-Kashtan O, Civin CI. Fas ligand as a tool for immunosuppression and generation of immune tolerance[J]. Stem Cells,2004,22(6):908-924.
[5]Bonham CA, Peng L, Liang X, et al. Marked prolongation of cardiac allograft survival by dendritic cells genetically engineered with NF-kappa B oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA4-Ig[J]. J Immunol,2002,169(6):3382-3391.
[6]Shortman K, Liu YJ. Mouse and human dendritic cell subtypes[J]. Nat Rev Immunol, 2002,2(3):151-161.
[7]Valdez Y, Mah W, Winslow MM, et al. Major histocompatibility complex class II presentation of cell-associated antigen is mediated by CD8alpha+ dendritic cells in vivo[J]. J Exp Med,2002,195(6):683-694.
[8]O'Connell PJ, Li W, Wang Z, et al. Immature and mature CD8alpha+ dendritic cells prolong the survival of vascularized heart allografts[J]. J Immunol,2002,168(1): 143-154.
[9]Chang CC, Ciubotariu R, Manavalan JS, et al. Tolerization of dendritic cells by T(S) cells:the crucial role of inhibitory receptors ILT3 and ILT4[J]. Nat Immunol,2002, 3(3):237-243.
[10]Gilliet M, Liu YJ. Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells[J]. J Exp Med,2002,195(6):695-704.
[11]Sato K, Yamashita N, Yamashita N, et al. Regulatory dendritic cells protect mice from murine acute graft-versus-host disease and leukemia relapse[J]. Immunity, 2003,18(3):367-379.
[12]Beriou G, Peche H, Guillonneau C, et al. Donor-specific allograft tolerance by administration of recipient-derived immature dendritic cells and suboptimal immunosuppression[J]. Transplantation,2005,79(8):969-972.
[13]Bonifaz L, Bonnyay D, Mahnke K, et al. Efficient targeting of protein antigen to the dendriticcell receptor DEC-205 in the steady stateleads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+T cell tolerance[J]. J ExpMed,2002,196(12):1627-1638.
[14]Morelli AE, Larregina AT, Shufesky WJ, et al. Internalization of circulating apoptotic cells by splenic marginal zone dendritic cells:dependence on complement receptors and effect on cytokine production[J]. Blood,2003,101(2):611-620.
[15]Verbovetski I, Bychkov H, Trahtemberg U, et al. Opsonization of apoptotic cells by autologous iC3b facilitates clearance by immature dendritic cells, down-regulates DR and CD86, and up-regulates CC chemokine receptor 7[J]. J Exp Med,2002, 196(12):1553-1561.
[16]Xu DL, Liu Y, Tan JM, et al. Marked prolongation of murine cardiac allograft survival using recipient immature dendritic cells loaded with donor-derived apoptotic cells[J]. Scand J Immunol,2004,59(6):536-544.