【作者】 顾晓冬；

【导师】 陈宗祐；

【作者基本信息】 复旦大学 ， 外科学， 2010， 博士

【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。更多还原

【Abstract】 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.PartⅠConstruction 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 IIEffects of blocking co-stimulatory pathway in vitro on alloantigen pulsed recipient dendritic cells pretreated by recombinant lentivirusObjective 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.PartⅢAnti-rejection effect of alloantigen pulsed recipient dendritic cells pretreated by recombinant lentivirus in murine heart transplantationObjective 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.更多还原