【作者】 何文涛；

【导师】 陈忠华；

【作者基本信息】 华中科技大学 ， 移植免疫， 2010， 博士

【摘要】 [目的]探讨galectin-9蛋白抗完全异基因小鼠心脏移植排斥的机制。[方法]构建含galectin-9基因的腺病毒,感染CHO细胞使其过表达galectin-9,免疫细胞化学和流式细胞染色确定其真核表达定位。以PMA+Ionomycin+BFA激活脾脏淋巴细胞,以流式细胞染色探讨活化前后CD4+淋巴细胞和CD8+T淋巴细胞亚群表达galectin-9的平均荧光强度。原核表达并纯化基因重组人galectin-9蛋白。流式分选CD4+CD25-T细胞,体外诱导其向Th17细胞分化,加或不加galectin-9蛋白,检测诱导的CD4+IL-17+T细胞比例。胶原酶消化法分离完全异基因小鼠移植心内浸润淋巴细胞,分析CD4+Tim-3+T细胞和CD8+Tim-3+T细胞的比例以及天然免疫细胞Tim-3+的比例。构建小鼠BALB/c→C57BL/6心脏移植模型,予galectin-9蛋白短期干预,观察移植物存活情况,探讨不同组别移植物引流淋巴结和小鼠外周血内CD4+IFN-γ+、CD4+IL-17+、CD8+IFN-γ+, CD8+IL-17+, CD4+Tim-3+、CD4+Tim-1+淋巴细胞的比例。对比各组别移植受者脾脏内CD4+CD25+Foxp3+T细胞(Treg)的比例。[结果]galectin-9表达于CHO细胞的胞浆,galectin-9在活化的CD4+和CD8+淋巴细胞内胞浆有表达,可能以分泌形式发挥作用。在体外,galectin-9可抑制Th17细胞的生成。完全异基因移植心脏物浸润细胞内的Tim-3+淋巴细胞以CD8+细胞为主,CD8+Tim-3+淋巴细胞占CD8+淋巴细胞的比例达到40.5%±5.2%。给予galectin-9短期治疗可显著延长完全异基因小鼠移植心的存活时间(MST=23.0±1.0天vs对照组7.2±0.4天),移植物存活延长的同时伴随引流淋巴结内CD8+IFN-γ+、CD8+IL-17+和外周血中CD4+Tim-3+淋巴细胞比例下调,而CD4+Tim-1+比例上调,但galectin-9治疗组小鼠体内的Treg比例并无明显上调。[结论]Galectin-9在活化的CD4+T和CD8+T细胞胞浆内可检测到,可能通过分泌形式发挥免疫负反馈抑制作用。在体外,galectin-9可抑制Th17细胞的生成。galectin-9延长小鼠移植心的存活与CD8+T细胞免疫下调,Th1和Th17免疫受抑制(CD4+Tim-3+)而Th2免疫(CD4+Tim-1+)占优势有一定关联。[目的]探讨galectin-9促进DC成熟的效应,用Rapamycin在体外能否抑制galectin-9的促成熟效应。探索利用小剂量rapamycin和galectin-9能否诱导小鼠移植心耐受。[方法]分离BALB/c→C56BL/6排斥心脏内浸润细胞,流式检测圈定单核细胞/DC细胞亚群进行Tim-3的表达水平分析,Western Blot检测移植心内Tim-3L(galectin-9)的表达。体外培养BALB/c小鼠的BMDC,检测Tim-3的表达情况。以一定浓度梯度的galectin-9处理DC,或联合一定浓度梯度的Rapamycin检测DC表面共刺激分子CD80/CD86的表达。建立BALB/c→C56BL/6小鼠移植模型,按照以下处理进行分组：A.同基因对照组(C56BL/6→C56BL/6);B.异基因移植galectin-9单独治疗组；C.异基因移植Rapamycin治疗组；D. galectin-9+Rapamycin联合治疗组；E.异基因移植PBS治疗组。观察小鼠移植心存活情况。ELISPOT检测长期存活小鼠对供者抗原或非相关第三方抗原反应分泌IL-4和IFN-γ的情况。[结果]移植心内单核细胞/DC细胞Tim-3的阳性率高达47.3%±5.6%。移植后第3天galectin-9的表达水平显著上调,而第7天时表达减少。培养的不成熟BMDC组成性表达Tim-3,阳性率约为6.7%左右。使用galectin-9可促进DC细胞上调共刺激分子Cd80/CD86,而在联合使用Rapamycin后DC表达的共刺激分子水平明显减少。体内联合galectin-9和Rapamycin可促进全部移植物长期接受(>180天,n=6)；而单用galectin-9治疗,仅短期延长移植物存活(MST=23.5±2.2天)；单用rapamycin的治疗组,4例移植心在分别在移植后不同时间点被排斥,2例存活大于180天,与galectin-9联合Rapamycin使用组差异有统计学意义(p<0.05)。联合治疗组移植物长期受者对供者抗原刺激分泌IFN-γ和IL-4明显弱于第三方,获得针对供者抗原特异的免疫低反应。[结论]移植物内天然免疫细胞高表达Tim-3和移植心早期上调galectin-9,两者可能参与了移植排斥的发生。Galectin-9具有刺激DC成熟的作用,而Rapamycin可抑制galectin-9引起的DC成熟,两者联合使用可诱导移植心获得耐受。联合治疗的受者针对供者抗原产生显著减弱的Th1和Th2免疫反应。[目的]探讨ST2825作用于TLR信号关键接头蛋白MyD88以减轻小鼠异基因心脏移植排斥的机制。[方法]①GM-SCF+IL-4诱导BALB/c小鼠骨髓造血干细胞分化为DC,使用多种TLR配体及坏死心肌匀浆刺激其活化。检测ST2825抑制MyD88对DC表达共刺激分子CD80/CD86的影响。②分离C57BL/6小鼠淋巴结细胞(以T细胞为主),以BALB/c来源的DC刺激其增殖。加或不加CpG(TLR9配体),检测ST2825对混培体系中T细胞激活的影响。③行BALB/c→C57BL/6心脏移植,术前两小时给予250mg/kgST2825,以后每日给药1次直到术后第6天,第7日获取不同组别的移植心和脾脏,并留取7例观察移植心完全停跳时间。实时定量PCR检测移植心内促炎因子IL-1β,IL-6和TNF-α的mRNA水平,HE病理分析淋巴细胞浸润和心肌完整性,流式细胞仪检测移植受者脾脏内CD4+IL-17+和CD4+IFN-y+淋巴细胞以及CD4+CD25+Foxp3+调节性T细胞比例。[结果]体外使用ST2825可显著抑制各种TLR配体及坏死心肌匀浆刺激DC引起的CD80/CD86上调(除TLR3配体外),ST2825呈剂量依赖性抑制DC刺激的T细胞活化。单独使用ST2825显著延长小鼠心移植物存活时间(MST=19.4±0.98天vs 7.2±0.4天)。ST2825治疗组心肌结构较为完整,淋巴细胞浸润减少,移植物内的IL-1β和IL-6转录显著下调,CD4+IL-17+和CD4+IFN-γ+淋巴细胞比例亦显著下调,而CD4+CD25+Foxp3+调节性T细胞比例略上调。[结论] ST2825可显著显著延长小鼠同种异基因心移植物存活时间,与ST2825抑制DC活化,进而抑制T细胞活化并与CD4+CD25+Foxp3+调节性T细胞的生成有关。[目的]探讨利用MyD88阻断剂ST2825阻断TLR信号,联合共刺激分子阻断剂anti-CD 154诱导皮肤移植耐受。[方法]建立BALB/c→C57BL/6小鼠皮肤移植模型,摸索ST2825阻断剂联合anti-CD 154体内给予的方案,观察移植皮片的存活时间。对移植皮肤长期存活的小鼠行ELISPOT检测对供者抗原的反应性：取受者脾细胞与供者脾细胞(BALB/c)或非相关第三方脾细胞(C3H)共孵育,检测IFN-γ和IL-4的斑点数。[结果]单独anti-CD 154干预组,单独ST2825干预组,CMC对照干预组异基因移植皮肤均不能获得保护,短期内即排斥。而联合使用anti-CD154+ST2825的治疗组,大多数皮肤移植物获得长期存活(>150天)。ELISPOT检测提示移植受者体内针对供者抗原呈现特异性低反应,脾细胞分泌IFN-γ的反应明显减弱,但分泌IL-4的反应保留。[结论]利用我们建立的anti-CD154+ST2825诱导方案可诱导完全异基因小鼠皮肤移植物长期接受。移植受者对供者抗原呈现特异性免疫低反应,可能与Th1免疫向Th2免疫偏移有关。更多还原

【Abstract】 [Aim] To explore the immunosuppressive effect of galectin-9 on fully allogeneic carcdiac allografts in mice.[Methods] CHO cells were engineerly overexpressed galectin-9 using adenovirus which contains the galecitn-9 gene. Immunocellular chemistry and flow cytometry staining were performed to ascertain the location of galectin-9. Preactivated or normal splenic CD4+or CD8+lymphocytes were subjected to flow cytometry analysis for mean fluoresence intensity. Galectin-9 protein was expressed and purified by pET E. Coli. FACS-sorted CD4+ CD25- T cells were cultured to differentiate to Th17 cells in the presence or absence of galectin-9. The percentage of Thl7 cells were detected three days later. BALB/c→C57BL/6 cervical cardiac transplant models were built. The recipients were treated with a short course of galectin-9 treantment. Compare the survival time of different recipients. The percentage of CD4+IFN-y+, CD4+IL-17+, CD8+IFN-γ+, CD8+IL-17+, CD4+Tim-3+and CD4+Tim-1+lymphocytes in the periphereal or draining lymphonode were analyzed by FACS. The percentage of CD4+CD25+Foxp3+T cells in spleen lymphocytes among different groups was compared.[Results] Galectin-9 was in the cytosol of CHO cells and actived CD4/CD8 cells. In vitro, Thl7 cell differnentitation was inhibited by the addition of galectin-9. The graft infiltrating cells were mainly CD8+T cells. The percentage of CD8+Tim-3+T lymphocytes in CD8+T lymphocytes was 40.5%±5.2%. A short course administration of galectin-9 promotes the survival of fully allogeneic cardiac allografs than the control group (MST= 23.0±1.0 days vs 7.2±0.4 days, respectively.). The prolonged survival was related with the reduction of CD8+IFN-γ+and CD8+IL-17+lymphocytes in the draining lymphonodes and the deviation from Th1/Th17(CD4+Tim-3+) to Th2(CD4+Tim-1+) immune responses.[Conclusion] Galectin-9 is in the cytosol of activated CD4+and CD8+lymphocytes. It may negatively regulate the overactivation of CD4+and CD8+lymphocyte immune responses. In vitro, galectin-9 inhibits the production of Thl7 cells. The immunosuppressive effect of galectin-9 on allografts is related with the reduced CD8+T cell responses and deviation from Th1/Th17 immune responses to Th2 immune responses. [Aim] To explore whether the the maturation promoting effect of galectin-9 on DCs could be reversed by Rapamycin. To explore the cardiac allograft tolerance induction mediated by galectin-9 and Rapamycin.[Methods] Infiltrating cells in the allografts were islated to analyze the Tim-3 expression level on monocyte/DC subgroups. The level of Tim-3 ligand galectin-9 in the allografts was detected by Western Blot. Costimulatory molecules CD80/CD86 on galectin-9/ Rapamycin-treated BMDCs were evaluated by flow cytometry. BALB/c→C56BL/6 vascularized cardiac allograft transplant model was built. Five groups were included in this study:1) syngeneic transplant control (C56BL/6→C56BL/6); 2) galectin-9-treated allogeneic transplantation; 3) Rapamycin-treated allogeneic transplantation; 4) combined treatment of galectin-9 and Rapamycin in allogeneic transplantation; 5) PBS-treated allogeneic transplantation. The complete cessation of palpation of the grafts was considered as observation endpoint. The recipients which long-term survived grafts were subjected to donor-specific assays on IFN-y and IL-4 secretion detection using ELISPOT.[Results] The percentage of Tim-3 positive cells in monocyte/DC of allograft infiltrating cells was around 47.3%±5.6%. Immature BMDCs constitutively express Tim-3 (around 6.7%). Galectin-9 could promote the upregulation of CD80/CD86 on BMDC, while the addition of Rapamycin reversed this process. Combined treatment of galectin-9 with Rapamycin promotes the permernant acceptance of fully allogeneic grafts (>180 days, n= 6). However, the protocol that single administration of Rapamycin is not potent enough to induce tolerance to all of the allografts. Galectin-9 treatment only prolonged the survival of allografs (MST= 23.5±2.2 days). The tolerant state induced by galectin-9 and Rapamycin was related with the donor-specific low secretion of IL-4 and IFN-γ.[Conclusion] High proportion of Tim-3 positive innate immune cells in the allografts may be involved in the initiation of transplant rejection. Rapamycin could be used to inhibit the proinflammatory effect of galectin-9 on DCs. Combined treatment of galectin-9 with Rapamycin promote the transplant tolerance induction which was associated with the reduced Th1 and Th2 responses. [Aim] To explore the immunosuppressive effect of MyD88 inhibitor which is the essential TLR signaling adaptor on cardiac allograft rejection.[Methods]①BMDC from BALB/c mice were cultured in the medium containing GM-CSF and IL-4. Various TLR agonists and cardiac homogenate were used to stimulated the maturation of BMDC in the presence or absence of ST2825. CD80/CD86 expression level on BMDC was evaluated.②Lymphonode lymphocytes from C57BL/6 were isolated to stimulate by BMDCs from BALB/c mice. CpG were added to the mixed culture system with or without the addition of ST2825. T cell proliferation was detected by flow cytometry.③BALB/c→C57BL/6 cardiac transplantation model was built. The recipient mice were administered with ST2825 at the dose of 250mg/kg/day two hours prior to transplantation. The treatment started from day 0 (the day of transplantation) to day 6. The complete cessation of the grafts was considered as rejection. On day 7, cardiac grafts were harvested for qRT-PCR analysis for transcription level of IL-1β,IL-6 and TNF-αand histological analysis. The proportion of CD4+IFN-y+, CD4+IL-17+and Treg in the spleen lymphocytes were detected by flow cytometry.[Results] Except for TLR3 ligands, ST2825 can inhibit the upregulation of CD80/CD86 on the BMDCs stimulated by varous TLR ligands and cardiac homogenate. ST2825 dose-dependently inhibit the proliferation of T lymphocytes in the one-way mixed culture system. A short term treatment with ST2825 significantly prolonged the survival of fully allogeneic cardiac grafts (MST= 19.4±0.98 days vs control 7.2±0.4 days). ST2825 mediated protection on allografts was related with the reduction of inflammatory cytokine IL-1βand IL-6. Moreover, ST2825 inhibits the upregulation of CD4+IL-17+and CD4+ IFN-γ+lymphocytes while promotes the propagation of Tregs in the spleens.[Conclusion] ST2825 can significantly prolong the survival of fully allogeneic cardiac grafts in mice. This effect was related with the immunosppressive effect of ST2825 on DC activation. Thus, ST2825 can inhibit the activation of T lymphocytes while promote the survival of Tregs. [Aim] To explore the possibility that robust skin transplant tolerance could be achieved by combined TLR signaling inhibition using MyD88 inhibitor with anti-CD 154 blockade.[Methods] BALB/c to C57BL/6 skin transplant model was established. The recipients were treated with anti-CD 154+ST2825 for a short term. Graft survival was monitored daily. Splenic lymphocytes from the recipients which habor the long-survived skin grafts were stimulated with splenic cells from the donor (BALB/c) or the third-party (C3H). ELISPOT technique was employed to evaluate the cytokine secretion (IL-4 and IFN-y) responses of the tolerant state.[Results]Single administration with anti-CD 154, ST2825 or CMC control cannot promote the prolongation of skin allografts. However, combined therapy using anti-CD 154 and ST2825 promote the permanent acceptance of fully allogeneic skin grafts (>150 days). The tolerant state was associated with reduced donor-specific IFN-y secretion while intact IL-4 secretion.[Conclusion] Tolerance induction protocol based on anti-CD154 combined with ST2825 may provide new manipulation to induce robust skin transplant tolerance. This tolerant state may be related to the reduced Th1 immune responses.更多还原