摘要
目的：临床上胰岛移植的应用给I型糖尿病患者带来了希望,同时猪的器官在器官移植中的应用解决了捐赠器官严重短缺的问题。但是,无论是同种还是异种胰岛经门静脉移植,都面临着移植后早期快速的大量胰岛丢失的问题以及受体体内产生的免疫排斥反应最终导致移植物排斥。因此本研究：①利用siRNA对新生猪胰岛细胞团的TF目标基因进行特异性地表达沉默,分析TF对NICC与血液接触后发生的IBMIR的始动作用,探寻一种更为安全有效的措施来抑制IBMIR,减少胰岛的丢失提高胰岛的植入率；②应用NOD-SCIDIL2rγ-/-小鼠作为猪胰岛移植受体,研究注射人外周血单个细胞和体外扩增的nTregs后是否可以诱导以及如何诱导对胰岛移植物的免疫耐受,建立一种以经体外扩增培养的Tregs为基础的治疗方法用以抑制T细胞介导的异种移植排斥反应,从而减轻或消除免疫抑制治疗的需求,为人nTregs在临床移植治疗的实际应用提供了参考。
方法和结果：
1.新生猪胰岛细胞组织因子基因沉默
本研究通过在新生猪胰腺原位及分离培养后的NICC免疫组织化学检测均可见TF呈阳性染色,从而证实猪的胰岛细胞自身就有TF的表达而不是因为细胞分离过程所导致的TF表达。本研究运用siRNA在基因水平上阻断猪胰岛细胞TF的表达,以抑制细胞与血液接触后触发的IBMIR,从而减少猪胰岛的丢失。实验设计了5条siRNA,单独或两两组合或三三组合按最佳转染条件分别转染NICC,结果发现其中三条siRNA组合可以使NICC的TF基因沉默效率达53%-70%,平均60%,p<0.01,且TF蛋白表达水平下降近50%。同时流式细胞仪检测结果显示,siRNA转染后,NICC的活性未受明显的影响,排除了因细胞死亡所导致NICC蛋白表达水平的降低。上述结果提示,设计的5条siRNA中有3条siRNA组合可显著降低NICC在基因及蛋白水平TF的表达,以便于进一步研究TF在IBMIR发生中的作用。
2.新生猪胰岛细胞组织因子基因沉默体外抑制IBMIR
本研究将未转染siRNA的NICC,转染对照siRNA的NICC和转染TF siRNA的NICC与人外周血在体外tubing loops模型内共孵育60分钟后,观察血凝块的大小及重量,血细胞计数检测血细胞的消耗,ELISA检测血浆中TAT及C3a的浓度,免疫组织化学检测中性粒细胞浸润及IgG沉积情况,Real-time PCR检测各组NICC的α-Gal基因表达。结果显示,转染TFsiRNA的NICC与人外周血共孵育后,不仅其血凝块的形成较其他两组受到明显的抑制,血凝块的大小和体积均有明显减小,而且血细胞计数显示血液中血小板、白细胞和中性粒细胞的消耗显著减少,补体激活受到明显的抑制。结果还显示,TF在NICC上的表达受到抑制后,血浆中TAT的浓度显著降低,中性粒细胞的浸润明显减少。上述结果提示阻断TF在胰岛上的表达可以抑制凝血、补体激活和前炎症介质的产生,也就抑制了IBMIR的发生。同时免疫荧光染色结果显示,NICC的TF表达降低并不能明显减轻血凝块中IgG在NICC周围的沉积,Real-time PCR结果表明TF特异性的siRNA对NICC TF基因的沉默作用没有影响到细胞a-Gal基因的表达,提示抗体介导的凝血激活通路并不是体外Loops模型中IBMIR发生的主要启动因子。
3.人源化小鼠模型研究人调节性T细胞介导的异种胰岛移植耐受
本研究在体外用CD4+CD25+CD 127dim/- Treg分离试剂盒从健康自愿者外周血单个核细胞分离获得CD4+CD25+调节性T细胞,应用IL-2,抗CD3/CD28抗体磁珠和雷帕霉素体外扩增纯化CD4+CD25+ Tregs,扩增后的Treg经流式细胞仪检测具有典型Treg的表型,其中CD4+CD25+Foxp3+的细胞达到90%以上；同时在体外检测扩增后的Treg的抑制功能。混合淋巴细胞反应(mixed lymphocyte reaction,MLR)的结果显示,体外扩增的Tregs可以明显抑制针对猪异种抗原的效应性T细胞的增殖效应；收集MLR各孔细胞培养上清并检测各种细胞因子的浓度,结果发现体外扩增的Tregs可显著抑制效应性T细胞分泌效应性细胞因子如IL-2和[FN-γ,而明显促进抑制性细胞因子IL-10的分泌,但细胞培养上清中TGF-β的浓度没有明显的变化。上述结果提示,经体外扩增体系扩增的Tregs具备调节性T细胞的表型和抑制功能。本研究体内实验是将分离培养的NICC移植至NOD-SCIDIL2rγ-/-受体小鼠的肾被膜下,于移植后的7天内,将1×107去除CD25+细胞的人PBMC或PBMC与2×106体外扩增的nTregs共同经尾静脉注射给接受NICC移植的NOD-SCID IL2rγ-/-小鼠体内。免疫组化用以检测移植物的存活情况,流式细胞仪检测人细胞植入情况,FACS和Real-time PCR分析移植耐受或排斥小鼠体内人nTregs的表型和基因表达。结果显示,仅接受NICC移植的NOD-SCID IL2rγ-/-小鼠,移植物可存活100天以上,免疫组化检测结果显示移植胰岛的胰岛素,胰高血糖素和生长抑素染色呈阳性,说明NOD-SCID IL2rγ-/-小鼠可以耐受猪胰岛移植物。NOD-SCID IL2rγ-/-受体小鼠静脉接受1×107去除CD25+细胞的人PBMC后,28天内免疫组化检测NICC移植物几乎被完全排斥,同时有较多的细胞浸润在移植部位,这些浸润的细胞经免疫组化进一步检测绝大部分为人CD45+细胞,其中包括CD4+及CD8+细胞,免疫荧光双染仅见CD4+细胞,未见CD4+Foxp3+细胞,流式细胞仪检测移植物浸润细胞表型的结果同样显示浸润的CD4+细胞中几乎没有CD25+Foxp3+双阳性细胞；而同时接受1×107去除CD25+细胞的人PBMC与2×106体外扩增的nTregs(?)植入的宿主小鼠NICC移植物却未被排斥,其存活时间不仅延长至100天以上,而且具有分泌胰岛素,胰高血糖素和生长抑素的功能,HE染色显示细胞植入28天可见有细胞围绕在胰岛的周围,但未浸润入胰岛中。这些围绕在胰岛周围的细胞经过免疫组化检测绝大部分亦为人CD45+细胞,其中包括CD4+及CD8+细胞,但免疫荧光双染结果显示在CD4+阳性细胞中,可见CD4+Foxp3+双阳性细胞。流式细胞仪检测移植物周围的细胞表型结果显示浸润的CD4+细胞中有高达18.6±2.3%的CD25+Foxp3+双阳性细胞。上述结果提示移植NICC的NOD-SCID IL2rγ-/-受体小鼠静脉接受1×107去除CD25+细胞的人PBMC后,人PBMC即可对猪异种抗原产生免疫应答而导致NICC移植物被排斥,参与免疫排斥的细胞主要包括CD4+和CD8+T细胞；如果同时植入体外扩增的自体2×106Tregs后,NICC移植物的排斥延迟,Treg抑制了自体PBMC对猪异种抗原产生的免疫应答而保护了NICC移植物。Real-time PCR检测NOD-SCID IL2rγ-/-受体小鼠移植部位浸润细胞基因表达水平结果同样显示Treg与PBMC共植入小鼠的Foxp3,CTLA-4及IL-10的基因表达水平明显高于仅接受PBMC植入小鼠的表达水平,p<0.01,但IFN-γ的表达水平却显著低于仅接受PBMC植入小鼠的表达水平,p<0.01；受体小鼠血清各种细胞因子浓度的检测结果显示,Treg与PBMC共植入小鼠血清中的IL-10的浓度明显高于仅PBMC植入小鼠血清IL-10的浓度,p<0.01,而IFN-γ的表达水平显著低于仅PBMC植入小鼠的表达水平,p<0.01。上述结果提示,Treg与PBMC共植入小鼠体内,Tregs可通过抑制效应性细胞因子IFN-y的表达,促进抑制性细胞因子IL-10的表达,从而抑制了人PBMC针对猪异种抗原产生的免疫排斥反应。
结论：本研究应用TF siRNA特异性地抑制了NICC TF的表达,NICC TF的基因沉默能在体外抑制IBMIR的发生；人Tregs可抑制人PBMCs(去除CD25+细胞)介导的异种免疫排斥反应而保护NOD-SCID IL2rg-/-受体小鼠NICC移植物,且Tregs在体内的抑制功能与IL-10参与有关。拟人化NOD-SCID IL2rg-/-小鼠在胰岛异种移植中人Tregs介导的免疫耐受及其机制的研究中是一种非常合适的模型。
Clinical islet transplantation is a promising treatment for typeⅠdiabetes and using pig organs could solve the significant increasing shortage of donor organs for allotransplantation. However early innate immune destruction of islets and rejection are major problems preventing clinical trials. Therefore in this study,①we used small interfering RNAs (siRNAs)-mediated gene knockdown to specifically silence TF gene expression in neonatal porcine islet cell clusters (NICC). This gene targeting approach allowed us to not only analyze directly the importance of TF in the initiation of IBMIR mediated by NICC, but also to explore the potential targets for developing strategies that can enhance islet engraftment and survival;②Using NOD-SCID IL2rγ-/- mice as recipients for pig islet transplantation, we studied whether and how immune tolerance of islet grafts to T-cell mediated cellular rejection is induced by reconstitution of these mice with human PBMCs and nTregs and produced a cell-based therapy that can be developed ex vivo that will suppress the T cell mediated response to the xeno-graft and thereby reduce or eliminate the requirement for immunosuppression.
1. TF knockdown in NICC
TF is expressed on newly isolated NICC and can be detected on neonatal pig pancreas tissue prior to NICC production confirming that it is not a by product of the isolation procedure. In order to test the hypothesis that specific suppression of islet TF expression would lead to a significant inhibition of IBMIR, small interfering RNA (siRNA) was used to knockdown TF gene expression in NICC. 5 pairs of siRNA were designed and transfected into NICC respectively or with different combinations under the optimal conditions. A 60%(50%-70%) and a 50% reduction in TF gene and protein expression respectively was achieved with selected 3 pairs of siRNA. FACS results showed that siRNA transfection had no significant effect on the viability of NICC used in these experiments. These data suggested that siRNA transfection was sufficient to specifically knockdown TF gene and protein expression in NICC.
2. Suppressed TF expression in NICC led to inhibition of IBMIR in vitro
After transfection, non-transfected, control siRNA transfected and TF siRNA transfected NICC were cultured with ABO compatible human blood in loops tubing respectively for 60 mins. Clot size and weight and blood cell counts were determined. Plasma was analyzed to determine levels of thrombin-antithrombin complexes (TAT) and C3a using commercially available ELISA kits. Immunohistochemical staining for neutrophil elastase and human IgG were used to determine neutrophil infiltration and antibody deposit in clots.α-Gal gene expression as assessed by real-time PCR. The results showed that TF siRNA resulted in substantially reduced not only clot size and weight but also the consumption of platelets, white blood cells and neutrophils. Consistent with these findings TAT and C3a levels were significantly less in loops containing TF siRNA transfected NICC compared to those with non-transfected or control siRNA transfected NICC. When TF siRNA transfected NICC were included in the loops with human blood, the number of infiltrating neutrophils within the clot was decreased considerably. These data showed that TF expression was an important initiator of the NICC mediated inflammatory response and this response was inhibited by TF knockdown. Meantime immunofluorescence staining of NICC containing clots taken from the loops showed strong deposition of human IgG which was not diminished when TF siRNA transfected NICC were used and a-Gal gene expression as assessed by real-time PCR was similar in all three groups. These data suggested that antibody mediated activation of the alternative pathway of thrombosis was not the major initiating event in this study.
3. Islet xenograft protection by ex vivo-expanded human regulatory T cells in humanized NOD-SCID IL2rγ-/- mice
CD4+CD25+CD127 dim/- T cells were isolated from PBMC by using a CD4+CD25+CD127 dim/- regulatory T cell isolation kit. Fresh Treg cells were cultured with human IL-2, CD3/CD28 Dynabeads and rapamycin for expansion. The expanded cells had the typical phenotype of Tregs and the percentage of CD4+CD25+Foxp3+ triple positive cells was over 90%; Mixed lymphocyte reaction (MLR) was used to investigate the suppression function of expanded Tregs. The results showed that expanded human Treg cells were able to suppress antiporcine proliferative responses in vitro. The concentration of IL-2 and IFN-γwere reduced and the IL-10 level was upregulated in the co-cultured cells supernatant, but the concentration of TGF-βhad no change. These data demonstrated that expanded Treg cells retained the phenotype and suppressive activity. NICC were transplanted to NOD-SCID IL2rγ-/1 mice under renal capsule.1×10'CD25+cell-depleted human PBMCs were co-transferred with or without 2×106 expanded human nTregs into the NICC recipient NOD-SCID IL2rγ-/- mice within 7 days after transplantation. Graft survival was monitored by histology. Human cell engraftment was determined by FACS. Phenotype of CD25-human cells and expanded human Tregs and their gene expression were analysed by FACS and real-time PCR. The results showed that NICC xenograft was intact with insulin, glucogan and somatostatin positive staining in NOD-SCID IL2rγ-/- recipient mice without human cell transfer for at least 100 days after transplantation. While NICC grafts were rejected in NOD-SCID IL2rγ-/- recipient mice 28 days after transfer with 1x107 human CD25+ cell-depleted PBMCs in the absence of autologous Tregs. There were lots of cells infiltrated in the graft which were CD45+ cells including CD4+ and CD8+ cells detected by immunohistology and only CD4 positive staining cells were detected by immunofluorescence double staining. NICC graft infiltrating human CD4+ cells isolated from rejecting NOD-SCID IL2rg-/- recipients did not express Foxp3 and IL-10 but IFN-γdetermined by FACS. In contract, NICC xenografts in human Treg transferred NOD-SCID IL2rγ-/- recipients still remained intact up to at least 100 days with insulin, glucogan and somatostatin positive staining after rechallenge with CD25+ cell-depleted human PBMC. CD45+ cells which included CD4+ and CD8+ cells didn't infiltrate but surround the graft detected by immunohistology and CD4 positive staining cells and CD4+Foxp3+double positive cells were detectable by immuno-fluorescence double staining. A large number of human CD4+ T cells expressed CD25 and Foxp3 (18.6±2.3%) in human Tregs transferred NICC recipient NOD-SCID IL2ryγ-/- mice at 28 day until to 100 day after rechallenging with human PBMCs. These data suggested that transfer of NOD-SCID IL2rγ-/- recipients with 1 x 107 CD25+ cell-depleted human PBMCs was sufficient to cause NICC xenograft rejection and the human PBMC-mediated xenograft rejection was significantly suppressed by human expanded Tregs which led to a long-term NICC graft survival. High level expression of Treg (Foxp3, CTLA-4, IL-10) but not T effect cell (IFN-y) associated genes was detected in NICC grafts from human Treg transferred NICC recipient mice 28 days after human PBMCs rechallenge by real-time PCR and human Tregs transferred NICC recipients demonstrated reduced level of human IFN-y and elevated level of human IL-10 in their serum 28 days after rechallenge with CD25+ cell-depleted human PBMC detected by cytometric bead array. These data suggest that the prolonged xenograft survival in the human Treg transferred and PBMC rechallenged NOD-SCID IL2ryγ-/- recipients was associated with the presence of human Tregs coexpressing IL-10 and inhibited production of effector cytokine IFN-y in these mice.
Conclusions:Tissue factor expression in NICC can be reduced by siRNA mediated knockdown and TF knockdown can reduce IBMIR in vitro. Human expanded Tregs were capable of protecting NICC xenograft from rejection mediated by CD25+ cell-depleted human PBMC in NOD-SCID IL2rg-/- recipients and IL-10 was required for human Treg-mediated suppression of the human anti-pig xenogeneic response in vivo. Humanized NOD-SCID IL2rg-/- mice are a suitable mouse model to study human Treg-mediated tolerance and the mechanisms involved in islet xenotransplantation.
引文
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