雷帕霉素促进小鼠CD4~+CD25~+T细胞增殖和Foxp3表达的机制研究
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摘要
器官移植后的排斥反应仍然是影响移植物存活的主要障碍,积极预防排斥反应和并发症对移植脏器的存活时间和受者的生活质量十分重要。器官移植排斥反应分为宿主抗移植物反应(HVGR)和移植物抗宿主反应(GVHR),我们通常所说的移植排斥反应即HVGR。HVGR分为超急排斥反应、加速排斥反应、急性排斥反应和慢性排斥反应这4种类型。目前,在器官移植的临床治疗中仍然存在以下问题急待解决：一是虽然临床使用免疫抑制剂能有效控制急性排斥反应,但对于慢性排斥反应,无论从病因、发生机制、病理过程等均缺乏深刻的认识,目前仍只能以对症治疗为主,缺乏有效的防治措施和方法,一旦出现慢性排斥反应,不仅影响移植物的功能和存活,最终往往需要二次移植。另一方面,虽然环孢素A(CsA)、他克莫司(FK506)等新型免疫抑制剂发展和临床应用,有效地抑制了移植排斥反应,大大提高了移植物的存活期,推动了临床器官移植的发展,但这些免疫抑制剂往往费用昂贵且缺乏特异性,抑制人体的免疫系统的同时也导致了明显的毒副作用,诱发感染、骨髓抑制和肿瘤等各种并发症,严重影响患者的生活质量并加重患者和社会的负担。因此,深入研究免疫抑制药物抗排斥的作用机理,对于阐明移植排斥反应、控制慢性排斥反应、维持移植物的长期存活并尽可能地消除或减少药物的毒副作用,具有重要的科学意义和应用价值。
器官移植后的排斥反应,本质是受者免疫系统针对供者移植物抗原产生的免疫应答。同种器官移植后,移植物中残留有过客白细胞(passenger leukocyte),主要为成熟的DC和巨噬细胞等抗原提呈细胞(donor antigen-prsenting cells, dAPC)。这些细胞在移植物血管与受者血管接通后,可进入受者血液循环或局部引流淋巴组织或受者T细胞也可进入移植物中,将其表面MHC分子或抗原肽/MHC分子复合物(pMHC)通过直接识别(direct allorecognition)机制直接提呈给受者体内的同种反应性T细胞(主要为CD8+T细胞及CD4+T细胞)；移植物的脱落细胞或MHC抗原经受者APC加工和处理后,以间接识别(indirect allorecognition)方式将供者抗原肽(即同种MHC分子片段)／受者MHC分子复合物的形式提呈给受者CD4+T细胞,产生免疫应答。活化的T细胞分化为Thl细胞以及新近发现的Th17细胞,通过分泌细胞因子、趋化因子进一步活化CD8+CTL细胞、趋化巨噬细胞、中性粒细胞等炎性细胞产生炎症反应,介导对移植物的排斥反应。因此,临床上常用的免疫抑制剂虽然作用机制不一,但都是主要通过抑制免疫细胞T淋巴细胞的活化、分化、增殖发挥抗移植排斥的作用。近年来,大量的研究发现体内存在一群含量极少的CD4+CD25+Foxp3+T细胞(Treg细胞),以主动的方式参与免疫应答的调控,在对自身和外源性抗原免疫耐受的诱导和免疫耐受的稳定、维持中发挥重要作用,在调控免疫反应中的作用受到人们的重视,成为移植免疫学研究的热点。因此,研究Treg细胞的分化、增殖的机制以及体内外扩增以诱导移植免疫耐受的方法对于防治排斥反应、减少免疫抑制剂的使用具有重要的意义,尤其是研究目前临床广泛使用的移植排斥抑制剂如CsA、FK506、雷帕霉素(Rapamycin, Rapa)等对Treg细胞的作用,对于指导临床免疫抑制剂的使用、更有效地防治排斥反应意义重大。
Rapa最初发现时仅作为一种低毒性的抗真菌药物使用,自1977年发现其具有免疫抑制作用后,于1989年开始试用作治疗器官移植后排斥反应的新药。从目前动物实验及临床应用的效果看,Rapa是一种高效低毒的新型免疫抑制剂。最新的研究表明Rapa具有促进Treg细胞产生的作用,但对其促进Treg产生的分子机制仍未明确。本课题拟研究Rapa促进CD4+CD25+Treg细胞分化、产生和增殖的细胞及分子机制,为指导临床用药、减少免疫抑制剂的使用及更有效地防治排斥反应提供实验数据和研究依据。
第一部分雷帕霉素对体外培养的CD4+CD25+T细胞Foxp3和细胞因子表达的影响
目的：通过观察雷帕霉素与CD4+CD25+T细胞体外共培养后对其增殖情况及Foxp3、TGF-β等细胞因子表达的影响,探讨雷帕霉素对CD4+CD25+T细胞产生和Foxp3表达的影响与TGF-p之间的关系。方法：无菌条件下取C57BL/6小鼠脾脏,分离单个核细胞,磁珠分选CD4+CD25+T细胞,按对照组、刺激组、雷帕霉素组、环孢霉素A组分组,aCD28+aCD3和低剂量IL-2刺激共培养72h。采用流式细胞仪检测CFSE标记的CD4+CD25+T细胞增殖情况和胞内染色标记的Foxp3表达,通过实时荧光定量RT-PCR法和ELISA法检测Foxp3、TGF-β等细胞因子基因和蛋白表达水平。结果：与刺激组比较,FACS检测发现雷帕霉素处理组的CD4+CD25+T细胞出现更明显的增殖；Foxp3的mRNA及平均荧光强度均明显高于刺激组；环孢素A处理组的CD4+CD25+T的增殖、Foxp3的mRNA及平均荧光强度均低于刺激组,提示雷帕霉素具有促进CD4+CD25+T细胞的增殖和Foxp3表达的作用而环孢素A则起抑制作用。进一步检测细胞因子的表达发现,雷帕霉素处理后的CD4+CD25+T细胞表达TGF-βmRNA和蛋白水平明显高于刺激组(P<0.01),相反环孢素A处理后的CD4+CD25+T细胞表达TGF-βmRNA和蛋白水平低于刺激组(P<0.05),提示Rapa促进CD4+CD25+T细胞表达和分泌TGF-β,而环孢素A抑制CD4+CD25+T细胞TGF-β的表达和分泌。结论：雷帕霉素能诱导体外培养的CD4+CD25+T细胞增殖与Foxp3和TGF-β的表达,而环孢霉素A体外抑制CD4+CD25+Treg细胞的增殖与Foxp3和TGF-β的表达,提示雷帕霉素对CD4+CD25+T细胞产生和Foxp3表达的促进作用可能与诱导TGF-β表达分泌有关。
第二部分阻断TGF-β作用对雷帕霉素处理的CD4+CD25+T细胞Foxp3表达的影响
目的：通过TGF-β中和抗体和Smad3缺陷小鼠脾脏CD4+CD25+T观察阻断TGF-β信号通路途径后,雷帕霉素对CD4+CD25+T细胞Foxp3的表达的影响,研究TGF-β在雷帕霉素处理的CD4+CD25+T细胞中对Foxp3表达的作用。方法：无菌条件下取C57BL/6小鼠脾脏,分离单个核细胞,磁珠分选CD4+CD25+T细胞,按刺激组、雷帕霉素组、雷帕霉素+抗TGF-β组分组,aCD28+aCD3和低剂量IL-2刺激共培养72h。通过实时荧光定量RT-PCR法检测Foxp3 mRNA表达水平,采用流式细胞仪检测CD4+CD25+T细胞的Foxp3表达。同样在无菌条件磁珠分选Smad3缺陷小鼠脾脏CD4+CD25+T细胞,加入雷帕霉素、aCD28+aCD3和低剂量IL-2刺激共培养72h后,采用实时荧光定量RT-PCR法和流式细胞仪检测CD4+CD25+T细胞的Foxp3表达。结果：与刺激组相比,雷帕霉素处理组CD4+CD25+T细胞的Foxp3mRNA表达水平显著增高(P<0.01),雷帕霉素+抗TGF-β组则无明显差异(P>0.05),提示雷帕霉素对Foxp3的诱导作用被中和抗体所抑制；而各组CD4+CD25+T细胞的Foxp3的表达比例分别为刺激组87.24%、雷帕霉素组93.62%、雷帕霉素+抗TGF-β组47.36%,提示阻断TGF-β作用能抑制雷帕霉素对Foxp3表达的促进作用；雷帕霉素处理的Smad3缺陷小鼠脾脏CD4+CD25+T细胞foxp3 mRNA和蛋白表达均较野生型对照组明显减低(P<0.01),提示阻断TGF-p信号通路能抑制雷帕霉素对CD4+CD25+T细胞Foxp3的表达的促进作用。结论：阻断TGF-p作用能抑制雷帕霉素诱导体外培养的CD4+CD25+T细胞Foxp3的表达,雷帕霉素对CD4+CD25+T细胞Foxp3表达的促进作用可能与诱导TGF-p表达分泌及TGF-p信号通路有关。
第三部分雷帕霉素对同种心脏移植小鼠体内CD4+CD25+Foxp3+T细胞的影响以及与TGF-β信号通路的关系
目的：使用雷帕霉素或环孢霉素A治疗同种异位心脏移植小鼠,观察小鼠体内CD4+CD25+T细胞增殖情况和Foxp3、TGF-β表达及移植物生存时间,进一步探讨雷帕霉素对CD4+CD25+T细胞产生和Foxp3表达的影响与TGF-β之间的关系及其抗移植排斥效果。方法：应用Cuff法建立雄性BALB/C→C57BL/10小鼠颈部异位心脏移植模型,根据不同处理情况,将受体随机分为4组(n=6)；未手术组、移植对照组、移植雷帕霉素治疗组(1.5mg/kg/d)和移植环孢霉素A治疗组(20mg/kg/d)。流式细胞仪检测移植术后第14天小鼠体内胸腺、脾脏和淋巴结CD4+CD25+T细胞比例和foxp3表达情况,实时荧光定量RT-PCR检测脾脏细胞TGF-βmRNA水平；取不同时间点进行小鼠眼眶采血收集外周血通过ELISA方法检测外周血血清TGF-β分泌水平的变化；观察小鼠移植物生存期和病理改变。结果：雷帕霉素治疗14天后的小鼠脾脏、胸腺和淋巴结中的CD4+CD25+T细胞和Foxp3表达均明显高于环孢素A处理组、移植对照组和未手术组；脾脏细胞TGF-p的mRNA改变与流式结果一致。ELISA法检测发现血清中的TGF-p水平不但明显高于环孢素A处理组,也明显高于移植对照组和未手术组,提示雷帕霉素可促进移植术后小鼠体内CD4+CD25+T细胞增殖和foxp3的表达,增加TGF-β表达分泌；而环孢素A不仅抑制移植术后小鼠体内CD4+CD25+T细胞增殖和foxp3的表达,也抑制TGF-β表达分泌。移植术后各组移植物的存活时间分别为移植对照组6.67±0.81天、雷帕霉素治疗组20.17±3.31天和环孢素A治疗组2133±4.17天；病理切片HE染色可见移植对照组心肌间淋巴细胞浸润严重,而雷帕霉素治疗组和环孢素A治疗组心肌间淋巴细胞浸润程度相当且均较移植对照组明显减轻,提示雷帕霉素能显著延长移植物的存活时间和抑制移植物的炎症反应(P<0.01),与环孢素A效果相当(P>0.05)；结论：雷帕霉素可能通过诱导TGF-p表达和分泌来促进作用CD4+CD25+Foxp3+T细胞的产生。雷帕霉素对同种异体的小鼠心脏移植中具有明显的抗移植排斥效果,可能与其在体内促进CD4+CD25+T细胞的产生和Foxp3的表达有关。
Allograft rejection has been one of major problems for allograft survival. It is very important to actively prevent the transplant recipient from rejection and complications for their survival time and the quality of life. Organ transplant rejection includes the host anti-graft reaction (HVGR), which is commonly referred to as organ transplant rejection, and graft-versus-host reaction (GVHR). HVGR is divided into 4 types:hyperacute rejection, accelerated rejection, acute rejection and chronic rejection. At present, problems in clinical transplant rejection therapy remained to be solved. One of them is immunosuppressants such as CsA and FK506 can efficiently control acute rejection, but have poor efficiency for chronic rejection, in view that lack of gnosia on mechanism of chronic rejection devolpment and its pathology. One the other hand, the lifelong use of immunosuppressant drugs can cause adverse reactions, such as the bone marrow suppression, cancer, infection, chronic loss of the transplanted organ function, as well as their own drug side effects and high costs. Therefore, it is significant to study the mechanism of immunosuppressive drugs on rejection for controlling of chronic graft rejection and maintaining long-term survival of great.
Rejection after organ transplantation is the essence of the immunity system against the donor graft antigens in the immune response. After allogeneic organ graft operation, the passenger leukocytes in allograft are primarily mature DC and macrophages and other antigen presenting cells (donor antigen-prsenting cells, dAPC). These cells may enter to the blood circulation or in part lymphoid tissue when donor blood vessels were connected to blood vessels, and directly submit the surface MHC molecules or antigenic peptide/ MHC molecule complexes (pMHC) to the recipient allogeneic T cells (mainly CD8+ T cells and CD4+ T cells) by direct recognition mechanism; The donor antigen (the same kinds of MHC molecules fragment)/recipient MHC molecule complexes from graft exfoliated cells or MHC antigen after processing by APC were submitted to the recipient CD4+ T cells by indirect recognition mechanism to produce immune responses. Activated T cells differentiate into Thl cells and the newly discovered Th17 cells, through secretion of cytokines, chemokines and further activation of CD8+ CTL cells, chemotaxis of macrophages, neutrophils and other inflammatory cells cause inflammation and mediate on graft rejection. Although the mechanism of the immunosuppressant used in clinical could not be coincidence, the same point is that both of them can inhibit the activation, differentiation and proliferation of the T lymphocyte. In recent years, a large number of studies have found that a group of content little CD4+CD25+Foxp3+T cells (Treg cells), as an important immune regulatory function of T cells to mediate immune response in a proactive manner, played an extremely important role in the regulation of maintenance immune tolerance to exogenous antigens or their own. Therefore, it is very important to study the negative regulation mechanism of Treg cells in control of transplant rejection and the dose of immunosuppressant drugs. In particular, studies of the immunosuppressive drugs widely used in clinical such as CsA, FK506, Rapamycin (Rapa) on Treg cells is for guiding the clinical usage of immunosuppressive agents and more effectively control of rejection significant.
Rapa first discovered is only used as a low-toxicity antifungal. After immunosuppressive effect was found in 1977, Rapa began trial in the treatment of organ transplant rejection in 1989. From the effects of the present animal experiments and clinical application, Rapa is a highly efficient and low toxicity of new immunosuppressants. The latest research shows that Rapa can promote the generation of Treg cells, but the molecular mechanisms are not yet clear. To study the molecular mechanisms of Rapa that effects on the differentiation and proliferation of CD4+CD25+ Treg cells will provide the basis of experimental data for guiding clinical treatment and reducing the use of immunosuppressive agents and efficiently controling of rejection to improve the survival of organ graft.
Part one: The influence of Rapa on the expression of Foxp3 and TGF in CD4+CD25+ T cells in vitro
Objective:To observe the proliferation and expression of Foxp3, TGF-βand explore the relationship between TGF-βand the expression of Foxp3 in CD4+CD25+ T cells when Rapa and CD4+CD25+ T cells co-cultured in vitro. Methods:Take spleen from C57BL/ 6 mice under sterile conditions, isolate mononuclear cells and separate CD4+CD25+ T cells by immunomagnetic beads, divide into control group, stimulated group, Rapa group and CsA group; Cells are stimulated with anti-CD28, anti-CD3 and low-dose IL-2 for 72h under, then detect the proliferation of CD4+CD25+ T cells and the expression of Foxp3 by the flow cytometry, the expression of Foxp3 mRNA by Semi-quantitative RT-PCR and the Foxp3 by and ELISA, Results:The proliferation of CD4+CD25+ T cells and the average fluorescence intensity of Foxp3 in Rapa group have more obviously was significantly higher than the stimulated group by FACS detecting; howere, these indicators in CsA group were lower then the stimulated group. Our results suggest that Rapa can promote the proliferation and the expression of Foxp3 in CD4+CD25+T cell, but CsA inhibits these functiones. To further test the cytokines expression, we found that the CD4+CD25+ T cells in Rapa group express TGF-βmRNA and protein were significantly higher than that stimulated group (P<0.01); To contrast, the CD4+CD25+T cells in CsA group express TGF-βmRNA and protein were were lower than the stimulation (P<0.05). The results show that Rapa can promote the expression and the secretion of TGF-βin CD4+CD25+ T cell, while CsA inhibit the expression and the secretion of TGF-βin CD4+CD25+ T cell. Conclusion:Rapa could induce the CD4+CD25+ T cell proliferation and the Foxp3/TGF-βexpression in vitro; the results documented that the effect that Rapa promote CD4+CD25+ T cells proliferation and the Foxp3 expression may be related to the expression and secretion of TGF-β.
Part two:TGF-βblocking reduces Rapa induced Foxp3 expression in CD4+CD25+ T cells
Objective:To observe the expression of Foxp3 in Rapa-treated CD4+CD25+ T cells when blocking TGF-P by TGF-βneutralizing antibody and smad3-deficient mice, study the effect of TGF-βon the expression Foxp3 in Rapa-treated CD4+CD25+ T cells. Methods: Take spleen from C57BL/6 mice under sterile conditions, isolat mononuclear cells, separate CD4+CD25+ T cells by immunomagnetic beads, divide into stimulated group, Rapa group and CsA group, Rapa plus anti-TGF-βgroup; Cells are stimulated with anti-CD28, anti-CD3 and low-dose IL-2 for 72h under, then detect the expression of Foxp3 mRNA by Semi-quantitative RT-PCR and the expression of Foxp3 in CD4+CD25+ T cells by flow cytometry.Take spleenic CD4+CD25+ T cells in smad3-/- mice or WT mice under sterile conditions, stimulate with Rapa, anti-CD28, anti-CD3 and low-dose IL-2 for 72h, then detect expression the Foxp3 by Semi-quantitative RT-PCR and ELISA.
Results:The expression of Foxp3 mRNA in Rapa group was significantly higher than that in the stimulated group (P<0.01); but that in Rapa plus anti-TGF-βgroup was no significant difference (P>0.05), indicating that the induction of Rapa on Foxp3 was inhibited by TGF-P neutralizing antibodies; and the fluorescence intensities of Foxp3 in each group are respectively to 87.24% in stimulated group,93.62% in Rapa group, 47.36% in Rapa and anti-TGF-P group, suggesting that blocking TGF-βcan inhibit Rapa to promote the expression of Foxp3.The expression of foxp3 mRNA and protein in the spleenic CD4+CD25+ T cells from smad3-/- mice in Rapa treatment was significantly lower (P<0.01)than those of wild-type control group, suggesting that blocking TGF-βsignaling pathway can inhibit the Rapa on CD4+CD25+ T cells to promoe the expression of Foxp3.Conclusion:Blocking TGF-βcan inhibit Rapa-induced the expression of Foxp3 in CD4+CD25+ T cells in vitro; The effect of Rapa on to promotion of the expression of Foxp3 in CD4+CD25+ T cells may be related to induce the expression and secretion of TGF-p.
Part three: Rapa promotes CD4+CD25+Foxp3+ T cells generation in heart
transplantation mouse through TGF-signaling pathway
Objective:To further explore the effect of Rapa on the transplant rejection and the influence of TGF-signaling pathway on the relationship between Rapa and the expression of Foxp3 in CD4+CD25+ T cells after allogeneic heart transplantation. Methods:After performing cervical heterotopic heart transplantation with allograft heart from the male BALB/C mice, C57BL/10 recipients were randomly divided into 4 groups (n=6): Normal group, Control group, Rapa group (1.5mg/kg/d) and CsA group (20mg/kg/d) are observed the graft survival time and the pathological changes. After 14 days, the ratioes of CD4+CD25+ T cells in thymus, spleen and lymph node and Foxp3 expression were detected by flow cytometry. The expression of TGF-βmRNA in spleenic CD4+CD25+ T cell are detected by semi-quantitative RT-PCR and the peripheral blood were collected at different time points to determine TGF-βconcentration by ELISA. Results:After 14 days, the ratioes of CD4+CD25+T cells as well as the Foxp3 expression of Treg cells in thymus, spleen and lymph node of Rapa treatment mice were significantly higher than that in CsA treatment group or control group. The TGF-P mRNA expression in spleenic CD4+CD25+T cells were consistent with the changes of the popularation of CD4+CD25+ T cells as well as the expression of Foxp3. The serum level of TGF-P in Rapa treatment group was not only significantly higher than that of CsA treatment group, but also significantly higher than that of control group. The results show that Rapa can promote the proliferation of CD4+CD25+ T cells, the expression of foxp3 and the secretion of TGF-P in allograft mice. In contrast, CsA not only inhibits the proliferation of CD4+CD25+ T cells and the expression of foxp3, but also inhibits the secretion of TGF-β. The heart allograft survival time of Rapa treatment group were significantly prolonged (20.17±3.31 days) compared with the control group(6.67±0.81 days), and compariable with that of CsA treatment group (21.33±4.17 days). The results suggest that Rapa could inhibit graft inflammation and significantly prolong the allograft survival time. Conclusion:Rapa prolong allograft survival related to promote the generation of CD4+CD25+ T cells and the expression of Foxp3 through TGF-βsignaling pathway.
器官移植后的排斥反应,本质是受者免疫系统针对供者移植物抗原产生的免疫应答。同种器官移植后,移植物中残留有过客白细胞(passenger leukocyte),主要为成熟的DC和巨噬细胞等抗原提呈细胞(donor antigen-prsenting cells, dAPC)。这些细胞在移植物血管与受者血管接通后,可进入受者血液循环或局部引流淋巴组织或受者T细胞也可进入移植物中,将其表面MHC分子或抗原肽/MHC分子复合物(pMHC)通过直接识别(direct allorecognition)机制直接提呈给受者体内的同种反应性T细胞(主要为CD8+T细胞及CD4+T细胞)；移植物的脱落细胞或MHC抗原经受者APC加工和处理后,以间接识别(indirect allorecognition)方式将供者抗原肽(即同种MHC分子片段)／受者MHC分子复合物的形式提呈给受者CD4+T细胞,产生免疫应答。活化的T细胞分化为Thl细胞以及新近发现的Th17细胞,通过分泌细胞因子、趋化因子进一步活化CD8+CTL细胞、趋化巨噬细胞、中性粒细胞等炎性细胞产生炎症反应,介导对移植物的排斥反应。因此,临床上常用的免疫抑制剂虽然作用机制不一,但都是主要通过抑制免疫细胞T淋巴细胞的活化、分化、增殖发挥抗移植排斥的作用。近年来,大量的研究发现体内存在一群含量极少的CD4+CD25+Foxp3+T细胞(Treg细胞),以主动的方式参与免疫应答的调控,在对自身和外源性抗原免疫耐受的诱导和免疫耐受的稳定、维持中发挥重要作用,在调控免疫反应中的作用受到人们的重视,成为移植免疫学研究的热点。因此,研究Treg细胞的分化、增殖的机制以及体内外扩增以诱导移植免疫耐受的方法对于防治排斥反应、减少免疫抑制剂的使用具有重要的意义,尤其是研究目前临床广泛使用的移植排斥抑制剂如CsA、FK506、雷帕霉素(Rapamycin, Rapa)等对Treg细胞的作用,对于指导临床免疫抑制剂的使用、更有效地防治排斥反应意义重大。
Rapa最初发现时仅作为一种低毒性的抗真菌药物使用,自1977年发现其具有免疫抑制作用后,于1989年开始试用作治疗器官移植后排斥反应的新药。从目前动物实验及临床应用的效果看,Rapa是一种高效低毒的新型免疫抑制剂。最新的研究表明Rapa具有促进Treg细胞产生的作用,但对其促进Treg产生的分子机制仍未明确。本课题拟研究Rapa促进CD4+CD25+Treg细胞分化、产生和增殖的细胞及分子机制,为指导临床用药、减少免疫抑制剂的使用及更有效地防治排斥反应提供实验数据和研究依据。
第一部分雷帕霉素对体外培养的CD4+CD25+T细胞Foxp3和细胞因子表达的影响
目的：通过观察雷帕霉素与CD4+CD25+T细胞体外共培养后对其增殖情况及Foxp3、TGF-β等细胞因子表达的影响,探讨雷帕霉素对CD4+CD25+T细胞产生和Foxp3表达的影响与TGF-p之间的关系。方法：无菌条件下取C57BL/6小鼠脾脏,分离单个核细胞,磁珠分选CD4+CD25+T细胞,按对照组、刺激组、雷帕霉素组、环孢霉素A组分组,aCD28+aCD3和低剂量IL-2刺激共培养72h。采用流式细胞仪检测CFSE标记的CD4+CD25+T细胞增殖情况和胞内染色标记的Foxp3表达,通过实时荧光定量RT-PCR法和ELISA法检测Foxp3、TGF-β等细胞因子基因和蛋白表达水平。结果：与刺激组比较,FACS检测发现雷帕霉素处理组的CD4+CD25+T细胞出现更明显的增殖；Foxp3的mRNA及平均荧光强度均明显高于刺激组；环孢素A处理组的CD4+CD25+T的增殖、Foxp3的mRNA及平均荧光强度均低于刺激组,提示雷帕霉素具有促进CD4+CD25+T细胞的增殖和Foxp3表达的作用而环孢素A则起抑制作用。进一步检测细胞因子的表达发现,雷帕霉素处理后的CD4+CD25+T细胞表达TGF-βmRNA和蛋白水平明显高于刺激组(P<0.01),相反环孢素A处理后的CD4+CD25+T细胞表达TGF-βmRNA和蛋白水平低于刺激组(P<0.05),提示Rapa促进CD4+CD25+T细胞表达和分泌TGF-β,而环孢素A抑制CD4+CD25+T细胞TGF-β的表达和分泌。结论：雷帕霉素能诱导体外培养的CD4+CD25+T细胞增殖与Foxp3和TGF-β的表达,而环孢霉素A体外抑制CD4+CD25+Treg细胞的增殖与Foxp3和TGF-β的表达,提示雷帕霉素对CD4+CD25+T细胞产生和Foxp3表达的促进作用可能与诱导TGF-β表达分泌有关。
第二部分阻断TGF-β作用对雷帕霉素处理的CD4+CD25+T细胞Foxp3表达的影响
目的：通过TGF-β中和抗体和Smad3缺陷小鼠脾脏CD4+CD25+T观察阻断TGF-β信号通路途径后,雷帕霉素对CD4+CD25+T细胞Foxp3的表达的影响,研究TGF-β在雷帕霉素处理的CD4+CD25+T细胞中对Foxp3表达的作用。方法：无菌条件下取C57BL/6小鼠脾脏,分离单个核细胞,磁珠分选CD4+CD25+T细胞,按刺激组、雷帕霉素组、雷帕霉素+抗TGF-β组分组,aCD28+aCD3和低剂量IL-2刺激共培养72h。通过实时荧光定量RT-PCR法检测Foxp3 mRNA表达水平,采用流式细胞仪检测CD4+CD25+T细胞的Foxp3表达。同样在无菌条件磁珠分选Smad3缺陷小鼠脾脏CD4+CD25+T细胞,加入雷帕霉素、aCD28+aCD3和低剂量IL-2刺激共培养72h后,采用实时荧光定量RT-PCR法和流式细胞仪检测CD4+CD25+T细胞的Foxp3表达。结果：与刺激组相比,雷帕霉素处理组CD4+CD25+T细胞的Foxp3mRNA表达水平显著增高(P<0.01),雷帕霉素+抗TGF-β组则无明显差异(P>0.05),提示雷帕霉素对Foxp3的诱导作用被中和抗体所抑制；而各组CD4+CD25+T细胞的Foxp3的表达比例分别为刺激组87.24%、雷帕霉素组93.62%、雷帕霉素+抗TGF-β组47.36%,提示阻断TGF-β作用能抑制雷帕霉素对Foxp3表达的促进作用；雷帕霉素处理的Smad3缺陷小鼠脾脏CD4+CD25+T细胞foxp3 mRNA和蛋白表达均较野生型对照组明显减低(P<0.01),提示阻断TGF-p信号通路能抑制雷帕霉素对CD4+CD25+T细胞Foxp3的表达的促进作用。结论：阻断TGF-p作用能抑制雷帕霉素诱导体外培养的CD4+CD25+T细胞Foxp3的表达,雷帕霉素对CD4+CD25+T细胞Foxp3表达的促进作用可能与诱导TGF-p表达分泌及TGF-p信号通路有关。
第三部分雷帕霉素对同种心脏移植小鼠体内CD4+CD25+Foxp3+T细胞的影响以及与TGF-β信号通路的关系
目的：使用雷帕霉素或环孢霉素A治疗同种异位心脏移植小鼠,观察小鼠体内CD4+CD25+T细胞增殖情况和Foxp3、TGF-β表达及移植物生存时间,进一步探讨雷帕霉素对CD4+CD25+T细胞产生和Foxp3表达的影响与TGF-β之间的关系及其抗移植排斥效果。方法：应用Cuff法建立雄性BALB/C→C57BL/10小鼠颈部异位心脏移植模型,根据不同处理情况,将受体随机分为4组(n=6)；未手术组、移植对照组、移植雷帕霉素治疗组(1.5mg/kg/d)和移植环孢霉素A治疗组(20mg/kg/d)。流式细胞仪检测移植术后第14天小鼠体内胸腺、脾脏和淋巴结CD4+CD25+T细胞比例和foxp3表达情况,实时荧光定量RT-PCR检测脾脏细胞TGF-βmRNA水平；取不同时间点进行小鼠眼眶采血收集外周血通过ELISA方法检测外周血血清TGF-β分泌水平的变化；观察小鼠移植物生存期和病理改变。结果：雷帕霉素治疗14天后的小鼠脾脏、胸腺和淋巴结中的CD4+CD25+T细胞和Foxp3表达均明显高于环孢素A处理组、移植对照组和未手术组；脾脏细胞TGF-p的mRNA改变与流式结果一致。ELISA法检测发现血清中的TGF-p水平不但明显高于环孢素A处理组,也明显高于移植对照组和未手术组,提示雷帕霉素可促进移植术后小鼠体内CD4+CD25+T细胞增殖和foxp3的表达,增加TGF-β表达分泌；而环孢素A不仅抑制移植术后小鼠体内CD4+CD25+T细胞增殖和foxp3的表达,也抑制TGF-β表达分泌。移植术后各组移植物的存活时间分别为移植对照组6.67±0.81天、雷帕霉素治疗组20.17±3.31天和环孢素A治疗组2133±4.17天；病理切片HE染色可见移植对照组心肌间淋巴细胞浸润严重,而雷帕霉素治疗组和环孢素A治疗组心肌间淋巴细胞浸润程度相当且均较移植对照组明显减轻,提示雷帕霉素能显著延长移植物的存活时间和抑制移植物的炎症反应(P<0.01),与环孢素A效果相当(P>0.05)；结论：雷帕霉素可能通过诱导TGF-p表达和分泌来促进作用CD4+CD25+Foxp3+T细胞的产生。雷帕霉素对同种异体的小鼠心脏移植中具有明显的抗移植排斥效果,可能与其在体内促进CD4+CD25+T细胞的产生和Foxp3的表达有关。
Allograft rejection has been one of major problems for allograft survival. It is very important to actively prevent the transplant recipient from rejection and complications for their survival time and the quality of life. Organ transplant rejection includes the host anti-graft reaction (HVGR), which is commonly referred to as organ transplant rejection, and graft-versus-host reaction (GVHR). HVGR is divided into 4 types:hyperacute rejection, accelerated rejection, acute rejection and chronic rejection. At present, problems in clinical transplant rejection therapy remained to be solved. One of them is immunosuppressants such as CsA and FK506 can efficiently control acute rejection, but have poor efficiency for chronic rejection, in view that lack of gnosia on mechanism of chronic rejection devolpment and its pathology. One the other hand, the lifelong use of immunosuppressant drugs can cause adverse reactions, such as the bone marrow suppression, cancer, infection, chronic loss of the transplanted organ function, as well as their own drug side effects and high costs. Therefore, it is significant to study the mechanism of immunosuppressive drugs on rejection for controlling of chronic graft rejection and maintaining long-term survival of great.
Rejection after organ transplantation is the essence of the immunity system against the donor graft antigens in the immune response. After allogeneic organ graft operation, the passenger leukocytes in allograft are primarily mature DC and macrophages and other antigen presenting cells (donor antigen-prsenting cells, dAPC). These cells may enter to the blood circulation or in part lymphoid tissue when donor blood vessels were connected to blood vessels, and directly submit the surface MHC molecules or antigenic peptide/ MHC molecule complexes (pMHC) to the recipient allogeneic T cells (mainly CD8+ T cells and CD4+ T cells) by direct recognition mechanism; The donor antigen (the same kinds of MHC molecules fragment)/recipient MHC molecule complexes from graft exfoliated cells or MHC antigen after processing by APC were submitted to the recipient CD4+ T cells by indirect recognition mechanism to produce immune responses. Activated T cells differentiate into Thl cells and the newly discovered Th17 cells, through secretion of cytokines, chemokines and further activation of CD8+ CTL cells, chemotaxis of macrophages, neutrophils and other inflammatory cells cause inflammation and mediate on graft rejection. Although the mechanism of the immunosuppressant used in clinical could not be coincidence, the same point is that both of them can inhibit the activation, differentiation and proliferation of the T lymphocyte. In recent years, a large number of studies have found that a group of content little CD4+CD25+Foxp3+T cells (Treg cells), as an important immune regulatory function of T cells to mediate immune response in a proactive manner, played an extremely important role in the regulation of maintenance immune tolerance to exogenous antigens or their own. Therefore, it is very important to study the negative regulation mechanism of Treg cells in control of transplant rejection and the dose of immunosuppressant drugs. In particular, studies of the immunosuppressive drugs widely used in clinical such as CsA, FK506, Rapamycin (Rapa) on Treg cells is for guiding the clinical usage of immunosuppressive agents and more effectively control of rejection significant.
Rapa first discovered is only used as a low-toxicity antifungal. After immunosuppressive effect was found in 1977, Rapa began trial in the treatment of organ transplant rejection in 1989. From the effects of the present animal experiments and clinical application, Rapa is a highly efficient and low toxicity of new immunosuppressants. The latest research shows that Rapa can promote the generation of Treg cells, but the molecular mechanisms are not yet clear. To study the molecular mechanisms of Rapa that effects on the differentiation and proliferation of CD4+CD25+ Treg cells will provide the basis of experimental data for guiding clinical treatment and reducing the use of immunosuppressive agents and efficiently controling of rejection to improve the survival of organ graft.
Part one: The influence of Rapa on the expression of Foxp3 and TGF in CD4+CD25+ T cells in vitro
Objective:To observe the proliferation and expression of Foxp3, TGF-βand explore the relationship between TGF-βand the expression of Foxp3 in CD4+CD25+ T cells when Rapa and CD4+CD25+ T cells co-cultured in vitro. Methods:Take spleen from C57BL/ 6 mice under sterile conditions, isolate mononuclear cells and separate CD4+CD25+ T cells by immunomagnetic beads, divide into control group, stimulated group, Rapa group and CsA group; Cells are stimulated with anti-CD28, anti-CD3 and low-dose IL-2 for 72h under, then detect the proliferation of CD4+CD25+ T cells and the expression of Foxp3 by the flow cytometry, the expression of Foxp3 mRNA by Semi-quantitative RT-PCR and the Foxp3 by and ELISA, Results:The proliferation of CD4+CD25+ T cells and the average fluorescence intensity of Foxp3 in Rapa group have more obviously was significantly higher than the stimulated group by FACS detecting; howere, these indicators in CsA group were lower then the stimulated group. Our results suggest that Rapa can promote the proliferation and the expression of Foxp3 in CD4+CD25+T cell, but CsA inhibits these functiones. To further test the cytokines expression, we found that the CD4+CD25+ T cells in Rapa group express TGF-βmRNA and protein were significantly higher than that stimulated group (P<0.01); To contrast, the CD4+CD25+T cells in CsA group express TGF-βmRNA and protein were were lower than the stimulation (P<0.05). The results show that Rapa can promote the expression and the secretion of TGF-βin CD4+CD25+ T cell, while CsA inhibit the expression and the secretion of TGF-βin CD4+CD25+ T cell. Conclusion:Rapa could induce the CD4+CD25+ T cell proliferation and the Foxp3/TGF-βexpression in vitro; the results documented that the effect that Rapa promote CD4+CD25+ T cells proliferation and the Foxp3 expression may be related to the expression and secretion of TGF-β.
Part two:TGF-βblocking reduces Rapa induced Foxp3 expression in CD4+CD25+ T cells
Objective:To observe the expression of Foxp3 in Rapa-treated CD4+CD25+ T cells when blocking TGF-P by TGF-βneutralizing antibody and smad3-deficient mice, study the effect of TGF-βon the expression Foxp3 in Rapa-treated CD4+CD25+ T cells. Methods: Take spleen from C57BL/6 mice under sterile conditions, isolat mononuclear cells, separate CD4+CD25+ T cells by immunomagnetic beads, divide into stimulated group, Rapa group and CsA group, Rapa plus anti-TGF-βgroup; Cells are stimulated with anti-CD28, anti-CD3 and low-dose IL-2 for 72h under, then detect the expression of Foxp3 mRNA by Semi-quantitative RT-PCR and the expression of Foxp3 in CD4+CD25+ T cells by flow cytometry.Take spleenic CD4+CD25+ T cells in smad3-/- mice or WT mice under sterile conditions, stimulate with Rapa, anti-CD28, anti-CD3 and low-dose IL-2 for 72h, then detect expression the Foxp3 by Semi-quantitative RT-PCR and ELISA.
Results:The expression of Foxp3 mRNA in Rapa group was significantly higher than that in the stimulated group (P<0.01); but that in Rapa plus anti-TGF-βgroup was no significant difference (P>0.05), indicating that the induction of Rapa on Foxp3 was inhibited by TGF-P neutralizing antibodies; and the fluorescence intensities of Foxp3 in each group are respectively to 87.24% in stimulated group,93.62% in Rapa group, 47.36% in Rapa and anti-TGF-P group, suggesting that blocking TGF-βcan inhibit Rapa to promote the expression of Foxp3.The expression of foxp3 mRNA and protein in the spleenic CD4+CD25+ T cells from smad3-/- mice in Rapa treatment was significantly lower (P<0.01)than those of wild-type control group, suggesting that blocking TGF-βsignaling pathway can inhibit the Rapa on CD4+CD25+ T cells to promoe the expression of Foxp3.Conclusion:Blocking TGF-βcan inhibit Rapa-induced the expression of Foxp3 in CD4+CD25+ T cells in vitro; The effect of Rapa on to promotion of the expression of Foxp3 in CD4+CD25+ T cells may be related to induce the expression and secretion of TGF-p.
Part three: Rapa promotes CD4+CD25+Foxp3+ T cells generation in heart
transplantation mouse through TGF-signaling pathway
Objective:To further explore the effect of Rapa on the transplant rejection and the influence of TGF-signaling pathway on the relationship between Rapa and the expression of Foxp3 in CD4+CD25+ T cells after allogeneic heart transplantation. Methods:After performing cervical heterotopic heart transplantation with allograft heart from the male BALB/C mice, C57BL/10 recipients were randomly divided into 4 groups (n=6): Normal group, Control group, Rapa group (1.5mg/kg/d) and CsA group (20mg/kg/d) are observed the graft survival time and the pathological changes. After 14 days, the ratioes of CD4+CD25+ T cells in thymus, spleen and lymph node and Foxp3 expression were detected by flow cytometry. The expression of TGF-βmRNA in spleenic CD4+CD25+ T cell are detected by semi-quantitative RT-PCR and the peripheral blood were collected at different time points to determine TGF-βconcentration by ELISA. Results:After 14 days, the ratioes of CD4+CD25+T cells as well as the Foxp3 expression of Treg cells in thymus, spleen and lymph node of Rapa treatment mice were significantly higher than that in CsA treatment group or control group. The TGF-P mRNA expression in spleenic CD4+CD25+T cells were consistent with the changes of the popularation of CD4+CD25+ T cells as well as the expression of Foxp3. The serum level of TGF-P in Rapa treatment group was not only significantly higher than that of CsA treatment group, but also significantly higher than that of control group. The results show that Rapa can promote the proliferation of CD4+CD25+ T cells, the expression of foxp3 and the secretion of TGF-P in allograft mice. In contrast, CsA not only inhibits the proliferation of CD4+CD25+ T cells and the expression of foxp3, but also inhibits the secretion of TGF-β. The heart allograft survival time of Rapa treatment group were significantly prolonged (20.17±3.31 days) compared with the control group(6.67±0.81 days), and compariable with that of CsA treatment group (21.33±4.17 days). The results suggest that Rapa could inhibit graft inflammation and significantly prolong the allograft survival time. Conclusion:Rapa prolong allograft survival related to promote the generation of CD4+CD25+ T cells and the expression of Foxp3 through TGF-βsignaling pathway.
引文
1. 吕梅励,章崇杰.医学免疫学[M].第二版.北京：科学出版社,2007,169-177.
2. Rita Rezzani.Cyclosporine A and adverse effects on organs:histochemical studies [J].Progress in Histochemistry and Cytochemistry,2004,39(2):85-128.
3. Wood KJ, Sawitzki B. Interferon γ:a crucial role in the function of induced regulatory T cells in vivo [J]. Trends Immunol,2006,27(4):183-187.
4. Huang S, Li L, Liang S, et al. Conversion of peripheral CD4(+)CD25(-) T cells to CD4(+)CD25(+) regulatory T cells by IFN-gamma in patients with Guillain-Barre syndrome [J], J Neuroimmunol,2009,217(1-2):80-84.
5. Busse D, de la Rosa M, Hobiger K, et al. Competing feedback loops shape IL-2 signaling between helper and regulatory T lymphocytes in cellular microenvironments [J]. Proc Natl Acad Sci USA,2009,107(7):3058-3063.
6. Rossini AA, Greiner DL, Mordes JP. Induction of immunologic tolerance for transplantation [J]. Physiological Reviews,1999,79:99-130.
7. Alison Taylor, Johan Verhagen, Cezmi A, et al. T regulatory cells and allergy[J]. Microbes and I nfection,2005,7(7-8):1049-1055.
8. Hall BM, Tran G, Hodgkinson SJ. Alloantigen specific T regulatory cells in transplant tolerance [J]. Int Immunopharmacol,2009,9(5):570-574.
9. Doganci A, Karwot R, Maxeiner JH, et al. IL-2 receptor beta-chain signaling controls immunosuppressive CD4+ T cells in the draining lymph nodes and lung during allergic airway inflammation in vivo [J]. J Immune,2008,181(3):1917-1926.
10. Zorn E, Mohseni M, Kim H, et al. Combined CD4+ donor lymphocyte infusion and low-dose recombinant IL-2 expand Foxp3 regulatory T cells following allogeneic hematopoietic stem cell transplantation [J]. Biol Blood Marrow Transplant,2009,15(3):382-388.
11. Segundo DS, Ruiz JC, Izquierdo M, Fernandez-Fresnedo G et al. Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+Foxp3+ regulatory T cells in renal transplant recipients. Transplantation,2006,82(4):550-7.
12. Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+CD25+Foxp3+ regulatory T cells [J]. Blood,2005,105(12):4743-8.
13. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol.1995,155(3):1151-64.
14. Takahashi T, Tagami T, Yamazaki S, et al, Immunological selftolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen[J]. J Exp Med,2000,192(2):303-310.
15. Wood KJ, Sakaguehi S. Regulatory T cells in transplantation tolerance [J]. Nat Rev Immunol, 2003,3(3):199-210.
16. Feng NH, Wu HF, Wu J, et al. Transplantation tolerance mediated by regulatory Tcell in mice [J]. Chin Med J,2004,117:1184-1189.
17. Zeng D, Lan F, Hoffmann P, et al. Suppression of graft-versus-host disease by naturally occurring regulatory T cells [J]]. Transplantation,2004,77:S9-S11.
18. Wiesner RH, Demetris AJ, Belle SH, et al. Acute hepatic allograft rejection:incidence, risk factors, and impact on outcome [J]. Hepatology,1998,28:638-645.
19. Wang C, Sun J, Sheil AG, et al. A short course of methylprednisolone immunosuppression inhibits both rejection and spontaneous acceptance of rat liver allografts [J]. Transplantation, 2001,72:44-51.
20. Segundo DS, Ruiz JC, Izquierdo M, et al. Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+Foxp3+ regulatory T cells in renal transplant recipients [J]. Transplantation,2006,82(4):550-7.
21. Coenen JJ, Koenen HJ, van Rijssen E, et al. Rapamycin, not cyclosporine, permits thymic generation and peripheral preservation of CD4+CD25+Foxp3+ T cells [J]. Bone Marrow Transplant,2007,39(9):537-45.
22. Strauss L, Whiteside TL, Knights A, et al. Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin [J]. J Immunol,2007, 178(1):320-9.
23. Qu Y, Zhang B, Zhao L, et al. The effect of immunosuppressive drug rapamycin on regulatory CD4+CD25+Foxp3+T cells in mice[J]. Transpl Immunol,2007,17(3):153-61.
24. Keever-Taylor CA, Browning MB, Johnson BD, et al. Rapamycin enriches for CD4(+) CD25(+) CD27(+) Foxp3(+) regulatory T cells in ex vivo-expanded CD25-enriched products from healthy donors and patients with multiple sclerosis[J]. Cytotherapy,2007,9(2):144-57.
25. Chen YG, Liu F, Massague J. Mechanism of TGF-β receptor inhibition by FKBP12. EMBO J, 1997,16(13):3866-3876.
26. Dodge I L, Demirci G, Strom T B, et al. Rapamycin induces transforming growth factor-beta production by lymphocytes [J]. Transplantation,2000,70(7):1104-1106.
27. Marie JC, Letterio JJ, Gavin M, et al. TGF-betal maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells [J]. J Exp Med,2005,201(7):1061-1067.
28. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods 2001; 25:402-408.
29. Abraham RT, Wiederrecht GJ. Immunopharmacology of rapamycin. Annu Rev Immunol.1996, 14:483-510.
30. Allison AC. Immunosuppressive drugs:the first 50 years and a glance forward. Immuno-pharmacology.2000,47(2-3):63-83.
31. Gregori S, Casorati M, Amuchastegui S, et al. Regulatory T cells induced by 1 alpha, 25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance [J]. J Immunol,2001,167(4):1945-53.
32. Chen X, Murakami T, Oppenheim JJ, et al. Differential response of murine CD4+CD25+ and CD4+CD25- T cells to dexamethasone-induced cell death[J]. Eur J Immunol,2004, 34(3):859-869.
33. Chung IY, Dong HF, Zhang X, et al. Effects of IL-7 and dexamethasone:induction of CD25, the high affinity IL-2 receptor, on human CD4+ cells [J]. Cell Immunol,2004,232(1-2):57-63.
34. Nakamura K, Kitani A, Fuss I et al. TGF-beta 1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice. J Immunol,2004, 172(2):834-842.
35. Brunkow M E, Jeffery E W, Hjerrild K. A, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 2001,27(1):68-73.
36. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3[J]. Science,2003,299(5609):1057-1061.
37. Hori S, Sakaguchi S. Foxp3:a critical regulator of the development and function of regulatory T cells [J]. Microbes and Infection,2004,6:745-751.
38. Roncador G, Brown PJ, Maestre L, et al. Analysis of Foxp3 protein expression in human CD4+CD25+regulatory T cells at the single cell level [J]. Eur J Immunol,2005,35(6):1681-1691.
39. Liu Y, Zhang P, Li J, Kulkarni AB, et al. A critical function for TGF-beta signaling in the development of natural CD4+CD25+Foxp3+ regulatory T cells. Nat Immunol,2008,9(6):632-40.
40. Chen Z. A technique of cervical heterotopic heart transplantation in mice. Transplantation 1991, 52:1099-1107.
41. Qian S, Demeteris D, Murase N, et al. Murine liver allograft transplantation:tolerance and donor cell chimerism. Hepatology 1994,19(4):916-927.
42. Skoskiewicz M, Chase C, Winn JH, et al. Kidney transplants between mice of graded immunogenetic diversity. Tansplant Proc 1973,5(1):721-729.
43. Zheng Z, Linfu Z, Douglas Q, et al. Pattern of liver, kidney, heart, and intestine allograft rejection in different mouse strain combination. Transplantation 1996; 62:1267-1272.
44. Superina RA, Peugh WN, Wood KJ, et al. Assessment of primarily vascularized cardiac allografts in mice. Transplantation 1986; 49:226-227.
45. Corry RJ, Winn HJ, Russell PS. Primary vascularized allografts of hearts in mice:the role of H-2D, H-2K, and non H-2 antigens in rejection. Transplantation 1973; 16:343-351
46. Matsuura A, Abe T, Yasuura K. Simplified mouse cervical hearts transplantation using a cuff technique. Transplantation 1991; 51:896-901.
47. Yukihiro T, Zhang QW, Masahiro Y, et al. Improved technique of heterotopic cervical heart transplantation. Transplantation 1997; 64:1598-1601.
48. Wang Q, Liu Y, Li xk, Simplifitd technique for heerotopic vascu-lanaed cervical hean tranaplantation in rmice [J]. Microsurgery,2005,25(!):76-79
49. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4 +CD25+ regulatory T cells [J]. Nat Immunol,2003,4(4):330-336.
50. Khattri T, Cox SA, Yasayko F. An essential role for scurfm in CD4+CD25+ T regulatory cells[J]. Nat Immunol,2003,4:337-342.
51. Annacker O, Pimenta-Araujo R, Burlen-Defranoux O, et al. CD4+CD25+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10[J]. J Immunol,2001,166 (5):3008-3018.
52. Maloy KJ, Salaun L, Cahill R, et al. CD4+CD25+ Tr cells suppress innate immune pathology through cytokine-dependentme chanisms [J]. J Exp Med,2003,197(1):111-119.
1. 李卓娅.医学免疫学[M].第二版.北京：科学出版社,2007,88-96.
2.张彩苹,杜永成,许建英.钙／钙调神经磷酸酶一活化T细胞核因子信号通路与T细胞活化及支气管哮喘关系的研究进展[J].国际呼吸杂志,2009,29：417-421.
3.陈纳新.T细胞激活的分子机制[J].国外医学：免疫学分册,1996,19：299-303.
4. Lee HW, Nam KO, Park SJ, et al.4-1BB enhances CD8+T cell expansion by regulating cell cycle progression through changes in expression of cyclins D and E and cyclin-dependent kinase inhibitor p27kipl [J]. European Journal of Immunology, 2003,33:2133-2141.
5. Foo Y, Liew, Damo Xu, Elizabeth K, et al. Negative regulation of toll-like receptor mediated immune responses.[J] Nature,2005,5:446-456.
6. Akira S, Takeda K. Toll-like receptor signaling[J]. Nature Immunology, 2004,4:499-511.
7. Gwack Y, Feske S, Srikanth S, et al.Signalling to transcription:store-operated Ca2+ entry and NFAT activation in lymphocytes[J]. Cell Calcium,2007,42:145-56.
8. Martinez-Martinez S, Redondo JM. Inhhitors of thecalcineurin/NFAT pathway. Cur Med Chem,2004,11:997-1007.
9. Fujii Y, Fujii K, Iwata S, et al. Abnormal intracellular distribution of NFAT1 in T lymphocytes from patients with systemic lupus erythematosus and characteristic clinical features[J]. Clinical immunology,2006,119:297-306.
10. Liang Zhou, Jared E. Lopes, Mark M. W. Chong, et al.TGF-β-induced Foxp3 inhibits TH17 cell differentiation by antagonizing RORyt function[J]. Nature,2008, 453:236-240.
11.翟晓强曾甫清.CD4+CD25+Treg细胞在移植免疫耐受的研究进展[J].临床外科杂志,2009,17：56-58。
12. Janine L. Coombes, Karima R. Siddiqui, Carolina V, et al. Arancibia-Carcamo. A functionally specialized population of mucosal CD103+DCs induces Foxp3+ regulatory T cells via a TGF-P-and retinoic acid-dependent mechanism[J]. The Journal of Experimental Medicine,2007,204:1757-1764.
13. Valerie Dardalhon, Amit Awasthi, Hyoung Kwon,IL-4 inhibits TGF-β-induced Foxp3+ T cells and, together with TGF-β, generates IL-9+ IL-10+Foxp3-effector T cells[J]. Nature Immunology,2008,9:1347-1355.
14. ERARD F, CORTHESY P, N ABHOL Z M, et al. Interleukin 2 is both necessary and sufficient for the growth and differentiation of lectin-cytolytic T lymphocyte precursors[J]. Immuol,1985,134:1644-1652.
15. LI XC.IMA A, LI Y, et al.Blocking the commony-chain of Cytokine receptors induces T cell apoptosis and long-term islet allograft survival [J], Immunol, 2000,164:1193-1199. 16. CHURCH AC. Clinical advances in therapies targeting the interleukin-2 receptor[J]. QJM,2003,96:91-102. 17. RODIG SJ. MERAZ MA, WHITE JM, et al. Disruption of the Jak 1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic respondes[J]. Cell,1998,93:373-383.18.于哲,邢飞跃,王通。p38 MAPK的活化促进小鼠T细胞增殖[J].现代免疫学,2007,27：182-187。19.肖序仁,徐衍盛,敖建华.不同免疫抑制剂诱导外周血活化T淋巴细胞凋亡的研究[J]。中华器官移植杂志,2006,11：644-647.
20. Zhao W,Wang W, Shao Y. Effect of FK506 on expression at bcl-2, bcl-2 mRNA and veral apoptosis in reral preservation [J]. CHINESE JOURNAL OF EXPERIMENTAL SURGERY,2006,23:1566-1566.
21. Ke H, Huai Q. Structures of calcineurin and its complexes with immunophilins immunosuppressants[J]. Biochem Biophys ResCommun,2003,311:1095-1102.
22.李佳,白杨娟,王兰兰等.FK506对肝移植受者T细胞亚群上CD152和PD-1的调节研究[J].细胞与分子免疫学杂志,2008,1：54-57.
23.白杨娟,王兰兰,冯伟华等.FK506对肝移植受者外周血T细胞亚群及共刺激分子表达调节的研究[J].检验医学,2006,4：364-368.
24.白杨娟,王兰兰,邹远高。他克莫司与环孢菌素A对肝移植受者CD8+CD28-调节性T细胞的不同调节特性[J].免疫学杂志,2008,5：555-558.
25. Rezzani R. Cyclosporine A and adverse effects on organs histochemical studies[J], Prog Histochem Cytochem.2004,39(2):85-128.
26. Fiedler B.Wollert K C. Interference of antihypertrophic molecules and signaling pathways with the Ca2+-calcineurin-NFAT cascade in cardiac myocytes. [J]. Cardiovasc Res,2004,63:450-457.
27. Bayer A L, Yu A, Malek T R. Function of the II,2R for thymie and peripheral CD4+CD25+Foxp3+T regulatory cells[J]. J Immunol,2007,178:4062-4071.
28. WANG Hong-Jun, MA Hai-Xia, LI Zhi-xin, et al. The effect of cyclosporin A on CD4+CD25+T cells in TCR-transgenic DO 11.10 mice immunized with OVA[J]. CHINESE JOURNAL OF IMMUNOLOGY,2008,24:588-590.
29.贾瑞鹏,季曙明。雷帕霉素免疫抑制的分子机制与临床应用[J].肾脏病与透析肾移植杂志,2003,12：574-578.
30. Strauss L, Whiteside T L, Knights A, Bergmann C, et al. Survival of Naturally ocurring Human CD4+CD25+Foxp3+ Regulatory T Cells Cultured with Rapamycin[J]. J Immunol,2007,178:320-329.
31.郑永先,侯桂华,宋静等.雷帕霉素对同种移植耐受模型CD4+D25+细胞活性的影响[J].细胞与分子免疫学杂志,2007,23：327-330.
32.杨明珍,吴德沛,岑建农等。雷帕霉素诱导Balb/小鼠CD4+CD25+foxp3+调节性T细胞增殖[J].2007,23：795-799.
33.高杰,陈建飞,王子函等.雷帕霉素对诱导naive小鼠效应性T细胞转化为调节性T细胞的影响[J].中国医学科学院学报,2008,30：393-399.
2. Rita Rezzani.Cyclosporine A and adverse effects on organs:histochemical studies [J].Progress in Histochemistry and Cytochemistry,2004,39(2):85-128.
3. Wood KJ, Sawitzki B. Interferon γ:a crucial role in the function of induced regulatory T cells in vivo [J]. Trends Immunol,2006,27(4):183-187.
4. Huang S, Li L, Liang S, et al. Conversion of peripheral CD4(+)CD25(-) T cells to CD4(+)CD25(+) regulatory T cells by IFN-gamma in patients with Guillain-Barre syndrome [J], J Neuroimmunol,2009,217(1-2):80-84.
5. Busse D, de la Rosa M, Hobiger K, et al. Competing feedback loops shape IL-2 signaling between helper and regulatory T lymphocytes in cellular microenvironments [J]. Proc Natl Acad Sci USA,2009,107(7):3058-3063.
6. Rossini AA, Greiner DL, Mordes JP. Induction of immunologic tolerance for transplantation [J]. Physiological Reviews,1999,79:99-130.
7. Alison Taylor, Johan Verhagen, Cezmi A, et al. T regulatory cells and allergy[J]. Microbes and I nfection,2005,7(7-8):1049-1055.
8. Hall BM, Tran G, Hodgkinson SJ. Alloantigen specific T regulatory cells in transplant tolerance [J]. Int Immunopharmacol,2009,9(5):570-574.
9. Doganci A, Karwot R, Maxeiner JH, et al. IL-2 receptor beta-chain signaling controls immunosuppressive CD4+ T cells in the draining lymph nodes and lung during allergic airway inflammation in vivo [J]. J Immune,2008,181(3):1917-1926.
10. Zorn E, Mohseni M, Kim H, et al. Combined CD4+ donor lymphocyte infusion and low-dose recombinant IL-2 expand Foxp3 regulatory T cells following allogeneic hematopoietic stem cell transplantation [J]. Biol Blood Marrow Transplant,2009,15(3):382-388.
11. Segundo DS, Ruiz JC, Izquierdo M, Fernandez-Fresnedo G et al. Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+Foxp3+ regulatory T cells in renal transplant recipients. Transplantation,2006,82(4):550-7.
12. Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+CD25+Foxp3+ regulatory T cells [J]. Blood,2005,105(12):4743-8.
13. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol.1995,155(3):1151-64.
14. Takahashi T, Tagami T, Yamazaki S, et al, Immunological selftolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen[J]. J Exp Med,2000,192(2):303-310.
15. Wood KJ, Sakaguehi S. Regulatory T cells in transplantation tolerance [J]. Nat Rev Immunol, 2003,3(3):199-210.
16. Feng NH, Wu HF, Wu J, et al. Transplantation tolerance mediated by regulatory Tcell in mice [J]. Chin Med J,2004,117:1184-1189.
17. Zeng D, Lan F, Hoffmann P, et al. Suppression of graft-versus-host disease by naturally occurring regulatory T cells [J]]. Transplantation,2004,77:S9-S11.
18. Wiesner RH, Demetris AJ, Belle SH, et al. Acute hepatic allograft rejection:incidence, risk factors, and impact on outcome [J]. Hepatology,1998,28:638-645.
19. Wang C, Sun J, Sheil AG, et al. A short course of methylprednisolone immunosuppression inhibits both rejection and spontaneous acceptance of rat liver allografts [J]. Transplantation, 2001,72:44-51.
20. Segundo DS, Ruiz JC, Izquierdo M, et al. Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+Foxp3+ regulatory T cells in renal transplant recipients [J]. Transplantation,2006,82(4):550-7.
21. Coenen JJ, Koenen HJ, van Rijssen E, et al. Rapamycin, not cyclosporine, permits thymic generation and peripheral preservation of CD4+CD25+Foxp3+ T cells [J]. Bone Marrow Transplant,2007,39(9):537-45.
22. Strauss L, Whiteside TL, Knights A, et al. Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin [J]. J Immunol,2007, 178(1):320-9.
23. Qu Y, Zhang B, Zhao L, et al. The effect of immunosuppressive drug rapamycin on regulatory CD4+CD25+Foxp3+T cells in mice[J]. Transpl Immunol,2007,17(3):153-61.
24. Keever-Taylor CA, Browning MB, Johnson BD, et al. Rapamycin enriches for CD4(+) CD25(+) CD27(+) Foxp3(+) regulatory T cells in ex vivo-expanded CD25-enriched products from healthy donors and patients with multiple sclerosis[J]. Cytotherapy,2007,9(2):144-57.
25. Chen YG, Liu F, Massague J. Mechanism of TGF-β receptor inhibition by FKBP12. EMBO J, 1997,16(13):3866-3876.
26. Dodge I L, Demirci G, Strom T B, et al. Rapamycin induces transforming growth factor-beta production by lymphocytes [J]. Transplantation,2000,70(7):1104-1106.
27. Marie JC, Letterio JJ, Gavin M, et al. TGF-betal maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells [J]. J Exp Med,2005,201(7):1061-1067.
28. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods 2001; 25:402-408.
29. Abraham RT, Wiederrecht GJ. Immunopharmacology of rapamycin. Annu Rev Immunol.1996, 14:483-510.
30. Allison AC. Immunosuppressive drugs:the first 50 years and a glance forward. Immuno-pharmacology.2000,47(2-3):63-83.
31. Gregori S, Casorati M, Amuchastegui S, et al. Regulatory T cells induced by 1 alpha, 25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance [J]. J Immunol,2001,167(4):1945-53.
32. Chen X, Murakami T, Oppenheim JJ, et al. Differential response of murine CD4+CD25+ and CD4+CD25- T cells to dexamethasone-induced cell death[J]. Eur J Immunol,2004, 34(3):859-869.
33. Chung IY, Dong HF, Zhang X, et al. Effects of IL-7 and dexamethasone:induction of CD25, the high affinity IL-2 receptor, on human CD4+ cells [J]. Cell Immunol,2004,232(1-2):57-63.
34. Nakamura K, Kitani A, Fuss I et al. TGF-beta 1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice. J Immunol,2004, 172(2):834-842.
35. Brunkow M E, Jeffery E W, Hjerrild K. A, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 2001,27(1):68-73.
36. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3[J]. Science,2003,299(5609):1057-1061.
37. Hori S, Sakaguchi S. Foxp3:a critical regulator of the development and function of regulatory T cells [J]. Microbes and Infection,2004,6:745-751.
38. Roncador G, Brown PJ, Maestre L, et al. Analysis of Foxp3 protein expression in human CD4+CD25+regulatory T cells at the single cell level [J]. Eur J Immunol,2005,35(6):1681-1691.
39. Liu Y, Zhang P, Li J, Kulkarni AB, et al. A critical function for TGF-beta signaling in the development of natural CD4+CD25+Foxp3+ regulatory T cells. Nat Immunol,2008,9(6):632-40.
40. Chen Z. A technique of cervical heterotopic heart transplantation in mice. Transplantation 1991, 52:1099-1107.
41. Qian S, Demeteris D, Murase N, et al. Murine liver allograft transplantation:tolerance and donor cell chimerism. Hepatology 1994,19(4):916-927.
42. Skoskiewicz M, Chase C, Winn JH, et al. Kidney transplants between mice of graded immunogenetic diversity. Tansplant Proc 1973,5(1):721-729.
43. Zheng Z, Linfu Z, Douglas Q, et al. Pattern of liver, kidney, heart, and intestine allograft rejection in different mouse strain combination. Transplantation 1996; 62:1267-1272.
44. Superina RA, Peugh WN, Wood KJ, et al. Assessment of primarily vascularized cardiac allografts in mice. Transplantation 1986; 49:226-227.
45. Corry RJ, Winn HJ, Russell PS. Primary vascularized allografts of hearts in mice:the role of H-2D, H-2K, and non H-2 antigens in rejection. Transplantation 1973; 16:343-351
46. Matsuura A, Abe T, Yasuura K. Simplified mouse cervical hearts transplantation using a cuff technique. Transplantation 1991; 51:896-901.
47. Yukihiro T, Zhang QW, Masahiro Y, et al. Improved technique of heterotopic cervical heart transplantation. Transplantation 1997; 64:1598-1601.
48. Wang Q, Liu Y, Li xk, Simplifitd technique for heerotopic vascu-lanaed cervical hean tranaplantation in rmice [J]. Microsurgery,2005,25(!):76-79
49. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4 +CD25+ regulatory T cells [J]. Nat Immunol,2003,4(4):330-336.
50. Khattri T, Cox SA, Yasayko F. An essential role for scurfm in CD4+CD25+ T regulatory cells[J]. Nat Immunol,2003,4:337-342.
51. Annacker O, Pimenta-Araujo R, Burlen-Defranoux O, et al. CD4+CD25+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10[J]. J Immunol,2001,166 (5):3008-3018.
52. Maloy KJ, Salaun L, Cahill R, et al. CD4+CD25+ Tr cells suppress innate immune pathology through cytokine-dependentme chanisms [J]. J Exp Med,2003,197(1):111-119.
1. 李卓娅.医学免疫学[M].第二版.北京：科学出版社,2007,88-96.
2.张彩苹,杜永成,许建英.钙／钙调神经磷酸酶一活化T细胞核因子信号通路与T细胞活化及支气管哮喘关系的研究进展[J].国际呼吸杂志,2009,29：417-421.
3.陈纳新.T细胞激活的分子机制[J].国外医学：免疫学分册,1996,19：299-303.
4. Lee HW, Nam KO, Park SJ, et al.4-1BB enhances CD8+T cell expansion by regulating cell cycle progression through changes in expression of cyclins D and E and cyclin-dependent kinase inhibitor p27kipl [J]. European Journal of Immunology, 2003,33:2133-2141.
5. Foo Y, Liew, Damo Xu, Elizabeth K, et al. Negative regulation of toll-like receptor mediated immune responses.[J] Nature,2005,5:446-456.
6. Akira S, Takeda K. Toll-like receptor signaling[J]. Nature Immunology, 2004,4:499-511.
7. Gwack Y, Feske S, Srikanth S, et al.Signalling to transcription:store-operated Ca2+ entry and NFAT activation in lymphocytes[J]. Cell Calcium,2007,42:145-56.
8. Martinez-Martinez S, Redondo JM. Inhhitors of thecalcineurin/NFAT pathway. Cur Med Chem,2004,11:997-1007.
9. Fujii Y, Fujii K, Iwata S, et al. Abnormal intracellular distribution of NFAT1 in T lymphocytes from patients with systemic lupus erythematosus and characteristic clinical features[J]. Clinical immunology,2006,119:297-306.
10. Liang Zhou, Jared E. Lopes, Mark M. W. Chong, et al.TGF-β-induced Foxp3 inhibits TH17 cell differentiation by antagonizing RORyt function[J]. Nature,2008, 453:236-240.
11.翟晓强曾甫清.CD4+CD25+Treg细胞在移植免疫耐受的研究进展[J].临床外科杂志,2009,17：56-58。
12. Janine L. Coombes, Karima R. Siddiqui, Carolina V, et al. Arancibia-Carcamo. A functionally specialized population of mucosal CD103+DCs induces Foxp3+ regulatory T cells via a TGF-P-and retinoic acid-dependent mechanism[J]. The Journal of Experimental Medicine,2007,204:1757-1764.
13. Valerie Dardalhon, Amit Awasthi, Hyoung Kwon,IL-4 inhibits TGF-β-induced Foxp3+ T cells and, together with TGF-β, generates IL-9+ IL-10+Foxp3-effector T cells[J]. Nature Immunology,2008,9:1347-1355.
14. ERARD F, CORTHESY P, N ABHOL Z M, et al. Interleukin 2 is both necessary and sufficient for the growth and differentiation of lectin-cytolytic T lymphocyte precursors[J]. Immuol,1985,134:1644-1652.
15. LI XC.IMA A, LI Y, et al.Blocking the commony-chain of Cytokine receptors induces T cell apoptosis and long-term islet allograft survival [J], Immunol, 2000,164:1193-1199. 16. CHURCH AC. Clinical advances in therapies targeting the interleukin-2 receptor[J]. QJM,2003,96:91-102. 17. RODIG SJ. MERAZ MA, WHITE JM, et al. Disruption of the Jak 1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic respondes[J]. Cell,1998,93:373-383.18.于哲,邢飞跃,王通。p38 MAPK的活化促进小鼠T细胞增殖[J].现代免疫学,2007,27：182-187。19.肖序仁,徐衍盛,敖建华.不同免疫抑制剂诱导外周血活化T淋巴细胞凋亡的研究[J]。中华器官移植杂志,2006,11：644-647.
20. Zhao W,Wang W, Shao Y. Effect of FK506 on expression at bcl-2, bcl-2 mRNA and veral apoptosis in reral preservation [J]. CHINESE JOURNAL OF EXPERIMENTAL SURGERY,2006,23:1566-1566.
21. Ke H, Huai Q. Structures of calcineurin and its complexes with immunophilins immunosuppressants[J]. Biochem Biophys ResCommun,2003,311:1095-1102.
22.李佳,白杨娟,王兰兰等.FK506对肝移植受者T细胞亚群上CD152和PD-1的调节研究[J].细胞与分子免疫学杂志,2008,1：54-57.
23.白杨娟,王兰兰,冯伟华等.FK506对肝移植受者外周血T细胞亚群及共刺激分子表达调节的研究[J].检验医学,2006,4：364-368.
24.白杨娟,王兰兰,邹远高。他克莫司与环孢菌素A对肝移植受者CD8+CD28-调节性T细胞的不同调节特性[J].免疫学杂志,2008,5：555-558.
25. Rezzani R. Cyclosporine A and adverse effects on organs histochemical studies[J], Prog Histochem Cytochem.2004,39(2):85-128.
26. Fiedler B.Wollert K C. Interference of antihypertrophic molecules and signaling pathways with the Ca2+-calcineurin-NFAT cascade in cardiac myocytes. [J]. Cardiovasc Res,2004,63:450-457.
27. Bayer A L, Yu A, Malek T R. Function of the II,2R for thymie and peripheral CD4+CD25+Foxp3+T regulatory cells[J]. J Immunol,2007,178:4062-4071.
28. WANG Hong-Jun, MA Hai-Xia, LI Zhi-xin, et al. The effect of cyclosporin A on CD4+CD25+T cells in TCR-transgenic DO 11.10 mice immunized with OVA[J]. CHINESE JOURNAL OF IMMUNOLOGY,2008,24:588-590.
29.贾瑞鹏,季曙明。雷帕霉素免疫抑制的分子机制与临床应用[J].肾脏病与透析肾移植杂志,2003,12：574-578.
30. Strauss L, Whiteside T L, Knights A, Bergmann C, et al. Survival of Naturally ocurring Human CD4+CD25+Foxp3+ Regulatory T Cells Cultured with Rapamycin[J]. J Immunol,2007,178:320-329.
31.郑永先,侯桂华,宋静等.雷帕霉素对同种移植耐受模型CD4+D25+细胞活性的影响[J].细胞与分子免疫学杂志,2007,23：327-330.
32.杨明珍,吴德沛,岑建农等。雷帕霉素诱导Balb/小鼠CD4+CD25+foxp3+调节性T细胞增殖[J].2007,23：795-799.
33.高杰,陈建飞,王子函等.雷帕霉素对诱导naive小鼠效应性T细胞转化为调节性T细胞的影响[J].中国医学科学院学报,2008,30：393-399.
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