同种排斥CD4~+T细胞差异表达基因筛选及PAE抗移植排斥作用的研究
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摘要
器官移植排斥反应是一种复杂的病理生理过程,伴有大量基因的表达变化,并且各个基因之间相互影响,形成一个复杂的基因调控网络系统,特别是那些与移植排斥密切相关的低表达基因及其意义难以探测和研究。迄今为止,同种移植排斥的分子机理尚未被完全阐明,器官移植排斥反应目前为止仍是威胁患者和移植物长期存活的主要原因。近年来随着新型免疫抑制剂的不断涌现,一般情况下的临床急性移植排斥反应都得到有效控制。但是长期应用抗排斥药物,除高昂的费用外,所产生多种毒副作用,包括药物非特异性免疫抑制所致感染、肿瘤及药物的肝、肾及神经毒性是目前临床面临的严重问题。因此,深入研究并阐明移植排斥反应的分子机理,寻找特异性强,毒副作用小,效果更好的抗移植排斥药物,是移植免疫学领域迫切需要解决的问题。
既往研究显示,T淋巴细胞和巨噬细胞在同种移植排斥中发挥关键作用。其中CD4+T细胞,无论是直接识别途径还是间接识别途径,与CD8+T细胞相比在介导同种移植排斥方面都显得更为重要。实验证明,在无其它种类的淋巴细胞存在的条件下,CD4+T细胞单独就足以引起同种移植排斥；外周缺失CD4+T细胞可使移植皮片存活时间明显延长。因此CD4+T细胞已经成为研究同种移植排斥的关键靶细胞,CD4+T细胞的移植排斥相关基因的表达状况受到高度关注。
基因在组织器官中的差异表达是机体分化、发育、衰老等生命现象的分子基础,基因的差异表达特征为其功能研究提供了重要信息。应用RT-PCR及微阵列法对肾、肝、心脏、角膜及皮肤移植模型的研究证明,上述移植模型都存在基因表达的显著差异。以往研究报道,在同种移植排斥中,CD4+T细胞有多种基因异常表达,尽管研究表明这些基因与移植排斥密切相关,但是利用相关基因敲除小鼠或用特异性单克隆抗体阻断相关基因通路,对已知的移植相关免疫活化基因进行敲除或阻断,并不能完全阻断移植排斥,甚至某些条件下对移植物的生存无显著影响。研究结果提示,在同种移植排斥过程中除了已知的高拷贝表达基因外,可能还有一些低拷贝表达基因发挥重要作用。而以往的研究手段难以发现这些基因。主要原因一是目前常用的基因表达分析方法敏感度较低,不能发现低拷贝差异表达基因；二是大多数研究采用移植物组织或外周血细胞等多种细胞在内的复合组织为样本,由于多种细胞基因型及其表达谱的差异,目标细胞的基因表达情况被其他细胞亚群的数据干扰,许多有用的信息被淹没在背景信息之中。因此目前急需要构建一种能够限定目标细胞纯度,排除其他细胞及手术、创伤、感染等因素对目标细胞基因表达情况干扰的模型,并应用更敏感的基因表达检测手段进行分析,从而更好地筛选CD4+T细胞差异表达的移植排斥密切相关低拷贝基因,阐明CD4+T细胞介导移植排斥的确切机制。
基因表达序列分析(Serial Analysis of Gene Expression, SAGE)是一种能够快速、详细、准确地反映生物体在不同的发育期及生理、病理状态下基因表达水平的有效工具,它不需已知基因序列,对低丰度转录本敏感,可发现新基因。凭借这些独特优势,SAGE现已广泛应用到生命科学的各个领域,为了解正常作用机制、肿瘤发病机制、信号分子调控机制提供了有效工具,但其在移植领域的应用尚处于起步阶段。应用SAGE技术寻找同种移植排斥相关低拷贝基因,并进行功能确定、封闭或敲除,对于深入研究移植排斥反应分子机理,发现早期特异性预测指标,研制特异性抗排斥药物都有着深远的影响。
本课题第一部分利用SCID小鼠作为同种皮肤移植受体,采用CD4+T细胞过继性转输诱导同种移植排斥,有效排除手术、创伤、感染等因素的影响,避免CD4+T细胞的基因表达情况被其他类型T细胞亚群和B细胞的干扰,使背景更加清晰,并应用更敏感的SAGE技术建立基因表达文库,准确全面的反映同种移植排斥中CD4+T细胞基因表达情况,通过对差异表达基因进行功能分析,在基因组水平全面了解同种移植免疫排斥反应的基因表达的特点,为进一步探讨CD4+T细胞介导的同种移植排斥反应机理奠定坚实的实验和理论基础。
氧化应激是指机体内高活性分子如活性氧簇(reactive oxygen species,ROS)和活性氮簇(reactive nitrogen species, RNS)产生过多或消除减少,从而导致组织损伤。研究证明氧化应激是引起多种移植物损伤的主要因素,近来研究发现抗氧化治疗能够减轻环孢霉素A(CsA)引起的器官毒性。因此寻找一种既能有效诱导移植耐受又能对抗氧化应激的免疫抑制剂,具有重要的临床应用价值。近年发现,非洲臀果木提取物(Pygeum africanum extract, PAE)具有抗炎、抗肿瘤、抑制细胞增殖、抑制细胞迁移的作用。本课题首先应用大鼠糖尿病膀胱内氧化应激模型,证明PAE可从多个指标上改善氧化应激状态。
一氧化氮(Nitric oxide, NO)是活性氮簇之一,在免疫系统中发挥重要的生理病理作用。NO可作为一种重要的效应分子参与移植排斥。以往研究显示,人、小鼠、大鼠器官或组织移植后,血浆NO及组织NOS水平与移植排斥反应的严重程度呈正相关。特异性抑制NO生成,可以有效延长移植物存活时间。应用免疫抑制剂如CsA有效诱导移植耐受后,血浆NO水平也明显降低。研究证明,NO生成受多因素调节,巨噬细胞生成NO完全依赖于活化的CD4+T细胞,应用抗Thy1.2或抗CD4+T细胞的抗体能明显抑制浸润巨噬细胞产生NO,而用抗CD8+T细胞抗体后,NO生成无明显变化。CD4+T细胞依赖的NO生成可能与Th1／Th2平衡有关,Thl型细胞因子如IFN-γ,TNF-α促进诱生型一氧化氮合酶(inducible nitric oxide synthase, iNOS)的表达,而Th2型细胞因子则通过上调精氨酸活化,抑制L-Arg的生物利用度,抑制NO生成和巨噬细胞活化。
近年来干扰素调节因子家族(Interferon Regulatory Factors, IRFs)在机体免疫应答中的作用受到高度关注。IRF-1是γ-干扰素下游基因的转录激活因子,可以活化iNOS、IL-12、P40表达及Thl、CD8+T、NK细胞的免疫应答,在移植免疫领域受到高度关注。最初研究显示,IRF-2可作为拮抗剂与IRF-1竞争同一转录位点。但近来研究发现,IRF-2也是组蛋白H4、Bcl2、FasL的转录激活因子,IRF-2还与其他转录因子协同活化基因转录,例如与Statl形成复合物,参与细胞因子依赖的TAP1转录活化；与IRF-1协同活化CHTA和GBP1。此外,IRF-1和IRF-2基因缺失小鼠在Th1细胞分化缺陷,NK细胞发育异常和细胞因子分泌方面都有很大相似性,Bernd Elser等人研究结果显示IFN-γ通过IRF-1和IRF-2在转录水平上抑制IL-4表达,提示IRF-1和IRF-2是IFN-γ对抗Th2型免疫应答的重要调节因子。IRF-2在同种移植排斥中的作用尚未有报道。本研究建立的SAGE文库中,多个参与调节体内氧化应激水平的IFN-γ信号转导通路成员差异表达,同种移植排斥组干扰素调节因子-2明显升高(Interferon regulatory factor 2, IRF-2,同种移植排斥组：对照组=8：3,p=0.041),因此我们推测机体接受同种抗原刺激后,CD4+T细胞上调IRF-2表达,调节CD4+T向Th1漂移,进而活化巨噬细胞生成NO,NO作为效应分子参与同种移植排斥。
因此本课题第二部分在进一步验证IRF-2在同种移植排斥中的作用后,研究非洲臀果木提取物(Pygeum africanum extract, PAE)抗同种移植排斥反应的作用及机理。
第一部分同种排斥CD4+T细胞差异表达基因的筛选
目的：
应用CD4+T细胞过继转输-SCID小鼠的同种皮肤移植排斥模型,限定同种移植排斥中CD4+T细胞纯度,排除其他细胞亚群及手术、创伤、感染等因素对目标细胞基因表达情况干扰,并应用更敏感SAGE技术,准确全面的反映同种移植排斥中CD4+T细胞基因表达情况,为阐明CD4+T移植排斥反应机理奠定理论基础。
材料和方法：
建立CD4+T细胞过继输入-SCID小鼠同种皮肤移植排斥组和对照组模型。同种移植组发生完全排斥时取脾,制备脾细胞悬液,用CD4+T细胞纯化柱纯化CD4+T细胞,流式细胞术鉴定细胞CD4+表型,确定细胞纯度。提取CD4+T细胞总RNA,构建SAGE文库,筛选鉴定出小鼠同种移植排斥相关低拷贝基因,并应用EASE软件对差异表达基因进行功能分析。
结果：
1.以SCID小鼠为移植受者,B6小鼠或BABL／c小鼠为移植皮片供者,3周后皮片愈合良好。此时过继输入野生型BABL／c小鼠纯化CD4+T细胞,14天后B6-SCID小鼠移植皮片发生排斥,而BABL／c-SCID小鼠移植皮片无排斥发生,表明成功建立了CD4+T细胞介导的小鼠同种移植排斥模型和同系对照组模型。通过对同种排斥高峰期脾CD4+T细胞基因表达SAGE文库确定的185个差异表达基因进行功能分析,发现68个基因具有已知生物学功能,包括37个在同种移植组高表达的基因和31个在同种移植组低表达的基因。主要涉及抗原递呈、细胞增殖、细胞分化、细胞周期、细胞凋亡、细胞活化、防御反应、磷酸化、转录调节、细胞生长与维持、信号传导等功能,研究结果为从基因组水平深入研究移植排斥机理、寻找同种排斥关键基因,从根本上治疗和预防移植排斥奠定了实验基础。
2.功能分析显示,多个参与调节体内氧化应激水平的IFN-γ信号转导通路相关基因显著性差异表达,如Statl(10：1,p=0.003)、Jak2(1：5,p=0.047)、Irf2(8：3,p=0.041),提示氧化应激在同种移植排斥中具有重要意义。
结论及意义：
1.成功建立CD4+T细胞过继输入-SCID小鼠同种皮肤移植排斥模型和对照组模型,排除其他细胞亚群及手术、创伤、感染等因素对CD4+T细胞表达情况干扰,更准确的反映同种移植排斥中CD4+T细胞基因表达情况。
2.SAGE文库确定的185个排斥高峰期脾CD4+T细胞差异表达基因与多种生物学功能密切相关,主要涉及细胞凋亡、转录调节、细胞生长及维持、信号转导和氧化应激调节等。
3.参与IFN-γ信号转导通路的氧化应激相关基因在同种移植排斥中发挥重要的作用。
第二部分PAE抗移植排斥作用的研究
目的：氧化应激是引起多种移植物损伤的主要因素,抗氧化治疗可以减轻环孢霉素A等免疫抑制剂引起的肾脏毒性,延长移植物存活时间。本课题第一部分研究发现,同种移植排斥中,多个参与调节体内氧化应激水平的IFN-γ信号转导通路相关基因差异表达,提示氧化应激在同种排斥过程中具有重要意义。非洲臀果木提取物(PAE)具有抗炎、抗肿瘤、抑制细胞增殖、抑制细胞迁移的作用,推测该药物对同种移植排斥具有潜在的应用价值。但是该药物在移植免疫学领域的应用尚未见报道。本课题首次体内研究PAE对小鼠同种皮肤移植排斥的影响,并进一步探讨该药物对氧化应激模型和同种皮肤移植排斥过程中氧化应激相关基因IFN-γ、IRF-2、iNOS表达及活性的调节,旨在进一步探讨氧化应激相关基因在同种排斥中的作用及PAE的抗移植排斥机理。
材料和方法：
1.PAE对同种移植皮片存活状况的研究：建立脾细胞过继输入-SCID小鼠同种皮肤移植排斥模型和对照组模型,从建立模型第2天开始,药物组每天分别腹腔注射PAE 12.5 mg／kg、25 mg／kg、50 mg／kg、100 mg／kg,对照组给予相应剂量佐剂。逐日观察并记录移植物存活状态。
2.PAE对氧化应激的作用：Wistar大鼠经股静脉注射STZ方法构建糖尿病膀胱组织氧化应激模型,对照组大鼠注射相应剂量佐剂。成功建立模型4周后,药物组大鼠每天灌胃给予PAE 100 mg／kg,对照组给予相应剂量佐剂,药物应用4周后,各组分别检测PAE对氧化应激组织的iNOS表达,CAT和SOD活性的影响。
3.PAE抗移植排斥机理的研究：建立脾细胞过继转输-SCID小鼠同种皮肤移植排斥模型和对照组模型,药物组分别给予不同浓度PAE,对照组给予相应剂量佐剂,移植第14天时(此时对照组完全排斥),应用一氧化氮合成酶试剂盒,检测各组小鼠血清中总NOS活性；免疫组化检测移植皮片及脾脏iNOS表达状况；RT-PCR检测移植受者脾脏组织IRF-2、IFN-γmRNA表达情况；放射免疫分析法检测各组小鼠血清中TNF-α水平。
在同种移植对照组排斥高峰期处死各组小鼠,取脾,制备脾细胞悬液,体外培养24h,收集上清。在LPS刺激的腹腔巨噬细胞培养中,加入上述不同组脾细胞培养上清,24h后检测培养上清中总NOS活性。
结果：
1.给小鼠同种皮肤移植模型体内应用PAE,25mg／kg／day,连续14天,可明显延长移植皮片的存活时间,改善移植物生存状况(p<0.05)。
2.体内应用PAE,可明显降低氧化应激模型膀胱组织中iNOS蛋白表达,升高CAT及SOD活性(p<0.05)
3.与对照组相比,25mg／kg PAE处理同种移植模型小鼠14天后,血清中总NOS活性,移植皮片及脾脏组织中iNOS蛋白表达明显受到抑制；脾脏IFN-γ和IRF-2 mRNA表达显著下调,模型小鼠血清中TNF-α水平明显降低(p<0.05)体外实验表明,25mg／kg PAE处理组第14天的小鼠脾细胞培养上清能够明显抑制巨噬细胞总NOS活性(p<0.05)。
结论：25mg／kg／day PAE可以有效延长同种移植皮片存活时间；PAE抗排斥机理与其抗氧化应激作用有关,可下调同种移植排斥中氧化应激相关基因IFN-γ和IRF-2的表达,下调受体及移植物局部NOS表达及活性,抑制巨噬细胞分泌NOS和TNFα,有效改善移植物存活状态。研究结果为进一步阐明CD4+T细胞依赖的巨噬细胞分泌NO的机理奠定了基础,也为PAE应用于抗移植排斥提供了重要的实验依据。
结论和意义
1.本课题成功建立了CD4+T细胞过继转输-SCID小鼠同种皮肤移植排斥模型和对照组模型,排除其他细胞亚群及手术、创伤、感染等因素对CD4+T细胞基因表达情况干扰,更准确地反映同种移植排斥中CD4+T细胞基因表达情况。该模型构建的SAGE文库筛选出185个排斥高峰期脾CD4+T细胞差异表达基因,发现其具有多项生物学功能,最主要参与细胞凋亡,转录调节,细胞生长和维持,信号转导通路、氧化应激调节。研究结果为深入研究移植排斥反应分子机理,研制特异性抗排斥药物,从根本上治疗或预防移植排斥反应奠定了坚实的理论基础。
2.对差异表达基因进行功能分类表明,多个参与IFN-γ信号转导通路的氧化应激相关基因差异表达,表明该通路在同种移植排斥中发挥重要作用。
3.首次探讨了PAE体内应用对同种移植排斥反应的调节及机理,研究结果表明该药物可以明显延长移植物存活时间,其作用机理与通过降低氧化应激相关基因和蛋白,调控Th1／Th2相关细胞因子表达,抑制巨噬细胞生成NO有关。研究结果为进一步阐明CD4+T细胞依赖的巨噬细胞分泌NO的机理奠定了基础,也为PAE应用于抗移植排斥提供了重要的实验依据。
创新点
1.首次应用CD4+T细胞过继转输-SCID小鼠同种排斥模型建立SAGE文库；在基因组水平对同种移植排斥中CD4+T细胞进行差异基因表达筛选,筛选出185个排斥相关低拷贝基因。
2.筛选出的多数基因在移植领域迄今尚未见报道,表明SAGE是高灵敏、高通量基因转录组研究工具。筛选结果及功能分类研究丰富了同种排斥中CD4+T细胞基因表达信息资源,为今后确定同种移植排斥的关键基因提供了坚实基础,具有重要意义。
3.首次探讨了PAE对同种移植排斥反应的作用及机理,研究结果表明该药物可通过调节机体氧化应激水平抗移植排斥,为PAE应用于抗移植排斥提供了重要的实验依据。
Allotransplant rejection remains to be a main cause for the failure of the allograft. Transplant rejection is a complex process, accompanied by large changes in gene expression and the interactions between so many genes form a complex system, especially lowly expressed genes, organ transplant rejection response is still a threat to long-term graft survival. The long-term acceptance of allograft still requires life-long administration of immunosuppressive drugs. Besides their ability to prevent acute rejection episodes, conventional immunosuppressive drugs are less successful with many side effects, such as the high expence of immunosuppressive agents, increasing toxicity to liver, kidney, nervous and other systems. These limitations have driven researchers to in-depth study the mechanisms of allograft rejection, identify reliable, new biomarkers and search for a more effective and less toxic immunosuppressive agent to avoid organ rejection.
Transplant rejection is a complex process, T cells and macrophages play important role. The body contains various kinds of T cells, although CD8+ T cells alone seem to be able to initiate allorejection, numeral studies have shown that CD4+ T cells are necessary and sufficient to induce allograft rejection in the absence of other types of lymphocytes and depletion of peripheral CD4+ T cells can prolong the survival of skin allograft. The gene expression of CD4+ T cells in allotransplantation got more attention recently.
Gene expression profiling in different tissues and organs provides the molecular basis for the differentiation, development, aging and other phenomena of organism, and it provides important information for function studies. At present, a large number of researchers have found that there are significant differences for gene expression in kidney, liver, heart, corneal and skin transplantation using RT-PCR and microarray methods. There are so many genes differently expressed in CD4+ T cells in the process of rejection, even though studies have shown that these genes are very important, using gene knockout mice or specific antibodies cannot completely block transplant rejection, and even the survival of the graft had no significant improvement. This phenomenon shows that in addition to the highly expressed genes, there must be some lowly expressed genes which play more important role. Thus the sensitive and powerful laboratory method is needed to assess gene expression, find lowly expressed genes and prevent transplant rejection. Another problem is most of the research used transplant graft to study the gene profiling, as so many kinds of cells and gene expression information, there must be a lot of useful information merge in the background of gene expression. So it is needed to establish a model which can avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the researched cell, together with the sensitive and powerful laboratory method, we can identify more differently expressed genes and in-depth study the molecular mechanism of transplant rejection.
Serial Analysis of Gene Expression (SAGE) is a powerful technique that can quickly, exactly reflect expression profile of genes in a given sample in different situations. SAGE does not require prior knowledge of the genes of interest and it provides qualitative and quantitative data of potentially every transcribed sequence in a particular cell or tissue type. It can find out novel genes. Now SAGE has been widely applied in life sciences for understanding the mechanisms of tumor pathogenesis, signal transduction pathway, and inflammation, but it has not been wildly used in the field of organ transplantation. Application of SAGE technology is meaningful for in-depth study of the molecular mechanism of transplant rejection to find special biomarkers, develop effective immunosuppressant and avoid organ rejection.
In the first part of current study, we used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, thus avoid the inference of inflammation, operation and other kinds of cells such as B cells and other T cells to the gene expression of the target cell, together with the sensitive and powerful SAGE method, we find lowly expressed genes important for allograft rejection and we determined their functional categories. This study identified the underlying genetic mechanisms regulate the process using SAGE method. Reactive oxygen species (ROS) and reactive nitrogen clusters (RNS) participates in the oxidative injury to the tissue. It has been proven that oxidative stress in transplantation induced graft injury. Recent studies have shown that antioxidant treatment can reduce the organ toxicity caused by cyclosporine A (CSA). Finding an effective immunosuppressive agent which can also prevent or attenuate the toxicity of grafts has meaningful clinical value. Recently, pygeum africanum extract (PAE) been shown to have anti-inflammatory, anti-tumor, inhibiting cell proliferation, inhibiting cell migration functions. We evaluate the effect of PAE on oxidative stress and found that early treatment with PAE could effectively suppress the oxidative stress status in diabetic bladder. According to these findings, we speculate that PAE has potential value to prevent transplant rejection.
Nitric oxide (NO) is one of RNS and it plays an important role in the pathophysiology of immune system. NO is an important molecule involved in transplant rejection. Studies have shown that in the process of human, mouse and rat organ or tissue transplantation, and plasma NO level are related to post-transplant graft rejection closely and specifically inhibit the NO production can effectively prolong graft survival. Researchers found significantly lower NO levels when application of immunosuppressive agents such as CsA to effectively induce immune transplant tolerance. There are many factors regulate NO production and studies have shown that macrophages produce NO is totally dependent on activated CD4+ T cells, NO production was significantly inhibited when treat the graft recipients with mAb anti-Thy 1.2 or anti-CD4, while the treatment with mAb anti-CD8 did not have significant effect. It has been shown that the balance of Thl/Th2 cytokines can alter the level of NO, Thl cytokines have been shown to promote the expression of iNOS in several systems, while Th2 cytokines may be responsible for down-regulation of NO production by regulating L-arginine metabolism.
Recently, interferon regulatory factors (IRFs) get more attention in immune response. IRF-1 activates the transcription of many IFN-y-inducible genes which includes iNOS, IL12, P40. In addition, it is an important mediator for Thl, CD8, NK cell responses. IRF-1 has received attention in the field of organ transplantation. IRF-2 has originally been described as an antagonist of IRF-1-mediated transcription, however, more recent studies indicate that IRF-2 may act as a transcriptional activator for some genes, such as those encoding histone H4, Bcl-2 and FasL. It also has been shown that IRF-2 can cooperate with other transcription factors to activate the promoter. For example, IRF-2 activates the expression of TAP1 via IRF-2/STAT1complex on TAP1 promoter; in addition, IRF-2 cooperates with IRF-1to activate the CIITA and GBP1. Using gene knockout mice, researchers found that IRF-2 gene knockout mice, similar with IRF-1knockout mice, have defects in Thl and natural killer cell development and function, cytokine production. Bernd Elser et al. have found that IFN-y attenuates IL-4 expression via IRF-land IRF-2 pathways. These findings indicated that IRF-land IRF-2 play Key role in the mechanism of IFN-y represses Th2 response, but the role of IRF-2 in the process of transplant rejection has not been well studied. In the present results, there are some oxidative stress-related genes participated in IFN-gamma signal transduction pathway differently expressed, interferon regulatory factor 2 (IRF-2, allo:iso,8:3, p= 0.041) was over-expressed in the allograft rejection group. Thus, we speculated that IRF-2 is up-regulated in CD4+T cells after antigen stimulation, and this gene inhibits Thl immune response, thereby activating macrophages to produce NO, which is an effect molecule in transplant rejection.
Thus, the second part of the current study was to further identify the mechanism of IRF-2 participate in transplant rejection and the function and regulation of PAE on allotransplantation.
Part 1:Alternative Expressed Genes Identified in CD4+T Cells-Mediated Allorejection
Purpose:
Using CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, we avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the target cell, together with the sensitive and powerful SAGE method, we find lowly expressed genes important for allograft rejection and identify the underlying genetic mechanisms regulating the process using SAGE method.
Materials and methods: We used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, the isotransplanted SCID mice were used as the control. When the allotransplant skin were fully rejected, CD4+ T cells were harvested from the spleens by using the mouse CD4+ T cells enrichment columns, the cell purities were measured by flow cytometry. SAGE libraries were constructed using the total RNA of CD4+ T cells from allo-and iso-recipients. Our study identified hundreds of genes that significantly altered their express levels in the CD4+ T cells and Expression Analysis Systematic Explorer Program (EASE) version 2.0 was used to determine their functional categories.
Results:
1. The skin from C57BL/6 mouse was allotransplanted to SCID mouse and the skin from BABL/c mouse was isotransplanted to SCID mouse. After the transplant wound healed (21 days), we used BABL/c mouse naive CD4+T cell adoptive transfer to induce skin acute allograft rejection. Upon 14 days after adoptive transfer of CD4+T cells, the skin graft in the allotransplanted mouse was rejected but the skin graft in the isotransplanted mouse remained normal. Using SAGE method, we identified 185 differently expressed genes. Functional classification of these genes shows that these genes belongs to antigen presentation, apoptosis, cell activation, defense response, phosphorylation, transcription regulation, cell growth and maintenance, and signal transduction. This study provides a rich resource for the mechanism and function study in allograft rejection.
2. There are some oxidative stress-related genes participated in IFN-gamma signal transduction pathway differently expressed, such as Statl (10:1,p = 0.003), Jak2 (1:5, p = 0.047), Irf2 (8:3, p=0.041), these results suggest that this signal transduction pathway play a crucial role in the process of allograft rejection.
Conclusions:
1. We used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, thus avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the target cell, this study provide a rich resource to the true gene expression information in allorejection.
2. The SAGE libraries we constructed for CD4+ T cells which mediated allograft rejection identified 185 genes differently expressed between two libraries. Functional classification of these genes shows that apoptosis, transcription regulation, and cell growth and maintenance, signal transduction, oxide stress regulation frequently changed.
3. Oxidative stress-related genes participated in IFN-y signal transduction pathway play a crucial role in the process of allograft rejection.
Part 2:Study of the Anti-allorejection Effect of PAE
Antioxidant treatment can reduce the renal toxicity caused by cyclosporine A. The first part of this study identifies that oxidative stress-related genes participated in IFN-y signal transduction pathway play a crucial role in the process of allograft rejection. Pygeum africanum extract (PAE) has been shown to have anti-inflammatory, anti-tumor, inhibiting cell proliferation, inhibiting cell migration effects. Thus we speculate that PAE has potential application value to prevent transplant rejection. PAE has not been concerned in the field of transplantation. In this study, we first evaluate the effect of PAE skin on allograft survival, and further identify the effect of PAE on oxidative stress status in diabetic and transplant recipients, oxidative stress-related genes IFN-y, IRF-2, iNOS expression and Thl cytokine production. The purpose of the current study was to further identify the mechanism of IRF-2 participated in transplant rejection and the regulation and mechanism of PAE.
Materials and methods:
1. We used mice spleen cells adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, the isotransplantated SCID mice were used as control. The next day after cell adoptive transferring, PAE was given intraperitoneally to transplant recipients at various doses (12.5 mg/kg,25 mg/kg,50 mg/kg, and 100 mg/kg) in sterile saline solution; control group was given sterile saline solution. The survival conditions of the skin grafts were observed daily.
2. Wistar rats received an i.v. injection with STZ to induce diabetes oxide stress medol. After diabetes induction, PAE groups were fed with PAE (100 mg/kg, orally) in peanut oil. And control groups were fed with same dose peanut oil. The levels of iNOS expression, CAT, SOD activity were measured.
3. We used mice spleen cells adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, the isotransplantated SCID mice were used as control. The next day after cell adoptive transferring, PAE was given intraperitoneally to transplant recipients at various doses (12.5 mg/kg,25 mg/kg,50 mg/kg, and 100 mg/kg) in sterile saline solution; control group was given sterile saline solution. Serum NOS activity was assessed using a NOS activity assay kit, the expression of iNOS in the grafted skin and spleen was detected by immunohistochemistry, the expression of IRF-2 and IFN-y mRNA was measured by RT-PCR, serum levels of TNF-a were measured with specific radioimmunoassay.
The splenocytes from control and PAE+allotransplantat group were cultured in vitro for 24h, and the supernatant were used to culture macrophage stimulated with LPS for 24h, the NOS activity was assessed using a NOS activity assay kit.
Results:
1. Application of 25mg/kg PAE after transplantation can effectively prolong allograft survival (p< 0.05).
2. Compared with diabetic group, PAE decreased iNOS expression significantly and increased CAT and SOD (p<0.05).
3. Compared with the control group, serum NOS activity was decreased, expression of iNOS protein, IFN-y and IRF-2 mRNA in the spleen tissue was significantly decreased, serum TNF-a level significantly decreased after application of 25 mg/kg PAE (p< 0.05). And 50 mg/kg Pygeum africanum extract also can improve the oxidative stress status in some extent. The splenocytes supernatant from 25 mg/kg PAE group can downregulate NOS level of macrophage (p< 0.05).
Conclusions:
Application of 25mg/kg/day PAE in vivo can effectively prolong allograft survival. And in vivo and vitro study indicate the mechanism is suppress the oxidative stress status, through down-regulating the expression of IRF-2,suppressing Thl-type cytokines levels, shifting Thl/Th2 cytokines balance, then decrease NOS activity and expression, it will be a new effective immunosuppressive agents to prevent allograft rejection. This research provides the foundation for further illustrate the mechanism of NO production dependent on CD4+ T cells, also provides important experimental basis for application of PAE on transplantation.
Conclusions and Significances
1. We used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, thus avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the target cell, this study provide a rich resource to the true gene expression information in allorejection. The SAGE libraries we constructed for CD4+ T cells which mediated allograft rejection identified 185 genes differently expressed between two libraries. Functional classification of these genes shows that apoptosis, transcription regulation, and cell growth and maintenance, signal transduction, oxide stress regulation frequently changed. This study provides a rich resource for the mechanism and function study in allograft rejection.
2. Through functional classification, we found that several oxidative stress-related genes participated in IFN-y signal transduction pathway were differently expressed, these results suggest that IFN-y signal transduction pathway play a crucial role in the process of allotransplantat rejection.
3. We evaluated the regulation and mechanism of PAE on skin allograft survival conditions first time, and application of PAE after transplantation can effectively prolong allograft survival, through down-regulating the expression of oxidative stress-related gene and protein, shifting Th1/Th2 cytokines balance. This research provides the foundation for further illustrate the mechanism of NO production dependent on CD4+ T cells, also provides important experimental basis for application of PAEon transplantation.
Points of Innovation
1. We used CD4+T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection to construct SAGE libraries. The SAGE libraries for CD4+ T cells which mediated allograft rejection identified 185 genes differently expressed between two libraries.
2. Most of the 185 genes we identified in SAGE libraries have not been studied in previous transplantation studies. The SAGE method is a sensitive and powerful tool for gene expression study. And the SAGE libraries have potential research value in transplantation. It is useful for us to find the effective way to prevent allorejection.
3. We evaluated the regulation and mechanism of PAE on skin allograft survival conditions for the first time, and this research provides important experimental basis for application of PAE on transplantation.
既往研究显示,T淋巴细胞和巨噬细胞在同种移植排斥中发挥关键作用。其中CD4+T细胞,无论是直接识别途径还是间接识别途径,与CD8+T细胞相比在介导同种移植排斥方面都显得更为重要。实验证明,在无其它种类的淋巴细胞存在的条件下,CD4+T细胞单独就足以引起同种移植排斥；外周缺失CD4+T细胞可使移植皮片存活时间明显延长。因此CD4+T细胞已经成为研究同种移植排斥的关键靶细胞,CD4+T细胞的移植排斥相关基因的表达状况受到高度关注。
基因在组织器官中的差异表达是机体分化、发育、衰老等生命现象的分子基础,基因的差异表达特征为其功能研究提供了重要信息。应用RT-PCR及微阵列法对肾、肝、心脏、角膜及皮肤移植模型的研究证明,上述移植模型都存在基因表达的显著差异。以往研究报道,在同种移植排斥中,CD4+T细胞有多种基因异常表达,尽管研究表明这些基因与移植排斥密切相关,但是利用相关基因敲除小鼠或用特异性单克隆抗体阻断相关基因通路,对已知的移植相关免疫活化基因进行敲除或阻断,并不能完全阻断移植排斥,甚至某些条件下对移植物的生存无显著影响。研究结果提示,在同种移植排斥过程中除了已知的高拷贝表达基因外,可能还有一些低拷贝表达基因发挥重要作用。而以往的研究手段难以发现这些基因。主要原因一是目前常用的基因表达分析方法敏感度较低,不能发现低拷贝差异表达基因；二是大多数研究采用移植物组织或外周血细胞等多种细胞在内的复合组织为样本,由于多种细胞基因型及其表达谱的差异,目标细胞的基因表达情况被其他细胞亚群的数据干扰,许多有用的信息被淹没在背景信息之中。因此目前急需要构建一种能够限定目标细胞纯度,排除其他细胞及手术、创伤、感染等因素对目标细胞基因表达情况干扰的模型,并应用更敏感的基因表达检测手段进行分析,从而更好地筛选CD4+T细胞差异表达的移植排斥密切相关低拷贝基因,阐明CD4+T细胞介导移植排斥的确切机制。
基因表达序列分析(Serial Analysis of Gene Expression, SAGE)是一种能够快速、详细、准确地反映生物体在不同的发育期及生理、病理状态下基因表达水平的有效工具,它不需已知基因序列,对低丰度转录本敏感,可发现新基因。凭借这些独特优势,SAGE现已广泛应用到生命科学的各个领域,为了解正常作用机制、肿瘤发病机制、信号分子调控机制提供了有效工具,但其在移植领域的应用尚处于起步阶段。应用SAGE技术寻找同种移植排斥相关低拷贝基因,并进行功能确定、封闭或敲除,对于深入研究移植排斥反应分子机理,发现早期特异性预测指标,研制特异性抗排斥药物都有着深远的影响。
本课题第一部分利用SCID小鼠作为同种皮肤移植受体,采用CD4+T细胞过继性转输诱导同种移植排斥,有效排除手术、创伤、感染等因素的影响,避免CD4+T细胞的基因表达情况被其他类型T细胞亚群和B细胞的干扰,使背景更加清晰,并应用更敏感的SAGE技术建立基因表达文库,准确全面的反映同种移植排斥中CD4+T细胞基因表达情况,通过对差异表达基因进行功能分析,在基因组水平全面了解同种移植免疫排斥反应的基因表达的特点,为进一步探讨CD4+T细胞介导的同种移植排斥反应机理奠定坚实的实验和理论基础。
氧化应激是指机体内高活性分子如活性氧簇(reactive oxygen species,ROS)和活性氮簇(reactive nitrogen species, RNS)产生过多或消除减少,从而导致组织损伤。研究证明氧化应激是引起多种移植物损伤的主要因素,近来研究发现抗氧化治疗能够减轻环孢霉素A(CsA)引起的器官毒性。因此寻找一种既能有效诱导移植耐受又能对抗氧化应激的免疫抑制剂,具有重要的临床应用价值。近年发现,非洲臀果木提取物(Pygeum africanum extract, PAE)具有抗炎、抗肿瘤、抑制细胞增殖、抑制细胞迁移的作用。本课题首先应用大鼠糖尿病膀胱内氧化应激模型,证明PAE可从多个指标上改善氧化应激状态。
一氧化氮(Nitric oxide, NO)是活性氮簇之一,在免疫系统中发挥重要的生理病理作用。NO可作为一种重要的效应分子参与移植排斥。以往研究显示,人、小鼠、大鼠器官或组织移植后,血浆NO及组织NOS水平与移植排斥反应的严重程度呈正相关。特异性抑制NO生成,可以有效延长移植物存活时间。应用免疫抑制剂如CsA有效诱导移植耐受后,血浆NO水平也明显降低。研究证明,NO生成受多因素调节,巨噬细胞生成NO完全依赖于活化的CD4+T细胞,应用抗Thy1.2或抗CD4+T细胞的抗体能明显抑制浸润巨噬细胞产生NO,而用抗CD8+T细胞抗体后,NO生成无明显变化。CD4+T细胞依赖的NO生成可能与Th1／Th2平衡有关,Thl型细胞因子如IFN-γ,TNF-α促进诱生型一氧化氮合酶(inducible nitric oxide synthase, iNOS)的表达,而Th2型细胞因子则通过上调精氨酸活化,抑制L-Arg的生物利用度,抑制NO生成和巨噬细胞活化。
近年来干扰素调节因子家族(Interferon Regulatory Factors, IRFs)在机体免疫应答中的作用受到高度关注。IRF-1是γ-干扰素下游基因的转录激活因子,可以活化iNOS、IL-12、P40表达及Thl、CD8+T、NK细胞的免疫应答,在移植免疫领域受到高度关注。最初研究显示,IRF-2可作为拮抗剂与IRF-1竞争同一转录位点。但近来研究发现,IRF-2也是组蛋白H4、Bcl2、FasL的转录激活因子,IRF-2还与其他转录因子协同活化基因转录,例如与Statl形成复合物,参与细胞因子依赖的TAP1转录活化；与IRF-1协同活化CHTA和GBP1。此外,IRF-1和IRF-2基因缺失小鼠在Th1细胞分化缺陷,NK细胞发育异常和细胞因子分泌方面都有很大相似性,Bernd Elser等人研究结果显示IFN-γ通过IRF-1和IRF-2在转录水平上抑制IL-4表达,提示IRF-1和IRF-2是IFN-γ对抗Th2型免疫应答的重要调节因子。IRF-2在同种移植排斥中的作用尚未有报道。本研究建立的SAGE文库中,多个参与调节体内氧化应激水平的IFN-γ信号转导通路成员差异表达,同种移植排斥组干扰素调节因子-2明显升高(Interferon regulatory factor 2, IRF-2,同种移植排斥组：对照组=8：3,p=0.041),因此我们推测机体接受同种抗原刺激后,CD4+T细胞上调IRF-2表达,调节CD4+T向Th1漂移,进而活化巨噬细胞生成NO,NO作为效应分子参与同种移植排斥。
因此本课题第二部分在进一步验证IRF-2在同种移植排斥中的作用后,研究非洲臀果木提取物(Pygeum africanum extract, PAE)抗同种移植排斥反应的作用及机理。
第一部分同种排斥CD4+T细胞差异表达基因的筛选
目的：
应用CD4+T细胞过继转输-SCID小鼠的同种皮肤移植排斥模型,限定同种移植排斥中CD4+T细胞纯度,排除其他细胞亚群及手术、创伤、感染等因素对目标细胞基因表达情况干扰,并应用更敏感SAGE技术,准确全面的反映同种移植排斥中CD4+T细胞基因表达情况,为阐明CD4+T移植排斥反应机理奠定理论基础。
材料和方法：
建立CD4+T细胞过继输入-SCID小鼠同种皮肤移植排斥组和对照组模型。同种移植组发生完全排斥时取脾,制备脾细胞悬液,用CD4+T细胞纯化柱纯化CD4+T细胞,流式细胞术鉴定细胞CD4+表型,确定细胞纯度。提取CD4+T细胞总RNA,构建SAGE文库,筛选鉴定出小鼠同种移植排斥相关低拷贝基因,并应用EASE软件对差异表达基因进行功能分析。
结果：
1.以SCID小鼠为移植受者,B6小鼠或BABL／c小鼠为移植皮片供者,3周后皮片愈合良好。此时过继输入野生型BABL／c小鼠纯化CD4+T细胞,14天后B6-SCID小鼠移植皮片发生排斥,而BABL／c-SCID小鼠移植皮片无排斥发生,表明成功建立了CD4+T细胞介导的小鼠同种移植排斥模型和同系对照组模型。通过对同种排斥高峰期脾CD4+T细胞基因表达SAGE文库确定的185个差异表达基因进行功能分析,发现68个基因具有已知生物学功能,包括37个在同种移植组高表达的基因和31个在同种移植组低表达的基因。主要涉及抗原递呈、细胞增殖、细胞分化、细胞周期、细胞凋亡、细胞活化、防御反应、磷酸化、转录调节、细胞生长与维持、信号传导等功能,研究结果为从基因组水平深入研究移植排斥机理、寻找同种排斥关键基因,从根本上治疗和预防移植排斥奠定了实验基础。
2.功能分析显示,多个参与调节体内氧化应激水平的IFN-γ信号转导通路相关基因显著性差异表达,如Statl(10：1,p=0.003)、Jak2(1：5,p=0.047)、Irf2(8：3,p=0.041),提示氧化应激在同种移植排斥中具有重要意义。
结论及意义：
1.成功建立CD4+T细胞过继输入-SCID小鼠同种皮肤移植排斥模型和对照组模型,排除其他细胞亚群及手术、创伤、感染等因素对CD4+T细胞表达情况干扰,更准确的反映同种移植排斥中CD4+T细胞基因表达情况。
2.SAGE文库确定的185个排斥高峰期脾CD4+T细胞差异表达基因与多种生物学功能密切相关,主要涉及细胞凋亡、转录调节、细胞生长及维持、信号转导和氧化应激调节等。
3.参与IFN-γ信号转导通路的氧化应激相关基因在同种移植排斥中发挥重要的作用。
第二部分PAE抗移植排斥作用的研究
目的：氧化应激是引起多种移植物损伤的主要因素,抗氧化治疗可以减轻环孢霉素A等免疫抑制剂引起的肾脏毒性,延长移植物存活时间。本课题第一部分研究发现,同种移植排斥中,多个参与调节体内氧化应激水平的IFN-γ信号转导通路相关基因差异表达,提示氧化应激在同种排斥过程中具有重要意义。非洲臀果木提取物(PAE)具有抗炎、抗肿瘤、抑制细胞增殖、抑制细胞迁移的作用,推测该药物对同种移植排斥具有潜在的应用价值。但是该药物在移植免疫学领域的应用尚未见报道。本课题首次体内研究PAE对小鼠同种皮肤移植排斥的影响,并进一步探讨该药物对氧化应激模型和同种皮肤移植排斥过程中氧化应激相关基因IFN-γ、IRF-2、iNOS表达及活性的调节,旨在进一步探讨氧化应激相关基因在同种排斥中的作用及PAE的抗移植排斥机理。
材料和方法：
1.PAE对同种移植皮片存活状况的研究：建立脾细胞过继输入-SCID小鼠同种皮肤移植排斥模型和对照组模型,从建立模型第2天开始,药物组每天分别腹腔注射PAE 12.5 mg／kg、25 mg／kg、50 mg／kg、100 mg／kg,对照组给予相应剂量佐剂。逐日观察并记录移植物存活状态。
2.PAE对氧化应激的作用：Wistar大鼠经股静脉注射STZ方法构建糖尿病膀胱组织氧化应激模型,对照组大鼠注射相应剂量佐剂。成功建立模型4周后,药物组大鼠每天灌胃给予PAE 100 mg／kg,对照组给予相应剂量佐剂,药物应用4周后,各组分别检测PAE对氧化应激组织的iNOS表达,CAT和SOD活性的影响。
3.PAE抗移植排斥机理的研究：建立脾细胞过继转输-SCID小鼠同种皮肤移植排斥模型和对照组模型,药物组分别给予不同浓度PAE,对照组给予相应剂量佐剂,移植第14天时(此时对照组完全排斥),应用一氧化氮合成酶试剂盒,检测各组小鼠血清中总NOS活性；免疫组化检测移植皮片及脾脏iNOS表达状况；RT-PCR检测移植受者脾脏组织IRF-2、IFN-γmRNA表达情况；放射免疫分析法检测各组小鼠血清中TNF-α水平。
在同种移植对照组排斥高峰期处死各组小鼠,取脾,制备脾细胞悬液,体外培养24h,收集上清。在LPS刺激的腹腔巨噬细胞培养中,加入上述不同组脾细胞培养上清,24h后检测培养上清中总NOS活性。
结果：
1.给小鼠同种皮肤移植模型体内应用PAE,25mg／kg／day,连续14天,可明显延长移植皮片的存活时间,改善移植物生存状况(p<0.05)。
2.体内应用PAE,可明显降低氧化应激模型膀胱组织中iNOS蛋白表达,升高CAT及SOD活性(p<0.05)
3.与对照组相比,25mg／kg PAE处理同种移植模型小鼠14天后,血清中总NOS活性,移植皮片及脾脏组织中iNOS蛋白表达明显受到抑制；脾脏IFN-γ和IRF-2 mRNA表达显著下调,模型小鼠血清中TNF-α水平明显降低(p<0.05)体外实验表明,25mg／kg PAE处理组第14天的小鼠脾细胞培养上清能够明显抑制巨噬细胞总NOS活性(p<0.05)。
结论：25mg／kg／day PAE可以有效延长同种移植皮片存活时间；PAE抗排斥机理与其抗氧化应激作用有关,可下调同种移植排斥中氧化应激相关基因IFN-γ和IRF-2的表达,下调受体及移植物局部NOS表达及活性,抑制巨噬细胞分泌NOS和TNFα,有效改善移植物存活状态。研究结果为进一步阐明CD4+T细胞依赖的巨噬细胞分泌NO的机理奠定了基础,也为PAE应用于抗移植排斥提供了重要的实验依据。
结论和意义
1.本课题成功建立了CD4+T细胞过继转输-SCID小鼠同种皮肤移植排斥模型和对照组模型,排除其他细胞亚群及手术、创伤、感染等因素对CD4+T细胞基因表达情况干扰,更准确地反映同种移植排斥中CD4+T细胞基因表达情况。该模型构建的SAGE文库筛选出185个排斥高峰期脾CD4+T细胞差异表达基因,发现其具有多项生物学功能,最主要参与细胞凋亡,转录调节,细胞生长和维持,信号转导通路、氧化应激调节。研究结果为深入研究移植排斥反应分子机理,研制特异性抗排斥药物,从根本上治疗或预防移植排斥反应奠定了坚实的理论基础。
2.对差异表达基因进行功能分类表明,多个参与IFN-γ信号转导通路的氧化应激相关基因差异表达,表明该通路在同种移植排斥中发挥重要作用。
3.首次探讨了PAE体内应用对同种移植排斥反应的调节及机理,研究结果表明该药物可以明显延长移植物存活时间,其作用机理与通过降低氧化应激相关基因和蛋白,调控Th1／Th2相关细胞因子表达,抑制巨噬细胞生成NO有关。研究结果为进一步阐明CD4+T细胞依赖的巨噬细胞分泌NO的机理奠定了基础,也为PAE应用于抗移植排斥提供了重要的实验依据。
创新点
1.首次应用CD4+T细胞过继转输-SCID小鼠同种排斥模型建立SAGE文库；在基因组水平对同种移植排斥中CD4+T细胞进行差异基因表达筛选,筛选出185个排斥相关低拷贝基因。
2.筛选出的多数基因在移植领域迄今尚未见报道,表明SAGE是高灵敏、高通量基因转录组研究工具。筛选结果及功能分类研究丰富了同种排斥中CD4+T细胞基因表达信息资源,为今后确定同种移植排斥的关键基因提供了坚实基础,具有重要意义。
3.首次探讨了PAE对同种移植排斥反应的作用及机理,研究结果表明该药物可通过调节机体氧化应激水平抗移植排斥,为PAE应用于抗移植排斥提供了重要的实验依据。
Allotransplant rejection remains to be a main cause for the failure of the allograft. Transplant rejection is a complex process, accompanied by large changes in gene expression and the interactions between so many genes form a complex system, especially lowly expressed genes, organ transplant rejection response is still a threat to long-term graft survival. The long-term acceptance of allograft still requires life-long administration of immunosuppressive drugs. Besides their ability to prevent acute rejection episodes, conventional immunosuppressive drugs are less successful with many side effects, such as the high expence of immunosuppressive agents, increasing toxicity to liver, kidney, nervous and other systems. These limitations have driven researchers to in-depth study the mechanisms of allograft rejection, identify reliable, new biomarkers and search for a more effective and less toxic immunosuppressive agent to avoid organ rejection.
Transplant rejection is a complex process, T cells and macrophages play important role. The body contains various kinds of T cells, although CD8+ T cells alone seem to be able to initiate allorejection, numeral studies have shown that CD4+ T cells are necessary and sufficient to induce allograft rejection in the absence of other types of lymphocytes and depletion of peripheral CD4+ T cells can prolong the survival of skin allograft. The gene expression of CD4+ T cells in allotransplantation got more attention recently.
Gene expression profiling in different tissues and organs provides the molecular basis for the differentiation, development, aging and other phenomena of organism, and it provides important information for function studies. At present, a large number of researchers have found that there are significant differences for gene expression in kidney, liver, heart, corneal and skin transplantation using RT-PCR and microarray methods. There are so many genes differently expressed in CD4+ T cells in the process of rejection, even though studies have shown that these genes are very important, using gene knockout mice or specific antibodies cannot completely block transplant rejection, and even the survival of the graft had no significant improvement. This phenomenon shows that in addition to the highly expressed genes, there must be some lowly expressed genes which play more important role. Thus the sensitive and powerful laboratory method is needed to assess gene expression, find lowly expressed genes and prevent transplant rejection. Another problem is most of the research used transplant graft to study the gene profiling, as so many kinds of cells and gene expression information, there must be a lot of useful information merge in the background of gene expression. So it is needed to establish a model which can avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the researched cell, together with the sensitive and powerful laboratory method, we can identify more differently expressed genes and in-depth study the molecular mechanism of transplant rejection.
Serial Analysis of Gene Expression (SAGE) is a powerful technique that can quickly, exactly reflect expression profile of genes in a given sample in different situations. SAGE does not require prior knowledge of the genes of interest and it provides qualitative and quantitative data of potentially every transcribed sequence in a particular cell or tissue type. It can find out novel genes. Now SAGE has been widely applied in life sciences for understanding the mechanisms of tumor pathogenesis, signal transduction pathway, and inflammation, but it has not been wildly used in the field of organ transplantation. Application of SAGE technology is meaningful for in-depth study of the molecular mechanism of transplant rejection to find special biomarkers, develop effective immunosuppressant and avoid organ rejection.
In the first part of current study, we used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, thus avoid the inference of inflammation, operation and other kinds of cells such as B cells and other T cells to the gene expression of the target cell, together with the sensitive and powerful SAGE method, we find lowly expressed genes important for allograft rejection and we determined their functional categories. This study identified the underlying genetic mechanisms regulate the process using SAGE method. Reactive oxygen species (ROS) and reactive nitrogen clusters (RNS) participates in the oxidative injury to the tissue. It has been proven that oxidative stress in transplantation induced graft injury. Recent studies have shown that antioxidant treatment can reduce the organ toxicity caused by cyclosporine A (CSA). Finding an effective immunosuppressive agent which can also prevent or attenuate the toxicity of grafts has meaningful clinical value. Recently, pygeum africanum extract (PAE) been shown to have anti-inflammatory, anti-tumor, inhibiting cell proliferation, inhibiting cell migration functions. We evaluate the effect of PAE on oxidative stress and found that early treatment with PAE could effectively suppress the oxidative stress status in diabetic bladder. According to these findings, we speculate that PAE has potential value to prevent transplant rejection.
Nitric oxide (NO) is one of RNS and it plays an important role in the pathophysiology of immune system. NO is an important molecule involved in transplant rejection. Studies have shown that in the process of human, mouse and rat organ or tissue transplantation, and plasma NO level are related to post-transplant graft rejection closely and specifically inhibit the NO production can effectively prolong graft survival. Researchers found significantly lower NO levels when application of immunosuppressive agents such as CsA to effectively induce immune transplant tolerance. There are many factors regulate NO production and studies have shown that macrophages produce NO is totally dependent on activated CD4+ T cells, NO production was significantly inhibited when treat the graft recipients with mAb anti-Thy 1.2 or anti-CD4, while the treatment with mAb anti-CD8 did not have significant effect. It has been shown that the balance of Thl/Th2 cytokines can alter the level of NO, Thl cytokines have been shown to promote the expression of iNOS in several systems, while Th2 cytokines may be responsible for down-regulation of NO production by regulating L-arginine metabolism.
Recently, interferon regulatory factors (IRFs) get more attention in immune response. IRF-1 activates the transcription of many IFN-y-inducible genes which includes iNOS, IL12, P40. In addition, it is an important mediator for Thl, CD8, NK cell responses. IRF-1 has received attention in the field of organ transplantation. IRF-2 has originally been described as an antagonist of IRF-1-mediated transcription, however, more recent studies indicate that IRF-2 may act as a transcriptional activator for some genes, such as those encoding histone H4, Bcl-2 and FasL. It also has been shown that IRF-2 can cooperate with other transcription factors to activate the promoter. For example, IRF-2 activates the expression of TAP1 via IRF-2/STAT1complex on TAP1 promoter; in addition, IRF-2 cooperates with IRF-1to activate the CIITA and GBP1. Using gene knockout mice, researchers found that IRF-2 gene knockout mice, similar with IRF-1knockout mice, have defects in Thl and natural killer cell development and function, cytokine production. Bernd Elser et al. have found that IFN-y attenuates IL-4 expression via IRF-land IRF-2 pathways. These findings indicated that IRF-land IRF-2 play Key role in the mechanism of IFN-y represses Th2 response, but the role of IRF-2 in the process of transplant rejection has not been well studied. In the present results, there are some oxidative stress-related genes participated in IFN-gamma signal transduction pathway differently expressed, interferon regulatory factor 2 (IRF-2, allo:iso,8:3, p= 0.041) was over-expressed in the allograft rejection group. Thus, we speculated that IRF-2 is up-regulated in CD4+T cells after antigen stimulation, and this gene inhibits Thl immune response, thereby activating macrophages to produce NO, which is an effect molecule in transplant rejection.
Thus, the second part of the current study was to further identify the mechanism of IRF-2 participate in transplant rejection and the function and regulation of PAE on allotransplantation.
Part 1:Alternative Expressed Genes Identified in CD4+T Cells-Mediated Allorejection
Purpose:
Using CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, we avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the target cell, together with the sensitive and powerful SAGE method, we find lowly expressed genes important for allograft rejection and identify the underlying genetic mechanisms regulating the process using SAGE method.
Materials and methods: We used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, the isotransplanted SCID mice were used as the control. When the allotransplant skin were fully rejected, CD4+ T cells were harvested from the spleens by using the mouse CD4+ T cells enrichment columns, the cell purities were measured by flow cytometry. SAGE libraries were constructed using the total RNA of CD4+ T cells from allo-and iso-recipients. Our study identified hundreds of genes that significantly altered their express levels in the CD4+ T cells and Expression Analysis Systematic Explorer Program (EASE) version 2.0 was used to determine their functional categories.
Results:
1. The skin from C57BL/6 mouse was allotransplanted to SCID mouse and the skin from BABL/c mouse was isotransplanted to SCID mouse. After the transplant wound healed (21 days), we used BABL/c mouse naive CD4+T cell adoptive transfer to induce skin acute allograft rejection. Upon 14 days after adoptive transfer of CD4+T cells, the skin graft in the allotransplanted mouse was rejected but the skin graft in the isotransplanted mouse remained normal. Using SAGE method, we identified 185 differently expressed genes. Functional classification of these genes shows that these genes belongs to antigen presentation, apoptosis, cell activation, defense response, phosphorylation, transcription regulation, cell growth and maintenance, and signal transduction. This study provides a rich resource for the mechanism and function study in allograft rejection.
2. There are some oxidative stress-related genes participated in IFN-gamma signal transduction pathway differently expressed, such as Statl (10:1,p = 0.003), Jak2 (1:5, p = 0.047), Irf2 (8:3, p=0.041), these results suggest that this signal transduction pathway play a crucial role in the process of allograft rejection.
Conclusions:
1. We used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, thus avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the target cell, this study provide a rich resource to the true gene expression information in allorejection.
2. The SAGE libraries we constructed for CD4+ T cells which mediated allograft rejection identified 185 genes differently expressed between two libraries. Functional classification of these genes shows that apoptosis, transcription regulation, and cell growth and maintenance, signal transduction, oxide stress regulation frequently changed.
3. Oxidative stress-related genes participated in IFN-y signal transduction pathway play a crucial role in the process of allograft rejection.
Part 2:Study of the Anti-allorejection Effect of PAE
Antioxidant treatment can reduce the renal toxicity caused by cyclosporine A. The first part of this study identifies that oxidative stress-related genes participated in IFN-y signal transduction pathway play a crucial role in the process of allograft rejection. Pygeum africanum extract (PAE) has been shown to have anti-inflammatory, anti-tumor, inhibiting cell proliferation, inhibiting cell migration effects. Thus we speculate that PAE has potential application value to prevent transplant rejection. PAE has not been concerned in the field of transplantation. In this study, we first evaluate the effect of PAE skin on allograft survival, and further identify the effect of PAE on oxidative stress status in diabetic and transplant recipients, oxidative stress-related genes IFN-y, IRF-2, iNOS expression and Thl cytokine production. The purpose of the current study was to further identify the mechanism of IRF-2 participated in transplant rejection and the regulation and mechanism of PAE.
Materials and methods:
1. We used mice spleen cells adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, the isotransplantated SCID mice were used as control. The next day after cell adoptive transferring, PAE was given intraperitoneally to transplant recipients at various doses (12.5 mg/kg,25 mg/kg,50 mg/kg, and 100 mg/kg) in sterile saline solution; control group was given sterile saline solution. The survival conditions of the skin grafts were observed daily.
2. Wistar rats received an i.v. injection with STZ to induce diabetes oxide stress medol. After diabetes induction, PAE groups were fed with PAE (100 mg/kg, orally) in peanut oil. And control groups were fed with same dose peanut oil. The levels of iNOS expression, CAT, SOD activity were measured.
3. We used mice spleen cells adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, the isotransplantated SCID mice were used as control. The next day after cell adoptive transferring, PAE was given intraperitoneally to transplant recipients at various doses (12.5 mg/kg,25 mg/kg,50 mg/kg, and 100 mg/kg) in sterile saline solution; control group was given sterile saline solution. Serum NOS activity was assessed using a NOS activity assay kit, the expression of iNOS in the grafted skin and spleen was detected by immunohistochemistry, the expression of IRF-2 and IFN-y mRNA was measured by RT-PCR, serum levels of TNF-a were measured with specific radioimmunoassay.
The splenocytes from control and PAE+allotransplantat group were cultured in vitro for 24h, and the supernatant were used to culture macrophage stimulated with LPS for 24h, the NOS activity was assessed using a NOS activity assay kit.
Results:
1. Application of 25mg/kg PAE after transplantation can effectively prolong allograft survival (p< 0.05).
2. Compared with diabetic group, PAE decreased iNOS expression significantly and increased CAT and SOD (p<0.05).
3. Compared with the control group, serum NOS activity was decreased, expression of iNOS protein, IFN-y and IRF-2 mRNA in the spleen tissue was significantly decreased, serum TNF-a level significantly decreased after application of 25 mg/kg PAE (p< 0.05). And 50 mg/kg Pygeum africanum extract also can improve the oxidative stress status in some extent. The splenocytes supernatant from 25 mg/kg PAE group can downregulate NOS level of macrophage (p< 0.05).
Conclusions:
Application of 25mg/kg/day PAE in vivo can effectively prolong allograft survival. And in vivo and vitro study indicate the mechanism is suppress the oxidative stress status, through down-regulating the expression of IRF-2,suppressing Thl-type cytokines levels, shifting Thl/Th2 cytokines balance, then decrease NOS activity and expression, it will be a new effective immunosuppressive agents to prevent allograft rejection. This research provides the foundation for further illustrate the mechanism of NO production dependent on CD4+ T cells, also provides important experimental basis for application of PAE on transplantation.
Conclusions and Significances
1. We used CD4+ T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection, thus avoid the inference of inflammation, operation and other kinds of cells to the gene expression of the target cell, this study provide a rich resource to the true gene expression information in allorejection. The SAGE libraries we constructed for CD4+ T cells which mediated allograft rejection identified 185 genes differently expressed between two libraries. Functional classification of these genes shows that apoptosis, transcription regulation, and cell growth and maintenance, signal transduction, oxide stress regulation frequently changed. This study provides a rich resource for the mechanism and function study in allograft rejection.
2. Through functional classification, we found that several oxidative stress-related genes participated in IFN-y signal transduction pathway were differently expressed, these results suggest that IFN-y signal transduction pathway play a crucial role in the process of allotransplantat rejection.
3. We evaluated the regulation and mechanism of PAE on skin allograft survival conditions first time, and application of PAE after transplantation can effectively prolong allograft survival, through down-regulating the expression of oxidative stress-related gene and protein, shifting Th1/Th2 cytokines balance. This research provides the foundation for further illustrate the mechanism of NO production dependent on CD4+ T cells, also provides important experimental basis for application of PAEon transplantation.
Points of Innovation
1. We used CD4+T cell adoptive transferring-allotransplanted SCID mice to induce skin allograft rejection to construct SAGE libraries. The SAGE libraries for CD4+ T cells which mediated allograft rejection identified 185 genes differently expressed between two libraries.
2. Most of the 185 genes we identified in SAGE libraries have not been studied in previous transplantation studies. The SAGE method is a sensitive and powerful tool for gene expression study. And the SAGE libraries have potential research value in transplantation. It is useful for us to find the effective way to prevent allorejection.
3. We evaluated the regulation and mechanism of PAE on skin allograft survival conditions for the first time, and this research provides important experimental basis for application of PAE on transplantation.
引文
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