【作者】 袁曙光；

【导师】 刘瑞洪；

【作者基本信息】 中南大学 ， 内科学， 2012， 博士

【摘要】 衰变加速因子(dcay accelerating factor,DAF,CD55)是一种补体调节蛋白(CRP),主要功能是抑制细胞表面的C3转化酶形成并加速其衰变, C3转化酶是补体激活链中的中心放大酶,DAF通过限制C3转化酶保护细胞免于自身补体介导的损伤。在移植免疫的研究中,DAF缺乏可加重缺血再灌注损伤,影响抗体介导的排斥反应(AMR),它在异种移植器官的表达可延长移植器官的生存期,在移植肾组织中DAF表达与肾功能及移植肾的生存期呈正相关。我们应用小鼠腹腔异位心脏移植模型,以Dafl基因敲除小鼠(Daf1-/-)研究DAF缺乏对同种异体移植排斥反应的作用,并探讨其对T细胞免疫影响。主要方法、结果如下：1.成功地建立小鼠腹腔异位心脏移植模型。分别吻合供心主动脉与受体腹主动脉、供心肺动脉与受体下腔静脉。同基因移植心脏平均存活60d以上；同种异基因移植心脏平均存活时间9d,移植心脏组织病理切片HE染色显示,弥漫性单个核细胞浸润于血管周围,急性细胞性排斥反应。2.供体缺乏DAF加速移植物排斥,增强直接途径识别的、抗供体特异性T细胞免疫。雄性BALB/c(H-2d)小鼠作为受体,移植供体为Daf1-/-(n=7)或WT(n=6)或C3-/-(n=6)的雄性、C57BL/6(B6,H-2b)小鼠的心脏,观察移植心脏的存活时间,结果显示,供体为C3-/-的移植心脏存活时间最长(平均14d),WT次之(平均9.5d),而供体为DAF缺乏的移植心脏存活时间最短(平均7d)结果有统计学意义(P<0.05)。以C3H(H-2k)雄性小鼠为受体重复上述实验,的结果相似,Daf1-/-供体心脏平均生存8d,WT平均17d,C3-/-平均25d,其中2只超过60d。差异有显著性(P<0.01)。在移植心脏排斥当天,收集受体小鼠脾脏细胞,应用ELISPOT检测受体脾脏细胞中抗供体抗原的、产生INF-γ的T细胞频数。结果显示,接受Dafl-/-心脏的受体小鼠的脾脏细胞中含有2倍以上的抗供体抗原的、分泌INF-γ的T细胞(与接受WT心脏的受体比较,p<0.05),相反,接受心脏的受体小鼠的脾脏细胞中产生抗供体抗原的、分泌INF-γ的T细胞频数较低,产生INF-γ的T细胞频数分别为：Daf1-/--235.5±60、WT98.5±15.3、 C3-/-56.3±16.7(/5xl04, x±S)o移植术后第6d(移植物尚未排斥),ELISPOT检测受体脾T细胞反应显示,接受Daf1-/-心脏的受体小鼠的脾脏中抗供体抗原的、分泌INF-y的T细胞明显高于接受WT心脏的受体,结果为：Daf1-/-269.5±48.2. WT126.0±19.3(/5×104,x±S)(p<0.0.)3.受体缺乏DAF对心脏移植生存时间、抗供体特异性T细胞免疫无明显影响。雄性BALB/c (H-2d)小鼠为供体,受体分别为Daf1-/-(n=4)或WT(n=5)雌性B6(H-2b)小鼠,受体为DAF缺乏的移植心脏存活时间平均10.5d,WT组平均1Od,结果无统计学差异(P>0.05)o ELISPOT检测受体抗-供体抗原T细胞数分别为Dafl-/-266.8±16.9、WT206.0±13.5(/5×104,x±S),两组无显著性差异(P>0.05)。4.DAF对移植排斥反应的调节不依赖于抗供体特异性抗体启动的补体经典途径激活。雄性BALB/c (H-2d)小鼠作为受体,移植供体为Daf-/-(n=3)或WT(n=3)雄性B6(H-2b)小鼠的心脏,移植后第6d,以供体(B6小鼠)胸腺细胞为抗原,流式细胞法检测受体血清中抗-供体抗体。结果显示,受体血清中抗供体特异性抗体两组间差异无显著性(p>0.05)。重度联合免疫缺陷(SCID)小鼠,其体内T、B淋巴细胞均缺失。将WT(n=3)或Daf1-/-(n=3) B6(H-2b)小鼠的心脏移植到异源的SCID/C3H(H-2k)小鼠体内,10d后,经小鼠尾静脉给受体注入来自同源的C3H小鼠脾脏的、未激活的T细胞6×106/只,注射过继转移T细胞9d后,Daf1-/-的心脏排斥,而WT的心脏存活时间明显延长(其中之一于第12d排斥,其中之二超过20d)。在SCID小鼠血清中没有检测到抗-供体的抗体。5.DAF影响CD8+效应T细胞(TCL)功能。经尾静脉注射给C3H (H-2k)小鼠注射B6(H-2b)小鼠脾细胞(每只注射15×106个细胞),2周后分离被致敏的C3H小鼠脾脏CD8+T细胞,分别以Daf1-/-WT、C3-/-的B6(H-2b)小鼠脾细胞为刺激细胞(内含APC),以ELISPOT检测被致敏的C3H小鼠T细胞再次接触相同抗原的反应。结果显示,以Daf1-/-脾细胞刺激的C3H小鼠T细胞反应最为强烈,其分泌INF-γ细胞数最高,为WT脾细胞刺激的2倍,C3-/-的最低。结果分别为：Daf1-/-878.5±270.9、WT389.8±53.5、C3-/-264.3±27.1(/1×105, x±S)(WT与C3-/-比较P<0.01;Daf1-/-与C3-/-或WT比较P<0.001)。6.DAF影响抗原提呈细胞(APC)对抗原的处理能力。源自WT或Dafl-/-的B6小鼠腹腔的巨噬细胞作为APC,与CD4+T细胞株及卵白蛋白OVA多肽(OVA323-339)或卵白蛋白OVA(浓度5-30μM/well)共培养24Hr后,离心取上清液,以CTLL分析检测上清液中T细胞活化后产生的IL-2的相对单位。结果如图所示,与WT相比,源于Daf1-/1小鼠巨噬细胞摄取、处理卵白蛋白OVA并提呈特异性抗原给T细胞产生IL-2的量,显著增高(P<0.0001),但对于卵白蛋白OVA多肽(OVA323-339),两者差异无显著性(P>0.05)。同样,以CD8+T细胞株与卵白蛋白OVA多肽(OVA257-264)或不同浓度的卵白蛋白OVA共培养24Hr后,离心取上清液,以CTLL分析检测上清液中T细胞活化后产生的IL-2的量,结果与CD4+T细胞株的相似。结论1.供体缺乏DAF不仅加速移植排斥反应,且明显增强抗供体特异性的T细胞反应。2.受体缺乏DAF对心脏移植生存时间、抗供体特异性T细胞免疫无明显影响。3.DAF影响CD8+效应T细胞(TCL)功能。当APC与T细胞相互作用时,APC缺乏DAF(不是T细胞本身)增强T细胞功能。4.DAF缺乏的APC加工处理抗原能力增强。5.DAF对移植排斥反应的调节不依赖于抗供体特异性抗体启动的补体经典途径激活。更多还原

【Abstract】 Decay-accelerating factor (DAF) is a cell surface complement regulatory protein that accelerates the dissociation of C3convertase, which plays a pivotal role in the ampification of complement activation, and thereby prevents the complement-mediated self-damaging effect on cell.In the context of transplantation, the deficiency of DAF, which has been thought to regulate antibody-mediated rejection, was detected to aggravate ischemia-reperfusion injury. Inversely, increased expression of DAF in renal allograft was associated with better graft function and longer graft survival.In addition, DAF also play a role in regulating cellular immunity and humoral immunity. Absence of DAF in patient with paroxysmal nocturnal hemoglobinuria increase C3b on the surface of erythrocyte and hemolysis. In contrast, overexpression of DAF on tumor cell results in immune escape. In the context of transplantation, the deficiency of DAF, which has been thought to regulate antibody-mediated rejection, was detected to aggravate ischemia-reperfusion injury. Inversely, increased expression of DAF in renal allograft was associated with better graft function and longer graft survival. We construct a mouse ventral heterotopic heart transplant model and use Daf1deficiency mice to study the effect of DAF deficiency on allograft rejection and T cell immunity.Methods and Results1.Construct a mouse ventral heterotopic heart transplant model.Hearts were transplanted heterotopically by anastomosing the donor aorta and pulmonary artery to the recipient abdominal aorta and inferior vena cava. Syngeneic heart mean survival time (MST) of60days, allograft MST of9days. Microscopic examination of H&E-stained tissue sections obtained at cessation of heartbeats revealed diffuse mononuclear cell infiltration and perivascular inflammation in both groups, typical of acute cellular rejection.2.The absence of donor Dafl accelerates graft rejection through augmentation of direct pathway mediated direct anti-donor T cell responses.To delineate the role of donor DAF in T cell mediated rejection of a vascularized allograft, we examined the survival of Dafl deficient (Daf1-/-) hearts, WT hearts and C3deficient hearts from male C57BL/6(B6, H-2b) mice following transplantation into fully allogeneic recipients (male BALB/c (H-2d) mice). In H-2d recipients, B6H-2b Dafl-/-hearts were rejected on7days posttransplantation, faster than WT and C3deficient hearts which were rejected on9.5and14days posttransplantation respectively. Results were statistically significant. Similar results were observed with C3H (H-2k) male recipients.(Daf1-/-MST of8days, WT MST of17days, C3-/-MST of25days with two>60days, n=6/group, p<0.01).IFN-y ELISPOY assays of direct anti-donor T cell response performed at the time of rejection revealed that spleens of recipients of Daf1-/-allografts contained almost2-fold more donor-reactive IFN-y producers (p<0.05vs WT recipients). In contrast, the total number of anti-donor IFN-y producing T cells in recipients of C3-/-allografts was low.(Daf1-/-235.5±60.3、WT98.5±15.3、C3-/-56.3±16.7(/5×104, X±S))Direct anti-donor T cell responses in recipients of Dafl-/-B6hearts assessed by ELISPOT assay6days posttransplantation were significantly higher than those detected in recipients of WT B6hearts.(Dafl-/269.5±48.2, WT126.0±19.3(/5×104, x±S)(p<0,01)).3.Recipient DAF deficiency has no effect on graft survival nor anti-donor T cell immunity.To assess the role of recipient DAF deficiency in T cell mediated rejection of a vascularized allograft. We examined the survival of heart from BALB/c (H-2d) following transplantation into Daf1-/-(n=4) and WT (n=5) B6(H-2b) female recipients. In Dafl-/-recipients, allografts were rejected10.5days posttransplantation and the results was not significantly different from WT recipient on day10posttransplantation.(p>0.05). The frequency of recipient IFN-y-producing T cells in Daf1-/-recipients was266.8±16.9, and in WT recipients was206.0±13.5(/5×104, χ±S). The statistics was not significant.4.Dornor DAF deficiency accelerated graft rejection independent of antibody initiated, classic complement pathway.We transplanted WT or Daf1-/-B6heart into allogenic BALB/c (H-2d) recipients. On day6posttransplantation, flow cytometry was performed to detect the donor-reactive alloantibodies directed to donor thymocyte. The titer of Ab was low and was not significantly different between recipients of WT (n=3)and Daf1-/-B6(n=3) heart grafts (p>0.05).We transplanted WT or.Daf1-/-B6heart into allogeneic SCID/C3H (H-2k) recipients. Ten days later we adminstered6×106T cells from naive C3H mice thymus gland i.v. through the tail vein and followed graft survival by palpation. The adoptively transferred T cells caused acute rejection of the Dafl-/-B6hearts by day9, while WT hearts exhibited significantly prolonged survival (one survive12days, two>20days) despite transfer of the same number of WT T cells. Besides, no anti-donor antibody was detected in serum of SCID/C3H (H-2k) recipients of both groups.5.DAF enhances function of alloreactive CD8effector T cells.We administered15×106splenic cell from B6(H-2b) mice to C3H (H-2k) mice i.v. Through the tail vein. Two weeks later, CD8+effector T cell from spleen of C3H mice was isolated and stimulated with splenic cell (with APC) from Daf1-/-、WT、C3-/-B6(H-2b) mice. IFN-γ ELIDPOT assays were performed to test the T cell response when they reencounter their target Ags. IFN-γ production and CLT activity were significantly greater when the primed T cells were challenged with Daf1-/-splenic cells than those to WT and C3-/-stimulators(Daf1-/-878.5±270.9、WT389.8±53.5、C3-/-264.3±27.1(/1×105, χ±S)).(WT vs. C3-/-P<0.01;Daf1-/-vsC3-/-WT P<0.001).6. DAF affects the antigen presentation ability of APCs.Peritoneal microphages (APC) from WT or Daf1-/-B6mice was co-cultured with CD+4effector T cells and ovoalbumin (OVA) or ovoalbumin polypeptide (OVA323-339) for24Hs. After centrifugation, supernatant was assessed by CTLL assay for IL-2produced by activated T cell. The production of IL-2was significantly greater by Daf1-/-originated macrophagy than WT when co-cultered with CT4+effector T cell and OVA (P<0.0001). However, there was not discernible difference in IL-2production between Daf1-/-originated and WT originated macrophagy when co-cultered with CT4+effector T cell and OVA323-339(p>0.05). Similar results were found in CT8+effector T cells when co-cultrured with peritoneal macrophagy and OVA/OVA323-339. Conclusion1. Donor DAF deficiency augment graft rejection and anti-donor T cell response.2. Recipient DAF deficiency has no effect on graft survival nor anti-donor T cell immunity.3. DAF enhances function of alloreactive CD8effector T cells. When APC interacts with T cell, APC DAF deficiency (not T cell DAF deficiency) enhance T cell function.4. APCs DAF deficiency enhance the antigen presentation ability.5. Dornor DAF deficiency accelerated graft rejection independent of antibody initiated, classic complement pathway.更多还原