急性移植排斥反应过程中MHC-I表达变化的实验研究与临床应用
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摘要
背景
器官移植是20世纪伟大的科学创举,器官移植的广泛开展挽救了无数终末期器官功能衰竭患者的生命。然而,器官移植技术仍存在诸多问题,使得移植后的器官不能正常发挥功能。移植后的急性排斥反应(AGR)是移植物功能降低甚至丧失的主要原因,也是导致慢性排斥反应的主要因素。尽早及时的发现急性排斥反应,并采取有效的措施加以控制,可挽救移植物或延长移植物寿命。
目前移植物的病理组织学检查仍然是判断急性排斥反应的金标准。然而,由于病理组织学检查存在创伤性、操作复杂、费用昂贵、不适合重复操作等缺点,不能作为临床常规检查手段。目前常用的一些无创性检查方法,包括影像学、生化学检查以及抗体检测等,由于其在准确性、敏感性、特异性方面的不足而没能广泛应用于临床。至今为止,没有一种用于判断急性排斥反应的简单、无创性的检查方法获得大家的认可。
主要组织相容性复合体(major histocompatibility complex, MHC),是位于哺乳动物某一染色体的一组紧密连锁的基因群,其产物的主要作用是参与抗原递呈和T细胞激活,在免疫应答和免疫调节中执行重要而广泛的功能。人类MHC又称人类白细胞抗原复合体(human leucocyte antigen, HLA)。经典的MHC分子具有高度的多态性,正是由于移植供受者间这种MHC分子的不匹配导致了移植排斥反应的发生。也就是说MHC是启动移植排斥反应的靶抗原,是移植免疫的主角。在移植免疫研究领域,人们往往把目光集中在移植物MHC分子以及宿主血清中针对移植物的抗体上。但作为体内表达MHC-Ⅰ类分子最丰富的细胞,淋巴细胞尤其是T淋巴细胞MHC-Ⅰ类分子的功能研究却较少见。已有研究表明,外周血淋巴细胞表面HLA-A、B mRNA以及其相应蛋白的表达随年龄老化而降低,可以作为机体免疫功能衰退的指标；CD4+T细胞表面MHC-Ⅰ类分子可以延长CD4+T细胞的存活时间；记忆性T细胞表面MHC-Ⅰ类分子的表达对其自身具有保护作用。我们前期工作利用RNAi技术封闭淋巴细胞表面MHC-Ⅰ分子,发现淋巴细胞的增殖和杀伤活性均降低。另外我们前期对近交系小鼠皮肤移植模型的研究发现,当发生急性排斥反应时小鼠外周血淋巴细胞MHC-Ⅰ基因中的H-2K、H-2D基因转录mRNA表达升高。那么MHC-Ⅰ蛋白作为行使功能的分子,与急性排斥反应的关系如何?以及其在临床上的应用如何?CD4+T细胞和CD8+T细胞哪个亚群细胞表面MHC-Ⅰ的表达更能代表急性排斥反应的发生?MHC-Ⅰ蛋白和MHC-ImRNA两者谁更利于急性排斥反应的早期发现?类似问题有待于我们进一步研究。
为了系统的研究MHC-Ⅰ分子在急性移植排斥反应全过程中的变化规律,本研究首先采用近交系小鼠皮肤移植模型,研究了移植前、移植后不同时间点小鼠外周血淋巴细胞MHC-ImRNA的表达及外周血CD4+、CD8+T细胞亚群MHC-K、移植物MHC-Ⅰ分子的表达变化,分析了MHC-Ⅰ分子的表达水平与移植排斥全过程的对应关系。进而动态监测了临床上包括移植术后稳定和移植术后发生急性排斥的共53例肾移植患者外周血CD4+、CD8+T细胞MHC-Ⅰ分子的表达情况,从动物和临床两方面探讨了MHC-Ⅰ作为判断急性移植排斥反应指标的可行性。
目的
1.构建近交系小鼠皮肤移植模型,从而获得急性移植排斥反应的全过程,研究急性移植排斥反应时外周血淋巴细胞MHC-Ⅰ的表达规律,探讨外周血淋巴细胞MHC-Ⅰ表达与急性移植排斥反应病理分级之间的对应关系。
2.对外周血淋巴细胞MHC-Ⅰ基因的mRNA和蛋白两个水平进行研究,分析两水平表达趋势是否一致,哪一个水平的表达变化更能反映急性移植排斥反应。
3.分别检测CD3+CD4+T细胞和CD3+CD8+T细胞表面MHC-Ⅰ分子的表达变化,分析MHC-Ⅰ分子的表达变化在两群细胞间是否存在差别。
4.与T细胞活化指标MHC-Ⅱ比较,探讨MHC-Ⅰ分子作为判断急性移植排斥反应的可行性。
5.动态检测肾移植患者术前和术后不同时间外周血单个核细胞HLA-I mRNA表达变化以及CD3+CD4+细胞和CD3+CD8+细胞HLA-Ⅰ蛋白水平的表达变化,对比排斥组和稳定组之间的差异,进一步探讨在临床肾急性移植排斥反应中HLA-I的诊断价值。
方法
1.实验小鼠分组：把270只BALB/C(简称BA)小鼠随机分为三组,分别是移植用药组(应用免疫抑制剂)、移植未用药组以及单纯感染组。用药组和未用药组又分为非手术组、同系移植组和异系移植组。用药非手术组即为用药术前对照组,未用药非手术组即为未用药术前对照组。用药组的设立是为了模拟临床采用了皮质激素(Horm)、环孢素A(CsA)及霉酚酸酯(MMF)中等剂量三联用药。采用肠球菌注射正常小鼠腹腔建立肠球菌小鼠腹膜炎模型既为细菌感染组。同系移植采用BA小鼠个体之间进行皮肤移植,异系移植以C57BL/6(简称C57)小鼠为供体,BA小鼠为受体,进行小鼠皮肤移植。每两天为一个时间点,分别为术后2、4、6天……,用药移植组小鼠观察到术后26天,未用药移植组小鼠观察到术后16天。
2.构建小鼠皮肤移植模型以及标本的留取：以C57小鼠为供体,BA小鼠为受体,采用经典的小鼠背—背皮肤移植手术方法,建立小鼠皮肤移植动物模型。移植后每隔一天(每两天)对小鼠状态进行全面分析,每个时间点6只小鼠,进行以下操作：记录移植前和移植术后各时间点小鼠移植皮片的肉眼变化；通过眼球取血采集小鼠血液到EDTA-K2抗凝血常规管中备用；采血的同时取移植皮片组织用4%多聚甲醛固定,用于组织病理学检测和免疫组化检测。对正常组和细菌感染组小鼠收集血液备用。
3.小鼠血液标本的检测：采用三色流式细胞技术检测小鼠外周血不同亚群T细胞：CD3+CD4+T细胞、CD3+CD8+T细胞表面MHC-Ⅰ、MHC-Ⅱ分子的表达；采用实时荧光定量RT-PCR技术检测小鼠外周血淋巴细胞MHC-ⅠmRNA,MHC-ⅡmRNA的表达变化；分析外周血淋巴细胞MHC-ⅠMHC-Ⅱ在AGR全过程中的变化规律,以及免疫抑制剂对上述指标表达水平的影响。
4.小鼠移植皮片标本的检查：对留取的所有时间点的皮肤组织进行常规病理切片,H-E染色后观察移植皮片淋巴细胞浸润等组织学变化,根据病理组织学改变将术后不同天数的移植物排斥反应发生的程度分为Ⅰ-Ⅳ级,记录移植物病理排斥动态变化。对比用药和不用药对移植物病理组织学改变和存活时间的影响。采用免疫组织化学染色法检测小鼠移植皮片中组织细胞和浸润淋巴细胞MHC-Ⅰ的表达,观察急性移植排斥反应发生时小鼠移植皮片中MHC-Ⅰ表达强度的变化。
5.临床肾移植患者：根据移植术后3个月之内是否发生过急性移植排斥反应,把肾移植患者分成稳定组和排斥组。收集肾移植稳定组患者术前和术后3天、7天……3月和大于3个月的血液标本和肾移植排斥组患者排斥前、排斥当天、抗排斥治疗后3天、7天、14天的血液标本,采用流式细胞技术对外周血中CD3+CD4+T细胞、CD3+CD8+T细胞表面的HLA-Ⅰ(MHC-Ⅰ)分子进行检测。采用实时荧光定量PCR技术对外周血淋巴细胞HLA-ImRNA进行检测。比较稳定组和排斥组的差异,初步从临床角度评价HLA-Ⅰ对于急性移植排斥反应的诊断价值。结果
1.移植皮片的肉眼及病理组织学结果
95%小鼠在实验过程中都有体重增长,其余5%在实验中被排除。同系移植组小鼠皮肤移植物生长良好,移植物缝合部位逐渐愈合,皮片柔软红润。在异系未用药组和异系用药组,移植物逐渐变暗、变黑,80%移植物坏死时发生在术后第10天和第22天。移植皮片经H-E染色,按照淋巴细胞浸润和皮肤组织坏死程度将急性排斥反应分为轻度(Ⅰ级)、中度(Ⅱ级)、重度(Ⅲ级)和极重度(Ⅳ级)四个等级。结果显示：随术后时间的延长,异系移植皮片淋巴细胞浸润逐渐增强。不用药情况下,病理Ⅰ-Ⅳ级排斥反应分别发生于术后2-4天、4-8天、8-12天和12-16天。用药情况下,淋巴细胞浸润时间延迟,病理排斥Ⅰ-Ⅳ级分别发生于2-8天、8-14天、14-20天、20-26天。同系移植皮片中很少淋巴细胞浸润,移植病理分级为0或Ⅰ级。
2.未用药情况下,移植术后MHC-Ⅰ的表达变化
同系移植组,术后各时间点外周血、皮肤移植物MHC-Ⅰ的表达变化不明显,与术前基本保持一致,而异系移植组在术后各时间点呈现出不同的变化。
外周血淋巴细胞MHC-Ⅰ的表达情况：CD3+CD8+细胞MHC-Ⅰ膜蛋白在术后6天表达明显升高,表达强度约为术前的1.3倍(MFI从术前219升高到290),升高一直持续到术后12天,术后14天MHC-Ⅰ膜蛋白表达回复到术前水平,升高的时段与病理Ⅱ-Ⅲ级对应；CD3+CD4+细胞MHC-Ⅰ膜蛋白升高开始的时间比CD3+CD8+细胞早2天,但其表达趋势和表达强度与CD3+CD8+细胞基本一致；实时荧光定量RT-PCR检测MHC-ImRNA表达与流式细胞术检测CD4+细胞MHC-Ⅰ膜蛋白表达规律一致。异系移植术后4天,外周血淋巴细胞MHC-Ⅰ表达明显升高,术后6天达峰值,约为术前的4倍(从术前1.38升高到5.96),表达升高一直持续到术后12天,术后14天恢复到术前水平。
免疫组化检测结果：所有小鼠移植皮片组织细胞、血管内皮细胞及浸润淋巴细胞均呈阳性表达,但当排斥反应发生时,移植物MHC-Ⅰ表达明显增强。3.应用免疫抑制剂情况下,移植术前及术后MHC-Ⅰ的表达变化
在移植手术前,与未用药相比,用药后MHC-ⅠmRNA及蛋白表达水平降低,说明免疫抑制剂对MHC-Ⅰ的表达有抑制作用。同系移植组,术后各时间点外周血、皮肤移植物MHC-Ⅰ的表达变化不明显,与术前基本保持一致。而异系移植组在术后各时间点呈现出不同的变化。
外周血淋巴细胞MHC-Ⅰ的表达情况：CD3+CD8+细胞MHC-Ⅰ膜蛋白在术后12天表达明显升高,术后16天达峰值,表达强度约为术前的1.3倍(MFI从术前169升高到228),升高一直持续到术后22天,术后24天MHC-Ⅰ膜蛋白表达回复到术前水平,升高的时段与病理Ⅱ-Ⅲ级对应；MHC-Ⅰ膜蛋白在CD3+CD4+T细胞和CD3+CD8+T细胞上表达趋势和表达强度基本一致,CD3+CD4+细胞MHC-Ⅰ升高开始的时间比CD3+CD8+细胞早4天；实时荧光定量RT-PCR检测mRNA表达与流式细胞术检测CD3+CD4+细胞膜蛋白表达趋势一致。异系移植术后8天,外周血淋巴细胞MHC-Ⅰ表达开始升高,术后16天达峰值,相对表达量约为术前的9倍(从术前0.58上升为5.76),然后逐渐下降到术后24天恢复到术前水平。
免疫组化检测结果：所有小鼠皮片组织细胞、血管内皮细胞及浸润淋巴细胞均呈阳性表达,与未用药组比较,表达强度减弱。但当急性排斥反应发生时,移植物MHC-Ⅰ表达增强。4.感染组小鼠外周血淋巴细胞MHC-Ⅰ、MHC-Ⅱ的表达变化
与正常对照组相比,感染组小鼠在腹腔注射肠球菌8小时时,外周血白细胞总数升高,外周血淋巴细胞MHC-ⅡmRNA和蛋白表达增强,但外周血淋巴细胞MHC-Ⅰ无论在mRNA水平还是蛋白水平的表达都没有明显的变化,说明感染性疾病不影响MHC-Ⅰ的表达。
5.外周血淋巴细胞MHC-Ⅰ与MHC-Ⅱ在急性移植排斥反应过程中表达变化的比较
外周血淋巴细胞MHC-ⅡmRNA表达：在同系移植组,术后各时间点MHC-Ⅱ表达与术前相比,差异无统计学意义。在未用药组,MHC-Ⅱ在术后4天开始升高,术后6天达峰值,约为术前的2.1倍(从术前1.29升高到2.71),术后14天降到术前水平。应用免疫抑制剂的情况下,MHC-Ⅱ在术后8天开始升高,术后24天恢复术前水平。与MHC-Ⅰ表达相比,MHC-Ⅱ升高幅度较小。外周血淋巴细胞MHC-Ⅱ膜蛋白的表达：在未用药组,仅在术后6天和8天时升高,在用药组,仅在术后12、14、16天表达升高,与MHC-Ⅰ相比,升高开始时间晚、升高持续时间短。
6.肾移植患者术前、术后以及发生移植排斥时MHC-Ⅰ表达变化稳定组：CD4+T淋巴细胞和CD8+T淋巴细胞HLA-Ⅰ膜蛋白表达趋势相同。术后一周内淋巴细胞HLA-Ⅰ表达稍有升高,升高幅度较小,约为术前的1.3倍,到术后2周时,恢复到术前水平。外周血淋巴细胞MHC-Ⅰ转录水平与膜蛋白水平趋势相同,但变化幅度较小。
排斥组：与排斥前最后一次随访检测的HLA-Ⅰ表达水平相比,在排斥发生的当天,HLA-Ⅰ表达明显升高(排斥当天308.2 vs.术前110.9),约为术前的2.7倍。应用免疫抑制剂冲击治疗3天后,HLA-Ⅰ表达明显下降,7天后,HLA-Ⅰ表达逐渐降低到术前水平。
反复发生排斥反应的患者HLA-Ⅰ始终处于高表达水平,免疫抑制剂冲击治疗对HLA-Ⅰ的表达影响较小。
结论
1.动物实验研究发现在急性移植排斥的早期阶段,病理显示Ⅱ-Ⅲ级时,外周血淋巴细胞MHC-Ⅰ蛋白和mRNA水平表达升高。与MHC-Ⅱ相比,MHC-Ⅰ表达升高开始时间早、升高持续时间长,升高变化幅度大。MHC-Ⅰ蛋白和(?)nRNA水平的表达变化均能及时预测急性移植排斥反应的发生。
2.临床应用进一步证实肾移植急性排斥反应发生时,外周血淋巴细胞HLA-Ⅰ蛋白和mRNA水平表达升高,尤其是膜蛋白水平表达升高更为明显。外周血淋巴细胞表面膜蛋白HLA-Ⅰ水平可作为诊断急性移植排斥反应发生的无创性检测指标。
BACKGROUND Organ transplantation is the most magnificent feat during 20th century. Extensive use of organ transplantation saved lives of numeric patients who had organ failures. However, there are still many problems unsolved. Among them,acute graft rejection (AGR)after transplantation is the main cause for that the graft function is decrease or even lose, and it's also the main cause for that result in chronic graft rejection. To identify and prevent AGR at the early stage of rejection can retrieve allograft.
At present, graft biopsy and histopathological examination remains the "golden standard" to diagnose AGR. It is hard to be used as routine method because of its Achilles'heel, such as recurrent error of sampling, complications and especially its invasive injuries. At the same time, some non-invasive monitoring markers, such as imaging examination, biochemistry examination and antibodies examination, have not yet been used for diagnosis of AGR in clinical practice because of their poor sensitivity and specificity. So there is an urgent need to develop more reliable and non-invasive early markers to monitor the immuno-state of transplanted patients and to predict the AGR.
MHC is a closely linked gene cluster located in one chromosome of mammals and codes a group of proteins, which are the key molecules participating in antigen presenting and activation of T lymphocytes, and MHC-Ⅰmolecule play important and extensive roles in immunoregulation. Classic MHC molecules have great polymorphism, and mismatched MHC gene between donor and receipt result in graft rejection after transplantation. In research area of transplantation, most researches focus on MHC of graft itself and antibody to graft in host serum. It is notable that T lymphocyte is one of the cells bearing most abundant MHC-Ⅰmolecules. Some researches argued that HLA-A and HLA-B transcription decreased with ageing in peripheral blood leucocytes and might be a monitor of failed immune function in aged person; MHC-Ⅰmolecule played a protective role for memory T cell; MHC-Ⅰmolecule prolonged the survival time of CD4 positive T cell. Our previous study showed that the expression of MHC-ⅠmRNA increased when acute rejection occurred. As a molecule that exercises the biological function, does MHC-Ⅰprotein also elevate when AGR occurs? And how is its application in clinic? Is MHC-ⅠmRNA or MHC-Ⅰprotein appreciable to predict AGR?
In order to investigate the value of MHC-Ⅰas a marker for diagnosis of AGR, we made inbred mouse skin transplantation model to monitored quantitatively and dynamically the MHC-Ⅰmolecules expression in peripheral blood lymphocytes (PBLs),grafts and infiltrating lymphocytes on different times during the whole process of AGR and studied the relationship among MHC-Ⅰexpression, the pathological changes of the grafts and immunosuppressive therapy firstly. Then we further serially monitored the HLA-Ⅰexpression in peripheral blood CD4 and CD8 T lymphocyte of 53 kidney transplant recipients who showed stable renal function or suffered from acute rejection at the different time points after transplantation, compared and analyzed the difference between stable patients and AGR patients and analyzed the level of HLA-Ⅰwith the state of immunotolerance. This research explored the feasibility of MHC-Ⅰas a marker for predicting AGR through both animal experiment and clinical study. OBJECTIVE
1. To establish the mouse skin transplantation model to observe the macroscopic and histopathological changes of skin graft after transplantation and classified rejection into 4 grades according to the scoring systems for acute mouse skin allograft rejection.
2. To explore the changes of MHC-ⅠmRNA expression and protein during the whole process of acute rejection and which is better for predicting AR?
3. Compared with the other noninvasive index of AR, MHC-II, to evaluate the value of MHC-I in PBLs as AR marker.
4. To serially measure respectively the HLA-I protein and mRNA expression in PBLs of kidney transplant recipients with stable renal function and with acute rejection at the different time points after transplantation. And to compare the difference between stable patients and those suffering from AGR, and analyze the level of HLA-I with the state of imrnunotolerance.
METHOD
1. Experimental groups:All 270 BALB/C recipient mice were randomly divided into three groups:untreated graft groups, immunosuppressive-treated graft groups and bacterial infection groups. Untreated graft groups and immunosuppressive-treated graft groups were further divided into non-transplant groups, syngeneic groups(group S and group SI) and allogeneic groups(group A and group AI). In groups with immunosuppresants, all the mice were administrated moderate dosage of three drugs including cyclosporine A (CsA), prednisolone (Horm) and mycophenolate mofetil (MMF) according to clinical therapeutic regimen. Enterococcus were injected into BALB/C mice enterocoelia to construct bacterical infectious group. Full-thickness skin from the backs of C57BL/6 mice (H-2b,donor) was transplanted onto that of Balb/C mice(H-2d,recipient) in allograft group(H-2b to H-2d) and in syngeneic graft group(H-2d to H-2d).
2. Establishment of mouse skin transplantation models and collection of samples: Full thickness dorsal skin derived from donor mice was transplanted onto the dorsal thoraxes of the recipients. Six recipient mice at each time point were analyzed systematically every two days as following:blood samples were collected and anticogulated by EDTA-K2; Skin grafts were obtained from the sacrificed mice at the same time as blood were collected, and then fixed in 4%formalin solution, ready for hematoxylineosin stain and immunohistochemistry detection. For normal control group and bacterical infectious groups, anticogulant blood sample were collected to be ready to use.
3. Detecting blood samples:To detect MHC-I, MHC-II MFI on different subtypes of lymphocytes (CD4+T, CD8+T) by FCM and MHC-I, MHC-II mRNA expression levels by FQ-PCR.
4. Examination of mice graft skin samples:all the graft skin samples were cut into 5-μm sections, and stained with hematoxylineosin(HE). The skin rejection was classified into 4 grades according to the extent of lymphocytes infiltration. The dynamic histological changes of graft rejection were recorded. MHC-I expression in the skin graft was examined by immunohistochemistry.
5. Serial monitoring the level of HLA-I expression:53 recipients (21-60 years old) who had undergone renal transplantations from March 2008 to September 2010 (33 cases with stable renal function as Group stable and 20 with acute rejection proved by kidney biopsy according to the Banff 2003 system as Group AR) were enrolled in present study. PBLs were collected both before transplant and the D3, D7, W2, W3, Ml, M2, M3+ after transplant. For the patients with AR symptoms, samples were obtained immediately before administration of anti-inflammatory agents and at D3, D7, more than W2. The level of HLA-I mRNA in the PBLs, HLA-I protein on the CD4+ and CD8+ T lymphocytes were measured by real-time RT-PCR and FCM respectively.
RESULTS
1. Skin grafts' macroscopy and pathological analysis All experimental mice gained weight during the trail process. The skin graft of mice in syngraft group grew well, no rejection was found until the end of observation. In A group and AI group, the graft gradually looked hard, black and shrank, and up to 80% graft necrosis took place at the 11th day and 22th day after transplantation respectively. In the syn grafts group (group C), there was no obvious infiltration in the epidermis, and could be considered as grade 0 or grade 1. In the whole process of trail in group A, the different degrees of infiltration of lymphocytes and monocytes were observed successively in the epidermis. During 2-4th,4-8th,8-12 th,12-16th day post-operation, pathological analysis showed grade 1,2,3,4 respectively. In group AI, pathological grade 1,2,3,4 was corresponding to 2-8th,8-14th,14-20th,20-26th day post-operation.
2. The expression of MHC-I in PBLs in bacterial infection group Compared with normal control group, at 8 hours after mice peritoneum were administrated with Enterococcus, the amount of white blood cells increased significantly, but the expression of MHC-I in PBLs, whether on mRNA level or protein level, has no changes.
3. The expression of MHC-I during AR in the absence of immunosuppressants
In Group S, no significant changes were observed in infiltrating lymphocyte and PBLs over the post-transplant period compared with the level of pre-operation, which is normal level. While in Group A, MHC-I expression varied significantly at different times post-operation.
Whether on protein level detected by FCM or on mRNA level detected by real time RT-PCR, MHC-I expression had the similar trends of variation during AGR. In group A, it increased rapidly at sixth day after operation and gradually decreased to the pre-operation level at D14. The time period when MHC-I increased was corresponding to pathological rejection Grade 2-4.
Positive MHC-I expression was observed in the skin graft and infiltrating lymphocytes during the whole process after transplantation. The MHC-I expression increased when AGR occurred.
4. The expression of MHC-I during AGR in the presence of immunosuppressants
In Group SI, no significant changes were observed in infiltrating lymphocyte and PBLs over the post-transplant period compared with the level of pre-operation. Compared with S group, Group SI showed a decreased MHC-I expression, which demonstrated that immunosuppressants could decrease MHC-I expression.
Whether on protein level detected by FCM or on mRNA level detected by real time RT-PCR, MHC-I expression has the similar trends of variation during AGR. In Group AI, it increased rapidly at eighth day after operation, reached a highest level at D16, and gradually decreased to the pre-operation level at D24. The time period when MHC-I increased was corresponding to pathological rejection Grade 2-4.
The intensity of MHC-I expression was lower in the skin graft and infiltrating lymphocytes during the whole process after transplantation in group SI than in group S. The MHC-Ⅰexpression increased when AR occurred.
5. The expression of MHC-Ⅰand MHC-Ⅱin PBLs during AGR
The expression of MHC-ⅡmRNA had the similar trend with MHC-ⅠmRNA during AGR, but the range of variation was smaller than that of MHC-ⅠmRNA. Compared with MHC-Ⅰprotein, The expression level of MHC-Ⅱprotein was lower and lasted shorter.
6. Variation of HLA-Ⅰexpression in patients after kidney transplantation
The level of HLA-Ⅰprotein on peripheral blood CD4+T lymphocyte and CD8+T lymphocyte had the similar trend. HLA-I on T lymphocyte showed a slight increase in the first week after operation and then rapidly decreased to the pre-operation level in the recipients with stable function. At the episodes of AGR, the level of HLA-Ⅰincreased significantly in all the patients of Group AR.
CONCLUSIONS
1. Animal experiment showed that at early stage of AGR, when is corresponding to pathological gradeⅠ-Ⅱ, the expression of both MHC-Ⅰprotein and mRNA increased, and lasted to pathological gradeⅣ. The expression of both MHC-Ⅰprotein and mRNA can predict AGR.
2. Clinical application further demonstrated that when AGR occurred in kidney transplantation, the expression of MHC-Ⅰin PBLs increased, especially on protein levels. The expression of MHC-Ⅰprotein on PBLs can be a marker for predicting AGR.
器官移植是20世纪伟大的科学创举,器官移植的广泛开展挽救了无数终末期器官功能衰竭患者的生命。然而,器官移植技术仍存在诸多问题,使得移植后的器官不能正常发挥功能。移植后的急性排斥反应(AGR)是移植物功能降低甚至丧失的主要原因,也是导致慢性排斥反应的主要因素。尽早及时的发现急性排斥反应,并采取有效的措施加以控制,可挽救移植物或延长移植物寿命。
目前移植物的病理组织学检查仍然是判断急性排斥反应的金标准。然而,由于病理组织学检查存在创伤性、操作复杂、费用昂贵、不适合重复操作等缺点,不能作为临床常规检查手段。目前常用的一些无创性检查方法,包括影像学、生化学检查以及抗体检测等,由于其在准确性、敏感性、特异性方面的不足而没能广泛应用于临床。至今为止,没有一种用于判断急性排斥反应的简单、无创性的检查方法获得大家的认可。
主要组织相容性复合体(major histocompatibility complex, MHC),是位于哺乳动物某一染色体的一组紧密连锁的基因群,其产物的主要作用是参与抗原递呈和T细胞激活,在免疫应答和免疫调节中执行重要而广泛的功能。人类MHC又称人类白细胞抗原复合体(human leucocyte antigen, HLA)。经典的MHC分子具有高度的多态性,正是由于移植供受者间这种MHC分子的不匹配导致了移植排斥反应的发生。也就是说MHC是启动移植排斥反应的靶抗原,是移植免疫的主角。在移植免疫研究领域,人们往往把目光集中在移植物MHC分子以及宿主血清中针对移植物的抗体上。但作为体内表达MHC-Ⅰ类分子最丰富的细胞,淋巴细胞尤其是T淋巴细胞MHC-Ⅰ类分子的功能研究却较少见。已有研究表明,外周血淋巴细胞表面HLA-A、B mRNA以及其相应蛋白的表达随年龄老化而降低,可以作为机体免疫功能衰退的指标；CD4+T细胞表面MHC-Ⅰ类分子可以延长CD4+T细胞的存活时间；记忆性T细胞表面MHC-Ⅰ类分子的表达对其自身具有保护作用。我们前期工作利用RNAi技术封闭淋巴细胞表面MHC-Ⅰ分子,发现淋巴细胞的增殖和杀伤活性均降低。另外我们前期对近交系小鼠皮肤移植模型的研究发现,当发生急性排斥反应时小鼠外周血淋巴细胞MHC-Ⅰ基因中的H-2K、H-2D基因转录mRNA表达升高。那么MHC-Ⅰ蛋白作为行使功能的分子,与急性排斥反应的关系如何?以及其在临床上的应用如何?CD4+T细胞和CD8+T细胞哪个亚群细胞表面MHC-Ⅰ的表达更能代表急性排斥反应的发生?MHC-Ⅰ蛋白和MHC-ImRNA两者谁更利于急性排斥反应的早期发现?类似问题有待于我们进一步研究。
为了系统的研究MHC-Ⅰ分子在急性移植排斥反应全过程中的变化规律,本研究首先采用近交系小鼠皮肤移植模型,研究了移植前、移植后不同时间点小鼠外周血淋巴细胞MHC-ImRNA的表达及外周血CD4+、CD8+T细胞亚群MHC-K、移植物MHC-Ⅰ分子的表达变化,分析了MHC-Ⅰ分子的表达水平与移植排斥全过程的对应关系。进而动态监测了临床上包括移植术后稳定和移植术后发生急性排斥的共53例肾移植患者外周血CD4+、CD8+T细胞MHC-Ⅰ分子的表达情况,从动物和临床两方面探讨了MHC-Ⅰ作为判断急性移植排斥反应指标的可行性。
目的
1.构建近交系小鼠皮肤移植模型,从而获得急性移植排斥反应的全过程,研究急性移植排斥反应时外周血淋巴细胞MHC-Ⅰ的表达规律,探讨外周血淋巴细胞MHC-Ⅰ表达与急性移植排斥反应病理分级之间的对应关系。
2.对外周血淋巴细胞MHC-Ⅰ基因的mRNA和蛋白两个水平进行研究,分析两水平表达趋势是否一致,哪一个水平的表达变化更能反映急性移植排斥反应。
3.分别检测CD3+CD4+T细胞和CD3+CD8+T细胞表面MHC-Ⅰ分子的表达变化,分析MHC-Ⅰ分子的表达变化在两群细胞间是否存在差别。
4.与T细胞活化指标MHC-Ⅱ比较,探讨MHC-Ⅰ分子作为判断急性移植排斥反应的可行性。
5.动态检测肾移植患者术前和术后不同时间外周血单个核细胞HLA-I mRNA表达变化以及CD3+CD4+细胞和CD3+CD8+细胞HLA-Ⅰ蛋白水平的表达变化,对比排斥组和稳定组之间的差异,进一步探讨在临床肾急性移植排斥反应中HLA-I的诊断价值。
方法
1.实验小鼠分组：把270只BALB/C(简称BA)小鼠随机分为三组,分别是移植用药组(应用免疫抑制剂)、移植未用药组以及单纯感染组。用药组和未用药组又分为非手术组、同系移植组和异系移植组。用药非手术组即为用药术前对照组,未用药非手术组即为未用药术前对照组。用药组的设立是为了模拟临床采用了皮质激素(Horm)、环孢素A(CsA)及霉酚酸酯(MMF)中等剂量三联用药。采用肠球菌注射正常小鼠腹腔建立肠球菌小鼠腹膜炎模型既为细菌感染组。同系移植采用BA小鼠个体之间进行皮肤移植,异系移植以C57BL/6(简称C57)小鼠为供体,BA小鼠为受体,进行小鼠皮肤移植。每两天为一个时间点,分别为术后2、4、6天……,用药移植组小鼠观察到术后26天,未用药移植组小鼠观察到术后16天。
2.构建小鼠皮肤移植模型以及标本的留取：以C57小鼠为供体,BA小鼠为受体,采用经典的小鼠背—背皮肤移植手术方法,建立小鼠皮肤移植动物模型。移植后每隔一天(每两天)对小鼠状态进行全面分析,每个时间点6只小鼠,进行以下操作：记录移植前和移植术后各时间点小鼠移植皮片的肉眼变化；通过眼球取血采集小鼠血液到EDTA-K2抗凝血常规管中备用；采血的同时取移植皮片组织用4%多聚甲醛固定,用于组织病理学检测和免疫组化检测。对正常组和细菌感染组小鼠收集血液备用。
3.小鼠血液标本的检测：采用三色流式细胞技术检测小鼠外周血不同亚群T细胞：CD3+CD4+T细胞、CD3+CD8+T细胞表面MHC-Ⅰ、MHC-Ⅱ分子的表达；采用实时荧光定量RT-PCR技术检测小鼠外周血淋巴细胞MHC-ⅠmRNA,MHC-ⅡmRNA的表达变化；分析外周血淋巴细胞MHC-ⅠMHC-Ⅱ在AGR全过程中的变化规律,以及免疫抑制剂对上述指标表达水平的影响。
4.小鼠移植皮片标本的检查：对留取的所有时间点的皮肤组织进行常规病理切片,H-E染色后观察移植皮片淋巴细胞浸润等组织学变化,根据病理组织学改变将术后不同天数的移植物排斥反应发生的程度分为Ⅰ-Ⅳ级,记录移植物病理排斥动态变化。对比用药和不用药对移植物病理组织学改变和存活时间的影响。采用免疫组织化学染色法检测小鼠移植皮片中组织细胞和浸润淋巴细胞MHC-Ⅰ的表达,观察急性移植排斥反应发生时小鼠移植皮片中MHC-Ⅰ表达强度的变化。
5.临床肾移植患者：根据移植术后3个月之内是否发生过急性移植排斥反应,把肾移植患者分成稳定组和排斥组。收集肾移植稳定组患者术前和术后3天、7天……3月和大于3个月的血液标本和肾移植排斥组患者排斥前、排斥当天、抗排斥治疗后3天、7天、14天的血液标本,采用流式细胞技术对外周血中CD3+CD4+T细胞、CD3+CD8+T细胞表面的HLA-Ⅰ(MHC-Ⅰ)分子进行检测。采用实时荧光定量PCR技术对外周血淋巴细胞HLA-ImRNA进行检测。比较稳定组和排斥组的差异,初步从临床角度评价HLA-Ⅰ对于急性移植排斥反应的诊断价值。结果
1.移植皮片的肉眼及病理组织学结果
95%小鼠在实验过程中都有体重增长,其余5%在实验中被排除。同系移植组小鼠皮肤移植物生长良好,移植物缝合部位逐渐愈合,皮片柔软红润。在异系未用药组和异系用药组,移植物逐渐变暗、变黑,80%移植物坏死时发生在术后第10天和第22天。移植皮片经H-E染色,按照淋巴细胞浸润和皮肤组织坏死程度将急性排斥反应分为轻度(Ⅰ级)、中度(Ⅱ级)、重度(Ⅲ级)和极重度(Ⅳ级)四个等级。结果显示：随术后时间的延长,异系移植皮片淋巴细胞浸润逐渐增强。不用药情况下,病理Ⅰ-Ⅳ级排斥反应分别发生于术后2-4天、4-8天、8-12天和12-16天。用药情况下,淋巴细胞浸润时间延迟,病理排斥Ⅰ-Ⅳ级分别发生于2-8天、8-14天、14-20天、20-26天。同系移植皮片中很少淋巴细胞浸润,移植病理分级为0或Ⅰ级。
2.未用药情况下,移植术后MHC-Ⅰ的表达变化
同系移植组,术后各时间点外周血、皮肤移植物MHC-Ⅰ的表达变化不明显,与术前基本保持一致,而异系移植组在术后各时间点呈现出不同的变化。
外周血淋巴细胞MHC-Ⅰ的表达情况：CD3+CD8+细胞MHC-Ⅰ膜蛋白在术后6天表达明显升高,表达强度约为术前的1.3倍(MFI从术前219升高到290),升高一直持续到术后12天,术后14天MHC-Ⅰ膜蛋白表达回复到术前水平,升高的时段与病理Ⅱ-Ⅲ级对应；CD3+CD4+细胞MHC-Ⅰ膜蛋白升高开始的时间比CD3+CD8+细胞早2天,但其表达趋势和表达强度与CD3+CD8+细胞基本一致；实时荧光定量RT-PCR检测MHC-ImRNA表达与流式细胞术检测CD4+细胞MHC-Ⅰ膜蛋白表达规律一致。异系移植术后4天,外周血淋巴细胞MHC-Ⅰ表达明显升高,术后6天达峰值,约为术前的4倍(从术前1.38升高到5.96),表达升高一直持续到术后12天,术后14天恢复到术前水平。
免疫组化检测结果：所有小鼠移植皮片组织细胞、血管内皮细胞及浸润淋巴细胞均呈阳性表达,但当排斥反应发生时,移植物MHC-Ⅰ表达明显增强。3.应用免疫抑制剂情况下,移植术前及术后MHC-Ⅰ的表达变化
在移植手术前,与未用药相比,用药后MHC-ⅠmRNA及蛋白表达水平降低,说明免疫抑制剂对MHC-Ⅰ的表达有抑制作用。同系移植组,术后各时间点外周血、皮肤移植物MHC-Ⅰ的表达变化不明显,与术前基本保持一致。而异系移植组在术后各时间点呈现出不同的变化。
外周血淋巴细胞MHC-Ⅰ的表达情况：CD3+CD8+细胞MHC-Ⅰ膜蛋白在术后12天表达明显升高,术后16天达峰值,表达强度约为术前的1.3倍(MFI从术前169升高到228),升高一直持续到术后22天,术后24天MHC-Ⅰ膜蛋白表达回复到术前水平,升高的时段与病理Ⅱ-Ⅲ级对应；MHC-Ⅰ膜蛋白在CD3+CD4+T细胞和CD3+CD8+T细胞上表达趋势和表达强度基本一致,CD3+CD4+细胞MHC-Ⅰ升高开始的时间比CD3+CD8+细胞早4天；实时荧光定量RT-PCR检测mRNA表达与流式细胞术检测CD3+CD4+细胞膜蛋白表达趋势一致。异系移植术后8天,外周血淋巴细胞MHC-Ⅰ表达开始升高,术后16天达峰值,相对表达量约为术前的9倍(从术前0.58上升为5.76),然后逐渐下降到术后24天恢复到术前水平。
免疫组化检测结果：所有小鼠皮片组织细胞、血管内皮细胞及浸润淋巴细胞均呈阳性表达,与未用药组比较,表达强度减弱。但当急性排斥反应发生时,移植物MHC-Ⅰ表达增强。4.感染组小鼠外周血淋巴细胞MHC-Ⅰ、MHC-Ⅱ的表达变化
与正常对照组相比,感染组小鼠在腹腔注射肠球菌8小时时,外周血白细胞总数升高,外周血淋巴细胞MHC-ⅡmRNA和蛋白表达增强,但外周血淋巴细胞MHC-Ⅰ无论在mRNA水平还是蛋白水平的表达都没有明显的变化,说明感染性疾病不影响MHC-Ⅰ的表达。
5.外周血淋巴细胞MHC-Ⅰ与MHC-Ⅱ在急性移植排斥反应过程中表达变化的比较
外周血淋巴细胞MHC-ⅡmRNA表达：在同系移植组,术后各时间点MHC-Ⅱ表达与术前相比,差异无统计学意义。在未用药组,MHC-Ⅱ在术后4天开始升高,术后6天达峰值,约为术前的2.1倍(从术前1.29升高到2.71),术后14天降到术前水平。应用免疫抑制剂的情况下,MHC-Ⅱ在术后8天开始升高,术后24天恢复术前水平。与MHC-Ⅰ表达相比,MHC-Ⅱ升高幅度较小。外周血淋巴细胞MHC-Ⅱ膜蛋白的表达：在未用药组,仅在术后6天和8天时升高,在用药组,仅在术后12、14、16天表达升高,与MHC-Ⅰ相比,升高开始时间晚、升高持续时间短。
6.肾移植患者术前、术后以及发生移植排斥时MHC-Ⅰ表达变化稳定组：CD4+T淋巴细胞和CD8+T淋巴细胞HLA-Ⅰ膜蛋白表达趋势相同。术后一周内淋巴细胞HLA-Ⅰ表达稍有升高,升高幅度较小,约为术前的1.3倍,到术后2周时,恢复到术前水平。外周血淋巴细胞MHC-Ⅰ转录水平与膜蛋白水平趋势相同,但变化幅度较小。
排斥组：与排斥前最后一次随访检测的HLA-Ⅰ表达水平相比,在排斥发生的当天,HLA-Ⅰ表达明显升高(排斥当天308.2 vs.术前110.9),约为术前的2.7倍。应用免疫抑制剂冲击治疗3天后,HLA-Ⅰ表达明显下降,7天后,HLA-Ⅰ表达逐渐降低到术前水平。
反复发生排斥反应的患者HLA-Ⅰ始终处于高表达水平,免疫抑制剂冲击治疗对HLA-Ⅰ的表达影响较小。
结论
1.动物实验研究发现在急性移植排斥的早期阶段,病理显示Ⅱ-Ⅲ级时,外周血淋巴细胞MHC-Ⅰ蛋白和mRNA水平表达升高。与MHC-Ⅱ相比,MHC-Ⅰ表达升高开始时间早、升高持续时间长,升高变化幅度大。MHC-Ⅰ蛋白和(?)nRNA水平的表达变化均能及时预测急性移植排斥反应的发生。
2.临床应用进一步证实肾移植急性排斥反应发生时,外周血淋巴细胞HLA-Ⅰ蛋白和mRNA水平表达升高,尤其是膜蛋白水平表达升高更为明显。外周血淋巴细胞表面膜蛋白HLA-Ⅰ水平可作为诊断急性移植排斥反应发生的无创性检测指标。
BACKGROUND Organ transplantation is the most magnificent feat during 20th century. Extensive use of organ transplantation saved lives of numeric patients who had organ failures. However, there are still many problems unsolved. Among them,acute graft rejection (AGR)after transplantation is the main cause for that the graft function is decrease or even lose, and it's also the main cause for that result in chronic graft rejection. To identify and prevent AGR at the early stage of rejection can retrieve allograft.
At present, graft biopsy and histopathological examination remains the "golden standard" to diagnose AGR. It is hard to be used as routine method because of its Achilles'heel, such as recurrent error of sampling, complications and especially its invasive injuries. At the same time, some non-invasive monitoring markers, such as imaging examination, biochemistry examination and antibodies examination, have not yet been used for diagnosis of AGR in clinical practice because of their poor sensitivity and specificity. So there is an urgent need to develop more reliable and non-invasive early markers to monitor the immuno-state of transplanted patients and to predict the AGR.
MHC is a closely linked gene cluster located in one chromosome of mammals and codes a group of proteins, which are the key molecules participating in antigen presenting and activation of T lymphocytes, and MHC-Ⅰmolecule play important and extensive roles in immunoregulation. Classic MHC molecules have great polymorphism, and mismatched MHC gene between donor and receipt result in graft rejection after transplantation. In research area of transplantation, most researches focus on MHC of graft itself and antibody to graft in host serum. It is notable that T lymphocyte is one of the cells bearing most abundant MHC-Ⅰmolecules. Some researches argued that HLA-A and HLA-B transcription decreased with ageing in peripheral blood leucocytes and might be a monitor of failed immune function in aged person; MHC-Ⅰmolecule played a protective role for memory T cell; MHC-Ⅰmolecule prolonged the survival time of CD4 positive T cell. Our previous study showed that the expression of MHC-ⅠmRNA increased when acute rejection occurred. As a molecule that exercises the biological function, does MHC-Ⅰprotein also elevate when AGR occurs? And how is its application in clinic? Is MHC-ⅠmRNA or MHC-Ⅰprotein appreciable to predict AGR?
In order to investigate the value of MHC-Ⅰas a marker for diagnosis of AGR, we made inbred mouse skin transplantation model to monitored quantitatively and dynamically the MHC-Ⅰmolecules expression in peripheral blood lymphocytes (PBLs),grafts and infiltrating lymphocytes on different times during the whole process of AGR and studied the relationship among MHC-Ⅰexpression, the pathological changes of the grafts and immunosuppressive therapy firstly. Then we further serially monitored the HLA-Ⅰexpression in peripheral blood CD4 and CD8 T lymphocyte of 53 kidney transplant recipients who showed stable renal function or suffered from acute rejection at the different time points after transplantation, compared and analyzed the difference between stable patients and AGR patients and analyzed the level of HLA-Ⅰwith the state of immunotolerance. This research explored the feasibility of MHC-Ⅰas a marker for predicting AGR through both animal experiment and clinical study. OBJECTIVE
1. To establish the mouse skin transplantation model to observe the macroscopic and histopathological changes of skin graft after transplantation and classified rejection into 4 grades according to the scoring systems for acute mouse skin allograft rejection.
2. To explore the changes of MHC-ⅠmRNA expression and protein during the whole process of acute rejection and which is better for predicting AR?
3. Compared with the other noninvasive index of AR, MHC-II, to evaluate the value of MHC-I in PBLs as AR marker.
4. To serially measure respectively the HLA-I protein and mRNA expression in PBLs of kidney transplant recipients with stable renal function and with acute rejection at the different time points after transplantation. And to compare the difference between stable patients and those suffering from AGR, and analyze the level of HLA-I with the state of imrnunotolerance.
METHOD
1. Experimental groups:All 270 BALB/C recipient mice were randomly divided into three groups:untreated graft groups, immunosuppressive-treated graft groups and bacterial infection groups. Untreated graft groups and immunosuppressive-treated graft groups were further divided into non-transplant groups, syngeneic groups(group S and group SI) and allogeneic groups(group A and group AI). In groups with immunosuppresants, all the mice were administrated moderate dosage of three drugs including cyclosporine A (CsA), prednisolone (Horm) and mycophenolate mofetil (MMF) according to clinical therapeutic regimen. Enterococcus were injected into BALB/C mice enterocoelia to construct bacterical infectious group. Full-thickness skin from the backs of C57BL/6 mice (H-2b,donor) was transplanted onto that of Balb/C mice(H-2d,recipient) in allograft group(H-2b to H-2d) and in syngeneic graft group(H-2d to H-2d).
2. Establishment of mouse skin transplantation models and collection of samples: Full thickness dorsal skin derived from donor mice was transplanted onto the dorsal thoraxes of the recipients. Six recipient mice at each time point were analyzed systematically every two days as following:blood samples were collected and anticogulated by EDTA-K2; Skin grafts were obtained from the sacrificed mice at the same time as blood were collected, and then fixed in 4%formalin solution, ready for hematoxylineosin stain and immunohistochemistry detection. For normal control group and bacterical infectious groups, anticogulant blood sample were collected to be ready to use.
3. Detecting blood samples:To detect MHC-I, MHC-II MFI on different subtypes of lymphocytes (CD4+T, CD8+T) by FCM and MHC-I, MHC-II mRNA expression levels by FQ-PCR.
4. Examination of mice graft skin samples:all the graft skin samples were cut into 5-μm sections, and stained with hematoxylineosin(HE). The skin rejection was classified into 4 grades according to the extent of lymphocytes infiltration. The dynamic histological changes of graft rejection were recorded. MHC-I expression in the skin graft was examined by immunohistochemistry.
5. Serial monitoring the level of HLA-I expression:53 recipients (21-60 years old) who had undergone renal transplantations from March 2008 to September 2010 (33 cases with stable renal function as Group stable and 20 with acute rejection proved by kidney biopsy according to the Banff 2003 system as Group AR) were enrolled in present study. PBLs were collected both before transplant and the D3, D7, W2, W3, Ml, M2, M3+ after transplant. For the patients with AR symptoms, samples were obtained immediately before administration of anti-inflammatory agents and at D3, D7, more than W2. The level of HLA-I mRNA in the PBLs, HLA-I protein on the CD4+ and CD8+ T lymphocytes were measured by real-time RT-PCR and FCM respectively.
RESULTS
1. Skin grafts' macroscopy and pathological analysis All experimental mice gained weight during the trail process. The skin graft of mice in syngraft group grew well, no rejection was found until the end of observation. In A group and AI group, the graft gradually looked hard, black and shrank, and up to 80% graft necrosis took place at the 11th day and 22th day after transplantation respectively. In the syn grafts group (group C), there was no obvious infiltration in the epidermis, and could be considered as grade 0 or grade 1. In the whole process of trail in group A, the different degrees of infiltration of lymphocytes and monocytes were observed successively in the epidermis. During 2-4th,4-8th,8-12 th,12-16th day post-operation, pathological analysis showed grade 1,2,3,4 respectively. In group AI, pathological grade 1,2,3,4 was corresponding to 2-8th,8-14th,14-20th,20-26th day post-operation.
2. The expression of MHC-I in PBLs in bacterial infection group Compared with normal control group, at 8 hours after mice peritoneum were administrated with Enterococcus, the amount of white blood cells increased significantly, but the expression of MHC-I in PBLs, whether on mRNA level or protein level, has no changes.
3. The expression of MHC-I during AR in the absence of immunosuppressants
In Group S, no significant changes were observed in infiltrating lymphocyte and PBLs over the post-transplant period compared with the level of pre-operation, which is normal level. While in Group A, MHC-I expression varied significantly at different times post-operation.
Whether on protein level detected by FCM or on mRNA level detected by real time RT-PCR, MHC-I expression had the similar trends of variation during AGR. In group A, it increased rapidly at sixth day after operation and gradually decreased to the pre-operation level at D14. The time period when MHC-I increased was corresponding to pathological rejection Grade 2-4.
Positive MHC-I expression was observed in the skin graft and infiltrating lymphocytes during the whole process after transplantation. The MHC-I expression increased when AGR occurred.
4. The expression of MHC-I during AGR in the presence of immunosuppressants
In Group SI, no significant changes were observed in infiltrating lymphocyte and PBLs over the post-transplant period compared with the level of pre-operation. Compared with S group, Group SI showed a decreased MHC-I expression, which demonstrated that immunosuppressants could decrease MHC-I expression.
Whether on protein level detected by FCM or on mRNA level detected by real time RT-PCR, MHC-I expression has the similar trends of variation during AGR. In Group AI, it increased rapidly at eighth day after operation, reached a highest level at D16, and gradually decreased to the pre-operation level at D24. The time period when MHC-I increased was corresponding to pathological rejection Grade 2-4.
The intensity of MHC-I expression was lower in the skin graft and infiltrating lymphocytes during the whole process after transplantation in group SI than in group S. The MHC-Ⅰexpression increased when AR occurred.
5. The expression of MHC-Ⅰand MHC-Ⅱin PBLs during AGR
The expression of MHC-ⅡmRNA had the similar trend with MHC-ⅠmRNA during AGR, but the range of variation was smaller than that of MHC-ⅠmRNA. Compared with MHC-Ⅰprotein, The expression level of MHC-Ⅱprotein was lower and lasted shorter.
6. Variation of HLA-Ⅰexpression in patients after kidney transplantation
The level of HLA-Ⅰprotein on peripheral blood CD4+T lymphocyte and CD8+T lymphocyte had the similar trend. HLA-I on T lymphocyte showed a slight increase in the first week after operation and then rapidly decreased to the pre-operation level in the recipients with stable function. At the episodes of AGR, the level of HLA-Ⅰincreased significantly in all the patients of Group AR.
CONCLUSIONS
1. Animal experiment showed that at early stage of AGR, when is corresponding to pathological gradeⅠ-Ⅱ, the expression of both MHC-Ⅰprotein and mRNA increased, and lasted to pathological gradeⅣ. The expression of both MHC-Ⅰprotein and mRNA can predict AGR.
2. Clinical application further demonstrated that when AGR occurred in kidney transplantation, the expression of MHC-Ⅰin PBLs increased, especially on protein levels. The expression of MHC-Ⅰprotein on PBLs can be a marker for predicting AGR.
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