【作者】 张弛；

【导师】 陈实；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 博士

【摘要】 建立一种以流式细胞术(FCM)为基础来检测人ABO天然血型特异性抗体的新技术。使用商品来源的标准化人源红细胞作为统一反应的靶细胞,与相对应的健康人血清样本作用后再加入特异性荧光标记的二抗,采用FCM检测特异性血型抗体滴度水平。通过检测所收集的52例健康人血清样本并对比以传统血液凝集法改进的酶标仪法,综合比较分析两种方法的检测结果显示：血型抗体(IgM为主)水平的变化趋势上两种方法关联性较好,但FCM新方法显示出更好的特异性、灵敏性和重复性,并能够半定量抗体及其分型(IgM、IgG及亚型)。FCM法是一种更为精确的天然血型抗体检测技术,将来很可能逐步替代传统的血液凝集法应用于ABO血型不合移植的血型抗体定量化及用于手术监测的全过程。绝大部分灵长类动物存在与人类相似的ABO血型系统,本部分的研究采用改良FCM法检测猕猴及食蟹猴血清中血型抗体水平及抗体的分布情况。同上一部分所述相同,以流式细胞术为基础,预先用人O型浓缩红细胞吸附猴血清中所含种属间非特异性抗体采用商品化来源的人源红细胞作为靶细胞统一抗原的标准表达量,并通过加入特异性荧光标记的抗人IgM或IgG二抗,对收集的实验用猕猴及食蟹猴的血清样本预先用人O型浓缩红细胞吸附猴血清中所含种属间非特异性抗体,然后再进行检测,以健康受试者的血清样本为对照,比较二者血型抗体水平的差异。实验结果显示,经过预先处理吸附猴血清中所含种属间非特异性抗体的改良步骤后,FCM法同样能够准确检测猴的血型抗体水平及分型。结果显示猴血清中天然血型抗体以IgM为主,滴度水平明显低于健康人(P<0.05)。由此我们得出改良后的FCM法同样适用于灵长类动物血清中血型抗体水平的检测,这为构建灵长类动物模拟人ABO血型不合器官移植模型的成功建立提供了关键技术保障。本部分采用钥孔戚血蓝蛋白耦联ABO血型抗原A特异表位(N一乙酰氨基半乳糖)(KLH-A)体内预致敏的方法,成功提高了食蟹猴(B血型)循环内的抗A血型抗体的水平。使用前述改良FCM法检测猴致敏前后血型抗体IgM和IgG水平的变化,并对比其差异。结果显示通过KLH-A成功致敏后显著升高食蟹猴血型抗体IgM水平,同时非血型预存抗体IgG的表达也有升高(P<0.001),致敏效果及升高后抗体水平的维持时间都存在一定的个体化差异。由此可得出通过KLH- A或B的预致敏不但能够特异刺激猴循环中针对血型抗原的抗体IgM的高表达,也提高了非特异抗体IgG的水平。通过预致敏后成功的升高了灵长类大动物的血型抗体水平,这也为模拟临床ABO血型不合肾移植模型的成功建立提供了良好的实验前提。该部分我们成功建立了预致敏后施行同种异体ABO血型不合的肾移植术,并由血型抗体介导激活补体杀伤最终导致的超急性排斥反应的模型。应用改良FCM法检测实验食蟹猴体内手术前预存天然ABO血型抗体滴度,结果显示血型抗体水平较低的对照猴,直接施行ABO血型不合的同种异体肾移植无法导致超急性排斥反应的免疫结果。病理研究显示对照组移植肾脏并未显示超急性排斥反应的证据,而是显示急性体液性和细胞性排斥混合的病理特征,免疫组化也未见血型抗体(IgM或IgG)及补体片段(C4d及C5b-9)的沉积。而在KLH-A预致敏的实验组(n=2),受体猴(血型B)接受KLH-A预致敏14天后,再接受来自供体猴(血型A)的肾脏移植,结果2只受体猴的移植肾均在血管吻合开放血流后1小时内由鲜红色变为黑紫色,其中一例(07cy05)于开放血流一小时内切取移植肾标本,另一例(09cy15)于术后第二日切取移植肾标本,病理和免疫组化的结果均显示明确的超急性排斥的。我们对实验组受体猴施行肾移植术后的发生超急性排斥的移植肾的病理学特点及免疫学变化进行了系统研究分析,进一步为模拟临床ABO血型不合肾移植术后的排斥诊疗和预防提供实验依据。更多还原

【Abstract】 To establish a standard method using Flow Cytometry in ABO blood group antibody (Ab) detection. Based on direct hemagglutination, binding of antibody was measured by indirect flow cytometry adding secondary isotype-specific fluoresce labeled ab, For validation, we analyzed sera samples from 52 blood donors and compared with the results of using ELISA Reader method. Changes in antibody measured by both methods correlated well with sera titers. FCM showed better specificity, sensitivity and reproducibility over ELISA Reader method, and allowed quantitative discrimination in the range of antibody levels. FCM is the most objective method to offer accurate detection of Natural ABO blood group ab and more suitable for ABO-incompatible transplantations and pre-and post-transplant monitoring, that may replace the traditional hemagglutination.Like humans, most of non-human primates also express the ABH specificites of the ABO histo-blood group system. In this study, we therefore developed a simple and rapid method of measuring the levels and distribution of monkey ABO antibody (Ab) using flow cytometry (FCM). Binding of standard human A or B RBCs and anti-A/B Ab was measured by adding secondary isotype-specific fluoresce labeled Ab, for validation, we analyzed all collective monkey blood sera samples and compared with the results of human healthy blood donor sera. The results revealed that, following adsorption on human type O RBCs to remove anti-human antibodies, FCM seems to be the objective and reliable technique to detect natural specific Ab levels in Monkey sera. Both sera were detected and the Monkey’s anti-A/B antibody titers were significantly lower than Human’s (P<0.05). We concluded that FCM shows an excellent method to offer accurate detection of natural ABO blood group Ab on non-human primates, and also can be the technique support for the further study.Hyperacute rejection (HAR), which occurs in ABO-incompatible renal transplantation or xenotranplantation with high titers of natural anti-donor specific antigen antibody, is a significant cause of allografts dysfunction and loss. More efficacious treatment modalities are needed to eliminate or curtail natural anti-ABO blood group antibody production and its deleterious effects on the kidney. The availability of animal models mimicking human HAR is essential to understand its pathophysiology and develop new treatment strategies. Using a non-human primate’s ABO-incompatible renal transplant model, we demonstrate that presensitization of recipients with KLH-A results in rejection of subsequent renal allografts. All presensitized monkeys (n=2) developed renal failure within the graft minutes to a few hours after activation of complement, with the vascular anastomosis, with subsequent arteriographic evidence of progressive cortical necrosis, necessitating removal of the grafts. The major histological findings that are associated with HAR are neutrophil and platelet margination in glomerular and peritubular capillaries, red blood cells stasis, fibrin deposition, and thrombosis within the microvasculature with sparing of larger blood vessels (showed in 07cy05; at least early in the process), and acute tubular injury and variable levels of cortical necrosis. Immunofluorescence (IF) studies demonstrated IgM (but not IgG) combination in glomerular and peritubular capillaries. Complement (C4d or C5b-9) deposition was diffuse and obvious in peritubular capillaries. Serum analysis demonstrated high levels of circulating anti-ABO blood group antibodies with strongly reactivity to blood group antigen. The clinical setting and histological findings of our models strongly resemble HAR, which was frequently associated with humoral rejection, a situation commonly encountered in human renal allograft recipients. These findings in our model established for HAR indicate that this non-human primate’s model may provide an excellent tool to explore the pathogenesis and immunological events involved in human renal allograft rejection. The development of this valuable model has the potential to advance understanding in several key areas including local and systemic immunological events that result in natural blood group antibody elaboration, its contribution to graft injury, and the effects of various potential therapeutic interventions, mechanisms of natural blood group antibody-mediated graft damage, and effective treatment strategies to interfere with these processes.更多还原