【作者】 田梅；

【导师】 吴雄飞；

【作者基本信息】 第三军医大学 ， 移植医学， 2009， 博士

【摘要】 晚期氧化蛋白产物(Advanced oxidation protein products,AOPPs)是Witko-Sarsat等于1996年首先报道的由尿毒症血液透析(Hemodialysis,HD)患者血浆中分离出的含有双酪氨酸结构的蛋白交联产物,系因患者体内过高的氧化应激水平导致各种蛋白质氧化损伤所形成的终末产物的总称。现已明确,AOPPs是一类炎症介质,能够激活单核细胞,刺激TNF-α、IL-6、IL-1等炎性因子的合成释放,促发单核细胞的炎症效应,是HD患者免疫功能紊乱、加速性动脉粥样硬化、透析相关性淀粉样变等长期并发症的重要致病环节。血浆中的AOPPs主要分为高分子量(约670kD)和低分子量(约70kD)两种形式,高分子量AOPPs是氧化白蛋白的聚合体,在HD患者中起主要作用的是高分子量的AOPPs,在本文中我们主要研究高分子量AOPPs。AOPPs一旦形成即不可逆,因其分子量大,目前各种透析模式均不能有效清除,成为HD患者的长期并发症的重要致病因素,严重影响依赖性透析病人的长期生存率和生存质量。目前AOPPs的检测主要是利用AOPPs在340nm波长有紫外吸收峰,以在同一波长有紫外吸收峰的氯胺-T作为替代品来定量检测,因氯胺T是一种有机化合物,并不能真实反映体内的AOPPs水平,缺乏特异性,容易出现假阳性。且该方法操作步骤烦琐,需13000rpm离心1小时,易受血脂、胆红素、溶血、药物的影响。近年的文献报道,高速离心后HD患者血清AOPPs值明显较未离心的AOPPs值低,且所测值与氧化应激的另一指标8-oxo-dG无关联,采用去脂剂沉淀甘油三酯后发现,所测AOPPs值较未用去脂剂前下降51%,但无论是高速离心还是去脂剂沉淀甘油三酯后,HD患者体内的AOPPs仍明显高于正常,特异、准确检测血浆AOPPs显得非常重要。研究表明,AOPPs与疾病的严重程度和疾病的预后相关。因此,应用AOPPs抗体通过免疫组化、ELISA、western blot等方法观察AOPPs在病变组织或体液中的表达情况对相关疾病的诊断、疗效观察、预后等具有重要意义。AOPPs可通过沉积于血管、组织而加重动脉粥样硬化,透析相关性淀粉样变,运用其相应抗体有可能阻断AOPPs的作用,从而为AOPPs的毒性作用提供一种新的治疗途径。另外,借助抗体与抗原特异性结合的特性,也可以将AOPPs抗体偶联透析器上,从而发挥靶向治疗的作用。为进一步研究AOPPs结合蛋白甚至受体奠定了坚实的基础。由上可见,AOPPs抗体具有广泛的应用前景和潜在的应用价值。目前,国内外仍无商品化的AOPPs抗体及这方面的研究报道。抗体制备中最关键的环节是制备的抗原一定要纯。目前国内外主要采用人血白蛋白(human serum albumin,HSA)与次氯酸(HOCl)孵育体外制备AOPPs-HSA作为研究的对象,它具备与体内AOPPs同样的生物学活性,但是这样制备出的AOPPs-HSA成分并不单一,能够满足生物学功能研究的要求,但却不适合抗体制作。凝胶层析是以各种凝胶为固定相,利用流动相中各组分的相对分子量不同而达到分离的一种层析方法。本文研究的重点是大分子量的AOPPs,Hitrap 26/60 sephacryl S-300 high Resolution是一种由分子量为4～20×104的葡聚糖交联聚合而成的葡聚糖凝胶,适于大分子的分离,其分离范围为10KD～1500KD。现有文献报道,AOPPs是一组不同分子量的复合物,为保证抗原的纯度及筛选到的抗体具有特异性,我们在免疫动物时取AOPPs-HSA纯化后的第一峰作为免疫原,制备AOPPs单克隆抗体,并以纯化后第一峰作为筛选抗体的检测源(以后在本文中免疫动物、筛选抗体的检测源、抗体特异性鉴定采用的AOPPs-HSA均为AOPPs-HSA纯化后的第一峰)。综上所述,本研究的目的是采用HSA与HOCl孵育体外制备AOPPs,通过葡聚糖凝胶Hitrap 26/60 sephacryl S-300得到AOPPs的相对纯品,取纯化后的第一峰作为免疫原,并以纯化后第一峰作为筛选抗体的检测源,制备AOPPs的单克隆抗体,建立抗体工作平台,应用AOPPs抗体通过Western blot、ELISA等方法观察AOPPs在病变组织或体液中的表达情况,对相关疾病的诊断、疗效观察、预后提供依据等。另外,为用抗体中和AOPPs毒性、将AOPPs抗体偶联透析器上,发挥靶向性治疗作用以及AOPP结合蛋白甚至受体的研究打下坚实的基础。方法:1.首先通过葡聚糖凝胶Hitrap 26/60 sephacryl S-300 high Resolution纯化HSA,经变性、非变性电泳确定纯化后白蛋白的分子量及纯度。取纯化的HSA与HOCL孵育体外制备AOPPs-HSA,再用Hitrap 26/60 sephacryl S-300纯化AOPPs-HSA。经变性、非变性电泳、分子筛蛋白标准确定纯化后AOPPs-HSA的分子量。并经单核细胞株(THP-1)TNF-α分泌实验鉴定,纯化后的AOPPs-HSA能否刺激单核细胞分泌TNF-α,促发单核细胞炎症反应的活性。2.经Sephacryl S 300柱纯化后AOPPs-HSA分为6个峰,取纯化后的AOPPs-HSA第1峰作为免疫原,免疫BALB/c小鼠,采用杂交瘤技术制备抗AOPPs-HSA的McAb,用纯化的AOPPs-HSA和HSA分别作为筛选抗体的检测源,选择与AOPPs-HSA反应呈强阳性而与HSA呈阴性的克隆,同时也选择与AOPPs-HSA和HSA反应呈强阳性克隆。亚类鉴定采用Sigma公司的免疫球蛋白标准亚类鉴定试剂盒。用Protein G柱纯化抗体。SDS-PAGE鉴定抗体的纯度。用间接ELISA、western blot鉴定抗体特异性。3.标记单克隆抗体,采用其中一株作为包被抗体,辣根过氧化物酶标记的另一株作为标记抗体,通过配对试验,筛选出最佳配对,建立双抗体夹心法用于定量检测人AOPPs抗原含量。优化包被抗体和酶标抗体的最适宜工作浓度,并进行准确度、精密度、线性范围、检测限等方法学评价试验。4.用此检测系统检测36例正常人,18例慢性肾衰但没有透析的患者及56例透析患者之间血清AOPPs含量有无区别,并与分光光度计检测的AOPPs比较。观察离心前后两种检测方法对AOPPs的影响,比较分光光度计法与双抗夹心法对AOPPs检出阳性率以及AOPPs与临床其它指标的相关性。结果:1.经Hitrap 26/60 sephacryl S-300 high Resolution纯化后HSA后分为3个峰,第三峰无论是变性电泳还是非变性电泳均只有一条带且此条带的分子量在55～72KD之间。表明从PAGE及SDS-PAGE看,经纯化后峰3为单一分子量的蛋白质,且其分子量约为67KD。用此纯化的HSA与HOCL孵育体外制备AOPPs-HSA并经Sephacryl S 300柱纯化后分为6个峰,PAGE及SDS-PAGE结果表明纯化后的1、2、3、4峰表现为胶顶部的复合物,说明我们在制作AOPPs的过程中用的白蛋白是比较纯的,而经过HOCL处理后白蛋白发生了结构上的变化,变成了一个大分子的结构,故出现在胶的顶部。但非变性电泳胶顶部的条带比变性电泳明显的多,而在变性电泳中,1、2、3、4峰在55～72KD处出现了比较明显的条带,说明此大分子的结构在变性电泳时会有部分解聚,变成55～72KD之间的蛋白,即解聚成白蛋白的单体,但大部分仍没有解聚。分子筛蛋白标准鉴定其分子量为670KD。2.经以上制备及纯化后AOPPs-HSA第一峰用分光广度计法检测其AOPPs含量为20.38μmol/l,远远高于对照HSA样本的3.14μmol/l。用纯化后的AOPPs-HSA可显著性诱导THP-1 TNF-a的分泌,未经氧化修饰的HSA对THP-1的分泌没有显著性影响。时间效应显示AOPPs-HSA刺激6小时,THP-1 TNFa的分泌量就已显著性升高,12h升至最高水平。3.取纯化后的AOPPs-HSA第1峰免疫BALB /c小鼠,共获得3株稳定分泌特异性抗AOPPs的单克隆杂交瘤细胞株,分别命名为1-C6、1-H1、3-A10,1株既抗HSA又抗AOPPs,命名为1-C5。亚类鉴定1-C5、1-C6、1-H1为IgG1, 3-A10株为IgG2b。间接ELISA交叉反应性分析显示,其中1-C5既与AOPPs又与HSA特异结合,其余三株均只与AOPPs特异结合,而与AOPPs-BSA、BSA、CD26亲和力低,经统计学分析,均有显著性差异(p < 0. 01)。western blot鉴定表明抗1-H1AOPPs-HSA抗体仅与AOPPs-HSA结合而不与HSA、AOPPs-BSA、AOPPs-RSA结合,且尿毒症患者血清中AOPPs的水平明显强于正常人。4.采用1-H1作为包被抗体,酶标抗体采用1-C5McAb时为最佳配对,成功建立了双抗夹心法检测人AOPPs抗原系统。5μg/ml为包被抗体的最佳浓度,1:500为检测抗体1-C5的最佳工作浓度。对标准曲线、方法检出限、方法回收率、方法的批内和批间变异进行了分析研究,结果显示本方法最小检出限为12 ng/ ml,标准曲线在20～300 ng/ ml范围内线性良好。批内变异系数为4.8% ,批间变异系数为7.17 %,平均加样回收率为98%,表明该方法准确性高,重复性好,且实验步骤简单,容易掌握,其敏感性完全能够满足临床应用的需要。5.分别用双抗夹心法、分光光度计法检测36例正常人,18例慢性肾衰但没有透析的患者及56例透析患者,发现HD组和CRF组血浆AOPPs水平显著高于正常对照组,HD组明显高于CRF组,P<0.001。18例CRF患者用分光光度计法检出16例高于正常,而用双抗夹心法仅检出10例AOPPs高于正常。56例HD患者用分光广度法检出51例高于正常,而用双抗夹心法仅检出42例高于正常。经X2检验,具统计学意义。分光光度计检出AOPPs阳性率明显高于双抗夹心法。高速离心后用分光光度计检测尿毒症患者血清AOPPs值明显较未离心的AOPPs值低,而高速离心对双抗夹心检测AOPPs无明显影响。两种检测方法呈明显相关(r=0.543, P<0.001, n=110),血浆AOPPs水平与血清肌配水平呈正显著正相关(r=0.664, P<0.001, n=110 ),与尿素氮呈显著正相关(r=0.606, P<0.001, n=110 ),与血色素呈显著负相关(r=-0.535, P<0.001, n=110 )。结论:1.经Hitrap 26/60 sephacryl S-300 high Resolution纯化后获得了纯品HSA,取此纯化后的HSA与HOCL孵育体外制备AOPPs-HSA并经Sephacryl S 300柱纯化后得到相对纯品AOPPs-HSA,经变性及非变性电泳鉴定表现为大分子的聚合物,经分子筛蛋白标准确定其分子量为670kD。此纯化蛋白能刺激单核细胞株THP-1分泌TNF-a。2.取纯化后的AOPPs-HSA第1峰免疫BALB /c小鼠,成功筛选出3株稳定分泌抗AOPPs-HSA,1株既抗HSA又抗AOPPs-HSA的McAb杂交瘤细胞株。所分泌的抗体亚型3株为IgG1型,1株为IgG2b。间接ELISA和Western blot实验证明我们制备的抗体可以特异地和天然或经HOCL与HSA处理后纯化的AOPPs结合。3.用此单克隆单体配对,建立了双抗夹心ELISA检测系统,对标准曲线、方法检出限、方法回收率、方法的批内和批间变异进行了分析研究,表明该方法准确性高,重复性好,且实验步骤简单,容易掌握,其敏感性完全能够满足临床应用的需要,可作为反映病人氧化蛋白损伤的一种检测方法。4.双抗夹心ELISA法检测发现HD组和CRF组血浆AOPPs水平显著高于正常对照组,HD组明显高于CRF组,P<0.001。分光光度计检出AOPPs阳性率明显高于双抗夹心法。经X2检验,具统计学意义。高速离心后用分光光度计检测尿毒症患者血清AOPPs值明显较未离心的AOPPs值低,而高速离心对双抗夹心检测AOPPs无明显影响。两种检测方法具有很好的相关性。鉴于以上结果,我们考虑分光光度计检出率高的原因可能是由于其缺乏特异性,导致假阳性,但还需要进一步的实验论证。5.本实验首次成功地大量纯化AOPPs,制备了其单克隆抗体,建立了AOPPs单克隆抗体的工作平台,建立了双抗夹心法检测人AOPPs系统,并应用于临床。目前国内外均尚未有相关工作的报道。为下一步用抗体中和AOPPs毒性、将AOPPs抗体偶联透析器上,发挥靶向性治疗作用以及分离AOPP结合蛋白甚至受体的研究打下坚实的基础。更多还原

【Abstract】 Advanced oxidation protein products (AOPPs) are dityrosine-containing protein crossing products isolated from the plasma of hemodialysis (HD) patients, which was firstly reported by Witko-Sarsat in 1996. AOPPs represent a generic name for final products of oxidative modification of proteins caused by oxidative stress in HD patients. AOPPs rise as novel uremic toxins with the progression of chronic renal failure (CRF) and oxidative stress and accumulate in HD patients’plasma and tissues. It HSA been clarified that AOPPs are a novel class of pro-inflammatory mediators with monocytes being their elective cellular target. AOPPs are able to trigger the oxidative burst of monocytes and the release of inflammatory cytokines, and take part in the process of systemic micro-inflammation of CRF patients, which is a crucial pathogenic link to long-term uremic complications, such as dysregulation of immune system, accelerated atherosclerosis and dialysis-related amyloid arthropathy. Plasma AOPPs are mainly composed of high molecular weight-AOPPs (HMW-AOPPs) and low molecular weight-AOPPs (LMW-AOPPs). HMW-AOPPs, aggregates of oxidized albumin, which play the main role in HD long-term complications, are our focal point. However, the formation of AOPPs is irreversible and their combination with proteins in vivo greatly hinders the removal through any hemodialysis modality, which results in the limitation of therapy.Up to now, AOPPs were measured by spectrophotometry on a microplate reader and were calibrated with chloramine-T solutions. But this method lacks sensitivity and specificity. Otherwise it will give us much false positive information. Compared with spectrophotometry, immunolocal method is specific, sensitive and HSA less false positive rate. Therefore, if a test of immunology can be developed, it may be a good succedaneum or supplement for spectrophotometry in the diagnosis of AOPPs relative oxidation stress.AOPPs serve as a pro-inflammatory mediator which participates in many pathologic and physiologic reactions. It HSA vital significance to observe AOPPs deposition situation in tissues and cells’plasma using the AOPPs antibody through the immunohistochemical methods and western blotting, ELISA and so on. AOPPs aggravate dysregulation of immune system, atherosclerosis and dialysis-related amyloid arthropathy through the deposition in the blood vessel. Therefore, it is possible to use its corresponding antibody coupled dialyzer to block AOPPs’function and provide one new treatment.According to the clues above, in order to study the AOPPs prevention therapeutic methods and AOPPs detection, we prepare its monoclonal antibody by using purified AOPPs as antigen and establishe a double sandwich enzyme immunoassay for quantitative detection of AOPPs.Methods1. To achieve purified human serum proteins (HSA), a gel filtration was performed on a GE Healthcare Hitrap 26/60 sephacryl S-300 high Resolution adapted to a FPLC system. AOPPs-HSA was prepared in vitro by incubation of 200 mg/ml purified HSA and 200mmol/l of hypochlorous acid (HOCl) for 30min. To achieve separations of AOPPs-HSA bound to HSA, a gel filtration was performed as HSA. Nondenaturing and denaturing electrophoresis was performed according to Laemmli’s procedure to identify HSA molecular weight and purity. The molecular weigh of AOPPs-HSA can be determined from known protein standard. In order to identify activity of purified AOPPs-HSA, we investigate the effect of AOPPs on the secretion of tumor necrosis factor a (TNF-a) in monocytes.2.The monoclonal antibodies against AOPPs were generated by fusion spleen cells from immunized BALB/c mouse with purified preparations of AOPPs antigen and SP2 /0 mouse myeloma cells. The culture of hybridoma lines were first screened by indirect ELISA, then mouse ascites were prepared. Immunoglobulin isotype was determined using a commercially available isotyping kit.The specificities of McAbs were characterized by indirect ELISA and Western Blotting.3. The purified monoclonal antibody specific to antigen AOPPs were labeled with HRP by periodinate sodium. Optimal paired McAbs were selected, which 1H1 McAb was employed as coated McAb and 1-C5 McAb as detecting McAb after HRP labeling. Following the optimization of working concentrations of coated and detecting mAbs, we successfully established ELISA for detecting AOPPs. Evaluation tests of methodology were done, including recovery test, repeatability test and linear range investigation.4. AOPPs of thirty-six control, eighteen undialyzed patients with advanced chronic renal failure and fifity-six HD patients were detected in plasma by sandwich enzyme immunoassay and spectrophotometry respectively. The influence of high speed gentrifugalism to AOPPs examination was observed in spectrophotometry and ELISA. Correlated data were analyzed by linear association test.Results1. Elution patterns obtained from HSA fractionation by size-exclusion chromatography were visualized as three peaks. Under both nondenaturing and denaturing conditions, fraction 3 was detected as a single band with molecular weight of around 67 kDa, which was purified HSA. AOPPs-HSA were prepared by exposure of purified HSA to HOCl. Elution patterns obtained from exposure products fractionation by size-exclusion chromatography were visualized as 6 peaks. Protein aggregation was clearly seen by the presence of a smear on top of the gels in PAGE and SDS-PAGE of fraction 1-4, presumably as a result of albumin cross-linking. But under denaturing condition, the smear was less intense than that under nondenaturing condition. Under nondenaturing and denaturing conditions, a single band of around 67 kDa in all the 6 fractions was detected, presumably as a result of non cross-linking albumin. The molecular weight of purified AOPPs-HSA by molecular sieve was 670KDa.2. AOPPs concentration in purified AOPPs-HSA was 20.38μmol/l examined by spectrophotometer, much higher than that in HSA. Purified AOPPs-HSA induced TNF-a secretion in monocytes, while HSA didn’t. Time effect showed that TNF-a secretion increased to the highest level after 12 hours of AOPPs-HSA stimulation.3. Four hybridoma cell lines were successfully achieved by hybridoma technique, using purified AOPPs-HSA fraction 1 to immune BALB /c mice. Of the four strains, three could stably secret McAbs with a high specificity to AOPPs, named as 1-C6, 1-H1 and 3-A10, one secreted McAb with a high specificity to AOPPs and HSA, named as 1-C5. All the strains had low affinity to AOPPs-BSA, BSA and CD26. 1-C5, 1-C6 and 1-H1 were identified as the immunoglobulin IgG1 isotype, 3-A10 as IgG2a.4. A sandwich enzyme immunoassay for quantitative detection of AOPPs contents was developed using 1-H1 as coating antibody and horseradish peroxidase (HRP) labelled 1-C5 as labelled antibody. The optimum dilutions for coating antibody and labeled antibody were 55μg/ml and 1:500 respectively. The lowest limitation of detection was 12ng/ ml and the linearity of the standard curve was preferable from 20 to 300 ng/ ml. The coefficients of variation for intra-assay and inter-assay precision were 4.8% and 7.17 % respectively. The mean sample recovery rate was 98%.5. AOPPs of thirty-six healthy controls, eighteen undialyzed patients with advanced chronic renal failure and fifity-six patients on hemodialysis were detected in plasma by sandwich enzyme immunoassay and spectrophotometry respectively.In vivo, plasma level of AOPPs was the highest in HD patients. Undialyzed patients with advanced chronic renal failure had higher level than healthy controls. 16 out of 18 serum samples (88.9 %) from undialyzed patients with advanced chronic renal failure were shown to be positive by spectrophotometry while 10 out of 18 serum samples (55.6 %) were shown to be positive by ELISA. In addition , 51 out of 56 serum samples (91.1 %) from HD patients were shown to be positive by spectrophotometry while 42 out of 56 (75 %) were shown to be positive by ELISA. High-speed centrifugation decreased apparently AOPPs level when using spectrophotometry,while had no influence on AOPPs level when using ELISA. AOPPs level detected by double-sandwished ELISA correlated positively with that by spectrophotometry(r=0.543, P<0.001, n=110). AOPPs level correlated positively with the level of creatinine (r=0.664, P<0.001) and BUN (r=0.606, P<0.001) and inversely with Hb (r= -0.535, P<0.001).Conclusion1. After the successful purification of human serun albumin and AOPPs-HSA, we collected purified AOPPs protein and successfully used it as an antigen to immunize BALB/c mice. Four hybridoma cell lines were achieved successfully with hybridoma technique, three of which could stably secret specific McAb against AOPPs, while one against AOPPs and HSA.2. A sandwich enzyme immunoassay for quantitative detection of AOPPs contents was developed by using 1-H1 as coating antibody and horseradish peroxidase (HRP) labelled 1-C5 as labelled antibody. The method can establish a rapid assay for the measurement of AOPPs antigen and obviates the disadvantages associated with spectrophotometer measurement.3. In vivo, plasma level of AOPPs was the highest in HD patients. Undialyzed patients with advanced chronic renal failure had higher levels than healthy controls. The level of AOPPs correlated positively with the level of creatinine and with BUN and inversely with Hb.4. All the methods developed in this study, such as antibody preparation and identity, and Sandwich ELISA, can not only be used as effective tools to monitor plasma AOPPs, but also facilitate the understanding of the disease related with AOPPs and antibody preventation therapy.更多还原