摘要
一研究目的
1. MICA (MHC class I related Chain A)是一种表达于细胞膜表面的多态性糖蛋白分子。为了探索MICA分子在器官移植免疫中的作用,我们对新分离的人体血管内皮细胞和其它细胞株的MICA-mRNA和蛋白质表达进行分析。
2.人体器官移植患者被发现有抗MICA同种异体(Allo)抗体存在。为了简明MICA抗体在器官移植免疫中的作用,我们对已经检测到的MICA抗体阳性标本进行血清学分型,并对其识别的抗原表位进行定位分析。
3.晚期肝病患者外周血中的可溶性MICA分子(sMICA)显着性地高于正常人群。为了研究sMICA分子对肝移植效果的可能影响作用,我们对受者血清sMICA浓度与肝移植术后发生的胆道并发症(BCS)相关性进行观察。
二实验材料和方法
1.建立高分辨的MICA等位基因分型方法
一次PCR反应从基因组DNA中扩增出包括外显子2,3,4和5在内的MICA基因序列(2.2 kb)。再按两段分别以4个正反方向反应的测序引物进行测序分析。MICA跨膜区(TM)不同的GCT重复序列产生的复合信号交由电脑程序进行分离处理。
2.血管内皮细胞的分离和培养
新生儿脐带静脉血管内皮细胞被分离和培养至4-6代。应用RT-PCR方法检测MICA mRNA在11例新分离培养的HUVEC和7种其它细胞株转录水平,应用细胞流式术检测细胞膜表面的MICA分子的表达情况；同时应用免疫印迹的方法对整个细胞MICA蛋白的表达水平进行检测和分析。
3.建立MICA等位基因稳定表达细胞株
将完整的MICA-cDNA克隆插入pEGFP-N1。再将构建的质粒用电穿孔的方法导入人B淋巴细胞株Hmy. C1R。经G418药物筛选后成功表达MICA的细胞呈现绿色的荧光,可在荧光显微镜下观察,并用细胞流式仪器进行分离纯化。经过培养后获得稳定过表达的转染细胞株可用单克隆抗体进行检测和分析。
4.应用杆状病毒-昆虫细胞蛋白表达系统制备MICA等位基因重组蛋白
将11个常见的MICA等位基因cDNA克隆至pFastBac-1质粒中,加上2种杂交和2种点突变的构建质粒DNA转化至预置杆状病毒的大肠杆菌DH10Bac,以获得带有MICA基因的重组病毒DNA。重组的杆状病毒能感染Sf9昆虫细胞,分泌大量的可溶性重组蛋白。通过His-tag与Ni离子蛋白纯化系统进行初步提纯,和经MICA单克隆抗体亲和柱进行第二次纯化。用化学的方法纯化的重组蛋白质分别标记到蛋白质液态芯片组件(微球)。建立一种能同时检测多种抗体的免疫学检测方法(Luminex flow cytometry)。根据血清抗体对多抗原液态芯片反应格局和MICA等位基因重组蛋白的氨基酸序列比对分析结果,判定MICA抗体的血清型及其识别的多态性MICA抗原表位。
5.血清中sMICA浓度定量分析与肝移植病例对象
对133例肝移植前和移植后血清sMICA的浓度进行了回顾性的调查分析。另外88例健康人群作为对照,试验方法为双抗体夹心ELISA。sMICA在正常人群的正态分布状况由正常人群血清中sMICA浓度经统计学处理获得。大于95%正常值范围的上限判定为高浓度的sMICA阈值。接受肝移植的病人所有的临床资料和BCS的诊断记录从病案资料中收集获得。
三结果
1.新分离的人血管内皮细胞膜表面有MICA的表达
RT-PCR结果显示MICA mRNA在所有检测细胞中有表达,流式细胞术和免疫印迹的检测结果显示HUVEC细胞膜或胞浆中MICA蛋白表达量很高,而人体淋巴细胞膜表面和胞浆未检出MICA分子的表达。结果表明MICA mRNA广泛表达于人体细胞,而MICA蛋白质在血管内皮细胞有很高的表达。
2.应用人抗MICA抗体特异性确定MICA抗原的多态性表位
我们确定了14种MICA抗原多态性表位,其中9种抗原表位与单个的氨基酸相关。1种与两个相邻的氨基酸有关,1种与3个氨基酸有关；有3种抗原表位比较复杂,仅从蛋白质的一级结构不能很好理解。我们发现了两种相互对立的血清型MICA抗体,这种组特异性抗体对杂交和点突变的重组蛋白表位的识别结果提示其与6个MICA组特异性的氨基酸相关联。
3.肝移植术后胆道并发症与患者血中高sMICA血症相关
sMICA的检测结果提示37.6%的肝病晚期患者的sMICA显着性高于正常值范围。在肝移植后仍出现高浓度的sMICA的病人中,有34.4%的受者出现供体肝胆道并发症状。而肝移植后处于正常sMICA浓度的病人中仅有17.3%的受者出现胆道并发症。胆道并发症发生的风险与受者在接受肝移植后存在的高浓度sMICA有着显着性的相关关系(P=0.0365)。进一步的分析发现患者在移植前有高浓度的sMICA。移植后原高sMICA血症的病人在sMICA浓度恢复正常后,其胆道并发症的发生率较移植后仍然维持高浓度的sMICA的患者显着降低(10.5%对38.7%,P=0.0302)。在对具有sMICA动态改变特征的病理分组的Log-rank检验分析揭示胆道并发症与sMICA的动态改变存在显着的相关关系(P=O.0188)。
四结论
1.人体血管内皮细胞表面表达MICA分子事实说明多态性MICA作为一种移植抗原参与体液免疫排斥反应成为可能。
2.我们在国际上率先报导了14种MICA抗体血清型和识别表位的分析结果。MICA抗原表位的确定对理解MICA抗体的产生和移植免疫中发挥的作用具有理论和现实意义。同时为致敏的受者选择合适的供者奠定MICA配型的实验基础。
4.我们发现肝移植术后高sMICA血症的患者比较容易发生胆道并发症的问题。结果提示sMICA在肝移植免疫中可能发挥一定的免疫调节作用。对病人血清中sMICA浓度的监视可作为一项肝移植后胆道并发症发生具有诊断意义评价指标。
1. Aims of Research
(1) MICA (MHC class I related Chain A) are polymorphic glycoproteins expressed on cell surface. In order to explore the role of polymorphic MICA molecules on organ transplantation, we analyzed the expression of MICA in freshly isolated human umbilical vein endothelial cells (HUVEC) and other cell lines.
(2) Patients who lose their graft have been found to have specific allo-antibodies against MICA antigens. To understand the development of MICA antibodies and their role in organ transplantation, we defined the serologic types of MICA antibodies from recipients with allografts.
(3) The patients with late liver diseases were found to have higher levels of serum sMICA than the healthy population. In order to observe the effect of sMICA on liver transplantation, we investigated whether high levels serum sMICA are associated with incidence of biliary cast sysdrome (BCS) following liver transplantation.
2. Methods and Materials
(1) Establishment of MICA allele-level typing method.
One PCR amplification was performed to obtain templates of 2.2 kb including exons 2,3,4, and 5 of MICA to be sequenced with two forward and two reverse primers. Overlay of nucleotide sequencing signals resulting from presence of different GCT repeats in exon 5 from two different MICA alleles can be identified by a computer-based analysis.
(2) Isolation and culture of HUVEC.
Human umbilical vein endothelial cells were isolated from umbilical cord veins and cultured within 4-6 passages. RT-PCR was used to analyze MICA mRNA that expressed on 11 HUEVCs and 7 culture cell lines. Surface MICA expression was examined by flow cytometry with monoclonal antibody staining. Total MICA proteins were accessed by Western blot.
(3)Establishment of MICA stable transfected cells.
The cDNA containing full-length MICA was cloned into plasmid pEGFP-N1. Then the constructs were transfected into human B cell line Hmy.C1R. After G418 selection, stable transfected cells were monitored by fluorescence microscopy and sorted by flow cytometry. Suface MICA expression was demonstrated by flow cytometry with the monoclonal antibodies 6B3 and 3.2H3.
(4) Production of MICA allele recombinants using a Bac-to-Bac Baculovirus Expression system.
11 MICA alleles were cloned into the plasmid pFastBac-1.2 hybrids and 2 mutants MICA molecules were made from two normal MICA alleles. The baculovirus with MICA gene was obtained from transformation of DH10Bac (E. coli) with the constructed-vectors. MICA recombinant proteins were produced in insect cells (Sf9) which were infected with baculovirus. Soluble MICA recombinant proteins fused with a His-tag (6 histidines) were purified by nickel-affinity agarose followed by mAb 6B3-affinity agarose. Using such materials, we have established the Luminex Flow Cytometry assay to identify the specificity of MICA antibodies. MICA serologic patterns of reactivity were determined with single MICA antigens which were bound to Luminex beads.
(5) Quantitation of serum sMICA and patients with liver transplantation.
Serum sMICA was retrospectively evaluated in pre-and post-transplant sera from 133 consecutive primary liver transplant patients and in sera from 88 healthy volunteers using sandwich ELISA. Normal distribution of serum sMICA was described by the data obtained from healthy population and sMICA concentration that was greater than the upper bound 95% normal range was considered as high levels of sMICA. Patient records were reviewed to identify patients who developed BCS.
3. Results
(1) MICA was expressed on the surface of human endothelial cells.
The results show that all of tested cells were found to have MICA mRNA expression by RT-PCR. HUEVCs have surface MICA expressed in high level, but MICA protein expression in T and B lymphocytes was not detected in flow cytometry and Western blot with MICA specifice monoclonal antibodies 6B3 and 1.7AD.
(2) Determination of the MICA epitopes that are recognized by human antibodies against MICA.
Human sera selected in this study were found to recognize up to 14 distinct MICA epitopes. Among these, nine epitopes correlated with a single unique amino acid:one shared two signature amino acids, one shared three signature amino acids in close proximity, and three epitopes involved multiple amino acids in a nonlinear sequence. Two groups of public epitopes (MICA-G1 and MICA-G2) were characterized and their epitopes were speculated to relate six amino acids.
(3) High level serum sMICA are associated with BCS following liver transplantation.
We show that 37.6% of patients with end-stage liver diseases had significantly higher pre-transplant serum sMICA than in healthy population.34.4% of recipients with post-transplant high levels of sMICA developed BCS. In contrast,17.3% of patients with post-transplant normal levels of sMICA developed BCS. The risk of BCS development is significantly associated with the presence of post-transplant high levels of sMICA (P=0.0365). Further analysis disclosed that patients with decreased post-transplant sMICA following liver transplantation had a lower incidence rate of BCS than those with remained high levels of sMICA after transplantation (10.5% vs.38.7%, P=0.0302). Furthermore, log-rank test showed that BCS occurrence was significantly associated with dynamic changes of sMICA among different groups (P=0.0188).
4. Conclusion
(1) The expression of MICA on the surface of endothelial cells makes this polymorphic molecule a possible target during the immune response of graft rejection in organ transplantation.
(2) We first reported 14 MICA epitopes that were indentified by human MICA antibodies. Those epitopes may help to understand the development of MICA antibodies and to identify the suitable donors for the sensitized transplant recipients.
(3) We found that biliary cast syndrome is more likely to develop in recipients who have post-transplant high levels of sMICA. The data suggested that sMICA might have some immunologic effect on BCS development following liver transplantation. Monitoring of serum sMICA could have a prognostic value in assessment of patients with liver transplantation.
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