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卵巢癌生物标志物的糖链谱研究进展
薛添1, 李艳红2, 李铮1     
1. 西北大学生命科学学院功能糖组学实验室,西安 710069;
2. 第四军医大学唐都医院妇产科,西安 710038
摘要: 糖类抗原125(CA125) 被认为是卵巢癌诊断的“金标准”,但在临床应用中普遍存在着特异性不高的问题.肿瘤形成和发展过程中常伴有糖基化修饰异常和糖链结构的改变,不同的肿瘤具有特异的异常糖链结构.近年来,借助凝集素芯片、多重质谱分析等糖蛋白组学和糖组学研究技术,发现不同来源CA125的O-糖链和N-糖链结构存在着明显的微观不均一性,以这些特征性糖链结构为标志物,可以显著提高CA125对卵巢癌的诊断特异性.在过去的10年,研究者们除对CA125糖链结构和糖基化模式做了深入的研究外,还利用糖组的研究方法,直接对来自卵巢癌患者血液、体液(腹水、囊泡液等)中糖蛋白的糖链做了精细的结构解析,结果显示,可有效鉴别卵巢癌患者和健康志愿者的特异性N-糖链结构,有可能成为灵敏度高和特异性好的卵巢癌生物标志物.卵巢癌生物标志物研究发展的总趋势是从传统的对蛋白质的定性和定量研究,逐步转向于对标志物糖基化修饰和特异性糖链结构的鉴定以及定量分析.本文从糖组学的视角,对卵巢癌标志物糖组学的研究现状及发展趋势进行了综述和展望.
关键词: 卵巢癌     糖组学     糖基化     标志物    
Progress in The Study of Glycosylation of Ovarian Cancer Biomarkers
XUE Tian1, LI Yan-Hong2, LI Zheng1     
1. Laboratory for Functional Glycomics, College of Life Sciences, Northwest University, Xi′an 710069, China;
2. Department of Obstetrics and Gynecology, Tangdu Hospital, The Fourth Military Medical University, Xi′an 710038, China
*This work was supported by a grant from The National Natural Science Foundation of China (81372365)
** Corresponding author: LI Zheng, Tel: 86-29-88304104, E-mail: zhengli@nwu.edu.cn
Received: April 10, 2017 Accepted: September 4, 2017
Abstract: CA125 is a gold standard for the diagnosis of ovarian cancer, however, its specificity is still relatively low in clinical application. Tumor formation and development process is often accompanied by abnormal glycosylation modification and changes in glycan structures. The different tumors have specifically abnormal glycan structures. In recent years, the technologies in glycoproteomics and glycomics, such as lectin microarrays and multiple mass spectrometry, are used to discover the small discrepancies of N-and O-linked glycan structures in CA125 of the different sources. These abnormal glycan structures could increase the specificity of CA125 for the diagnosis of ovarian cancer. In the past decade, the technologies of glycome were also used to directly analyze the subtle glycan structures in the serum and body fluid (ascites, vesicle fluid, etc.) from patients with ovarian cancer. The results showed that the N-linked glycan structures can effectively identify ovarian cancer patients and healthy volunteers, which may be new biomarkers with better sensitivity and specificity for the diagnosis of ovarian cancer. Trends in the development of biomarkers for ovarian cancer shifted from qualitative and quantitative studies of proteins, to identification and quantitative analysis of glycosylation modification and glycan structures of biomarkers. Taking the perspective of glycomics, the status and development trend of ovarian cancer biomarkers are reviewed and discussed in this paper.
Key words: ovarian cancer     glycomics     glycosylation     biomaker    

卵巢癌(ovarian cancer,OC)是常见的女性生殖系统肿瘤之一,与其他常见女性肿瘤相比较,卵巢肿瘤具有以下显著的特点:a.组织学类型繁多.在最新版《WHO Classification of Tumours of Female Reproductive Organs 》中将卵巢肿瘤分为14种组织学类型,而同一组织学类型的卵巢肿瘤中往往又同时存在恶性、交界性和良性3种不同的病理亚型[1].b.发病率、死亡率高,预后差、存活率低.卵巢癌在全球常见癌症中居第六位,常见女性癌症中居第五位,死亡人数在导致全球女性死亡的主要癌症中列第七位[2-5],近年来,卵巢癌发病率在中国呈上升趋势,预计2016年中国卵巢癌发病率将达到6.43/10万人[6].c.分布广泛,发达地区发病率是欠发达地区的2.2~3.7倍[7-9].d.缺乏明显的鉴别性体征和筛查方法、早期诊断困难.临床研究表明,早期阶段(Ⅰ/Ⅱ)确诊的卵巢癌患者5年生存率可达90%以上,然而,约70%~85%的患者在确诊时都已处于晚期阶段(Ⅲ/Ⅳ期),平均5年存活率仅有20%~35%[10-13],致使卵巢癌成为病死率最高的妇科恶性肿瘤[14].鉴于患者的生存率与确诊时的疾病分期密切相关,早期检测成为提高卵巢癌患者生存期的关键[15].因此,研究和发现新的早期检测标志物始终是卵巢癌研究的重要课题.

经多年不懈的研究探索,相继发现了包括糖类抗原125(CA125)、人附睾蛋白4(HE4)、Mesothelin[16]、Osteopontin[17]、Prostasin (KLK6)[18-20]等数十种卵巢癌血清肿瘤标志物[21].卵巢癌肿瘤标志物的使用,大幅降低了卵巢癌患者的死亡率[22].但在使用中发现,这些肿瘤标志物都有特异性不理想的问题.

在细胞恶变和肿瘤发展的过程中,糖基化异常是一个普遍现象,而且这种改变常早于糖蛋白表达量的变化[23].鉴于目前所发现的卵巢癌标志物都是糖蛋白,因此对其糖基化模式和糖链结构做精细的解析,从中寻找或发现新的肿瘤标志物,成为近年来卵巢癌标志物研究的新热点,并已取得不少研究成果.本文以糖组学的视角,对卵巢癌标志物糖组学的研究现状及发展趋势进行了综述和展望.

1 卵巢癌标志物CA125的糖链结构

CA125为高分子质量糖蛋白,有大量的N-糖链和O-糖链的修饰.CA125被美国FDA批准为2种卵巢癌标志物之一[24],被认为是卵巢癌诊断的“金标准”[25].但也存在特异性较差的问题[26-28],卵巢炎性疾病、卵巢良性肿瘤及某些卵巢外癌症均可引起CA125水平的异常升高[29-35].所以改进和提高CA125对卵巢癌的辨识能力具有重要意义.

1.1 CA125的O-糖链结构解析

CA125的O-糖链属黏蛋白型O-糖基化,糖链通过GalNAC与Thr/Ser连接,O-糖链结构比N-糖链简单,但连接方式比N-糖链复杂,因此O-糖链结构的解析也更困难和复杂,如图 1所示.利用凝集素结合和糖基序列分析[36]对不同来源CA125(pf-CA125:卵巢癌患者腹水;cl-CA125/ oc-CA125:卵巢癌细胞系;af-CA125:人羊膜液;pl-CA125:人胎盘来源的妊娠相关CA125;hs-CA125:人血清)做糖链结构解析,数据显示,O-糖链是CA125的优势糖链,多数为高唾液酸化和(/或)岩藻糖基化,最多携带2个岩藻糖,或2个唾液酸,或2个岩藻糖+2个唾液酸.糖链核心结构有2类,一类含单和双唾液酸化的核心1(Galβ1-3GalNAc),另一类含核心2组分(Galβ1-3(GlcNAcβ1-6) GalNAc),有单或双岩藻糖基化及唾液酸化等形式,也发现少量含硫酸化核心2的O-糖链,决定性结构为Galβ1-3GalNAc和(Galβ1-3Glc NAcβ1-6) GalNAc[37-40].

Fig. 1 Core schematic structure of O-glycan 图 1 O-糖链核心结构示意图

尽管卵巢癌患者和健康对照间O-糖链的基本结构无显著性差异[40],但不同来源CA125 O-糖链上存在不同糖型,有明显的异质性.如cl-CA125/oc-CA125 O-糖链的核心2上含核心1的单天线分支结构,此点与众不同,af-CA125 O-糖链除核心结构外,还带岩藻糖基化外臂,而pl-CA125携带的是α2, 3和α2, 6连接的单和双唾液酸的O-糖链,以及少量高甘露糖化O-糖链.另有研究报告,pf-CA125的O-糖链比正常卵巢细胞的O-糖链要短一些,推测pf-CA125上有截短型的O-糖链,唾液酸化程度也更高.此外,O-糖链基本结构相同的af-CA125与oc-CA125的糖类组成明显不同,甚至同一来源的CA125 O-糖链上也存在糖异构体[38, 41].

凝集素WGA和RCA(特异识别GlcNAc末端Gal)对af-CA125结合活性很强,对oc-CA125亲和力微弱[38].用一组识别不同唾液酸结构的凝集素(Siglec)对CA125做检测,结果显示,7种Siglec均可与非癌性的pl-CA125特异性结合,Siglec-2、Siglec-3优先识别pf-CA125,结合强度也远大于pl-CA125,Siglec-9、Siglec-10对cl-CA125有高度选择性[42],这种差异性显示了不同来源CA125 O-糖链结构的微观不均一性.

肿瘤抗原表位的改变时常会呈现蛋白骨架正常,而糖基化异常的现象[43-44].与正常卵巢CA125相比,pf-CA125 O-糖链典型的改变是:复杂型O-糖链分支核心2或4缺失、核心1(Neu5Acα2-3Galβ1-3GalNAcα,ST和Galβ1-3GalNAcα,T)过表达;产生核心1截短的O-糖链(Neu5Acα2-6GalNAcα,STn)和GalNAcα(Tn)[38, 41, 45].pf-CA125的糖基化异常,常见的还有唾液酸化和岩藻糖基化增加并携带岩藻糖基化的外臂[37, 46-48].

CA125的O-糖链的特征性差异,具有重要的意义,检测CA125的糖基化变化,可能会为卵巢癌患者提供特异性更强的生物标志物[40].

1.2 卵巢癌CA125的N-糖链结构解析

尽管O-糖链是卵巢癌CA125的优势糖链,但在卵巢癌CA125 N端亚单位,除典型的O-糖基化结构域外,也含有N-糖基化结构域,如图 2所示,具备N-糖基化条件[49-50].在pf-CA125和cl-CA125上可检测到高甘露糖型(Man5-Man9GlcNAc2)和复杂型(NeuAc0-1Fuc0-2 Hex5-7 HexNAc4-7)N-糖链.其中复杂型N-糖链占80%以上,并富含单岩藻糖.复杂型N-糖链以单天线和平分型双天线结构为主,也存在含岩藻糖基化核心的三/四天线结构糖链,在pf-CA125上还可检测到高唾液酸化的复杂型和杂交型N-糖链[37, 39-40, 51-52].而pl-CA125以多天线结构的N-糖链为主,高甘露糖型N-糖链含量低,核心结构中也缺乏GlcNAc,且不含复杂型或杂交型N-糖链[40, 51].

Fig. 2 General architecture of CA125 mucin 图 2 CA125结构示意图

表 1列出的是卵巢癌细胞系H0-8910培养上清液与正常卵巢组织CAl25分子的N-糖链结构的分析结果.可以看出,在卵巢癌来源CA125上多天线复杂型和平分型复杂型N-糖链明显减少,只有前者的1/10.无/有核心岩藻糖复杂型双天线N-糖链、高甘露糖型N-糖链及杂合型及含平分型GlcNAc残基结构的N-糖链则显著增加[53].

Table 1 N-glycan structural analysis and comparison of different resource CA125 表 1 不同来源CA125分子N鄄糖链的结构分析比较1)

糖基化异常是癌症发生的一个标志[54-57],异常的糖基化模式可以作为恶性卵巢肿瘤的诊断标志物[58-61].与健康对照相比,卵巢癌患者CA125分子N-糖链糖基化异常主要表现为双天线结构和高单唾液酸化N-糖链增加,糖链中岩藻糖基化核心水平升高,而非岩藻糖基化N-糖链减少.组织学研究也表明,相对于良性和交界性卵巢肿瘤,卵巢癌来源的CA125携带更多的Tn抗原,利用Tn-CA125诊断卵巢癌,ROC曲线下面积(AUC)值达到0.86,明显优于单纯检测CA125(AUC=0.70)[62].以CA125的N-糖链糖型为标志物不仅可以区分良性卵巢肿瘤和卵巢癌,还可明显提高诊断效率[52].

糖组的研究结果表明,以特征性糖链结构的异常作为提高现有血清肿瘤标志物的靶标,而不只是单纯地检测CA125的总蛋白浓度差异,不但可以有效地提升CA125对卵巢疾病的辨识能力、改善对卵巢癌诊断的特异性和敏感性,并且有助于卵巢癌的早期诊断[40, 58].

2 卵巢癌血液、体液标本中糖链标志物的糖链谱研究

在过去10年,研究者们除对CA125糖链结构和糖基化模式做了深入的研究外,还利用糖组学的研究方法,直接从卵巢癌患者血液、体液(腹水、囊泡液等)中分离和富集与卵巢癌相关的糖蛋白、糖肽或糖链,做精细的结构解析,希望能从中发现具有临床鉴别诊断价值的特征性糖链结构,作为新的卵巢癌肿瘤标志物.

2.1 卵巢癌患者血清中糖链标志物的筛选鉴定

研究揭示,释放于卵巢癌患者血清中的糖蛋白或糖链上可以普遍观察到糖基化的改变,由此生成了许多非常重要的肿瘤糖链生物标志物[63-66].Kim等[67-68]应用纳米高效液相色谱-芯片-飞行时间质谱技术(nanoHPLC-chip-TOF-MS time-of-flight mass spectrometer)[69-70],对健康女性(对照)、上皮性卵巢癌(EOC)和卵巢低恶性潜能肿瘤(LMP)患者的血清糖蛋白做糖组学研究中,鉴定出多种卵巢癌特异的N-糖链,并依据其结构特性细分为3组(图 3a).其中组1和组2 N-糖链在EOC患者中的表达水平降低,组1主要是高甘露糖和杂交型N-糖链,组2为至少含1个半乳糖残基的双天线N-糖链,有或没有岩藻糖和平分N-乙酰葡糖胺(GlcNAc)残基修饰,如果2个天线都是半乳糖基化的,则其中1个可被唾液酸修饰.

Fig. 3 Glycans structure and classification diagrams 图 3 糖链结构及分类示意图

留一法交叉验证,以特异性N-糖链为标志物,对卵巢癌的鉴别敏感度达86%、特异性为95.8%.此前,Karina等[71]在术前上皮性卵巢者血清中鉴定出1组特异性N-糖链,其中4种为高甘露糖型N-糖链,7种为复杂型岩藻糖基化N-糖链(图 3b).与健康对照相比较,EOC患者N-糖链天线含岩藻糖基化结构较多,在三天线和四天线唾液酸化糖链上尤为明显.这组N-糖链标志物对原发性EOC诊断的敏感度为97%、特异性达98.4%.而CA125检测的敏感度和特性分别为97%和88.9%.类似的研究结果见诸多篇文献[72-73].其中Kronewitter等[74]采用血清N-糖链分离、MALDI-FTICR质谱分析、Glycolyzer软件进行生物信息学评价的技术路线,从来自卵巢癌发现集(含1 410个糖链)中鉴定出了19种候选糖链标志物(图 3c).在卵巢癌患者血清中结构为(Hex3HexNAc4、Hex3HexNAc4Fuc1、Hex3HexNAc5、Hex3HexNAc5Fuc1) 的一组中性糖链、尤其是FA2糖链(Hex3HexNAc4Fuc1)[65]明显增加,这与卵巢癌患者血清IgG和全血清检测到结果一致[75].比较不同实验室的研究结果(图 3图 4a图 4b)可以看出,存在于卵巢癌患者血清中的N-糖链标志物在结构及其表达量(上调或下调)有高度的相似性,共同的特点是含唾液酸的糖链(Hex5HexNAc4NeuAc1) 表达上调,不含唾液酸的中性糖链(Hex5HexNAc4) 表达下调.在对单个N-糖链结构丰度的分析中,观察到四天线糖链(aF2A4G4、aF3A4G4) 的丰度和β1, 6-GlcNAc在卵巢癌血清中显著增加[52, 76].

Fig. 4 The C4BP, A1260 and N-glycan structural diagram(red rhombus: sialic acid, gray triangle: fucose) 图 4 C4BP、A1260及其N-糖链结构示意图(紫色菱形:唾液酸,红色三角:岩藻糖)

初步的研究显示,特异性的血清N-糖链结构,能够有效地鉴别卵巢癌和健康对照,有可能为卵巢癌的检测提供具有更高准确性、灵敏度和特异性的生物标志物[77-78].

2.2 卵巢癌患者腹水中糖链标志物的筛选鉴定

癌性腹水是卵巢恶性肿瘤最常见的并发症之一,据统计超过80%的卵巢癌晚期患者会产生胸腹水[79].在营养丰富的腹腔环境中癌细胞大量增殖.产生和分泌了大量糖蛋白代谢产物[80-81],其中包括CA125[82-83]、触珠蛋白[84-86]、纤连蛋白[87-88]、腓骨蛋白[89-90]、内腔蛋白[91]、α-1抗胰蛋白酶和α-1-抗胰凝乳蛋白酶[92]等多种与卵巢癌相关的糖蛋白.因此,卵巢患者腹水可以为糖链标志物的筛选研究提供来源方便且丰富的样本.

Miyamoto等[93]用凝集素ConA和WGA分别从卵巢癌患者腹水富集糖蛋白,经肽-N-糖苷酶F(PNGase F)酶解,释放糖链,nanoHPLC-chip-TOF or qTOF MS做糖链结构分析.发现在卵巢癌患者腹水中存在着大量高岩藻糖基化和唾液酸化的复杂型和杂合型糖链.并在两份卵巢癌患者腹水(Asc1和Asc412) 和对照血清(SS)中分别鉴别出77种、67种和51种结构不同的糖链.其中Asc1中有12种独特的糖链,Asc412有3种独特的糖链,有13种糖链是两种腹水中共有的,而在对照血清中未检测到,最终从中筛选出4种卵巢癌特异的N-糖链(H6N5、H6N5F1S1、H6N5F2S3和H7N6F1S1).

但当前的同类研究,多数还处于蛋白质水平[94-95].Garibaycerdenares等[96]采用2-D电泳分离、MALDI-TOF分析的技术路线,试图从卵巢癌腹水中发现能够作为肿瘤标志物的差异蛋白,结果发现在墨西哥卵巢癌患者腹水和癌组织中高岩藻糖基化的触珠蛋白α亚型数量增加,并与晚期卵巢癌相关,可以作为评测病程进展的生物标志物.Huang等[97]在先天性化疗抗性的卵巢癌患者腹水中成功鉴定出11种差异蛋白,其中3种蛋白上调,8种蛋白下调.验证实验证实,11种差异蛋白中,血浆铜蓝蛋白的表达在先天性化疗药物抗性和敏感的卵巢癌患者腹水样品间存在显著差异,可作为卵巢癌化疗药物治疗反应和预后监测的生物标志物.

从腹水样品中筛选卵巢癌的糖蛋白或糖链生物标志物,主要的优势是采样量大,静脉采血量一个人通常不超过4~5 ml,腹水的采集量可在几ml到1L以上,更重要的是,卵巢癌患者腹水蛋白种类超过了2 500种[94],因此腹水中的肿瘤抗原也远比血液中丰富,更有利于肿瘤生物标志物的分离和筛选.其缺点在于,腹水一般产生于病程的晚期,限制了在卵巢癌早期检测中的应用.另有文献报道,卵巢癌患者腹水中CA125的含量高出血清中CA125临界值(cut off=35U/ml)的20~100倍,但同时在良性卵巢肿瘤患者腹水样本中也检测到高滴度的CA125,且二者无显著性差异[95].

3 其他来源的卵巢癌糖链标志物 3.1 免疫球蛋白(Ig)

免疫球蛋白(Ig)是存在于人体血液和体液中最重要的高丰度糖蛋白之一,在机体获得性体液免疫中起着关键作用.已有研究发现,与Ig相关的糖基化异常与自身免疫病以及多种恶性肿瘤的发病机理密切相关[98-102],这种相关性也直接影响到卵巢癌糖链标志物的研究.

早期研究证明,卵巢癌患者血清中的肿瘤反应性免疫球蛋白中存在糖基化异常的IgG群,异常的糖基序被定位于IgG的Fc端上[103].N-糖链糖组学谱揭示,卵巢癌患者IgG群含不同程度唾液酸化和岩藻糖基化的三分支和四分支结构的N-糖链丰度显著增加,而平分型N-糖链水平明显降低,同时观察到N-糖链上无半乳糖基化结构水平升高,并且糖链上含独立的岩藻糖残基[104].

新近的研究对卵巢癌患者Ig糖基化异常做了更深入的解析,发现在IgG上有15个糖肽与上皮性卵巢癌(EOC)相关.N-糖链4N4F1、H5N4、H5N4F1、H4N5F1、H5N5F1和H5N4F1S1在EOC患者血清中降低,并存在无乳糖基化的N-糖链(H3N4F1),这些结果与先前的研究一致.同时还发现卵巢癌患者含单和双半乳糖基化N-糖链的IgG水平通常降低[75].

来自EOC患者血清的IgA有10种N-糖肽的水平异常.IgA1/2的N144 /131位点带1个或不带唾液酸,含无岩藻糖基化双天线N-糖链的几种糖肽水平分别增加,而双唾液酸化N-糖链的糖肽水平未同步发生显著的改变.在IgA2的N205位点上,含平分型N-乙酰葡糖胺(GlcNAc)双天线N-糖链的糖肽水平通常降低.在EOC患者血清中IgM有5个糖肽的水平增加.含2个高甘露糖型N-糖链结构(H7N2和H8N2) 的糖肽,在位点N439处只有1个高甘露糖表达异常.位点N209上携带双天线单或双唾液酸、带或不带平分型GlcNAc N-糖链结构的糖肽表达增强[105].

EOC患者血清中的免疫球蛋白IgA,IgG和IgM上存在多种差异表达的糖肽,显示其在EOC诊断中的潜力,其中IgG1N-糖链H5N5F1的检测对卵巢癌和对照组的鉴别力最强(分辨率88.6%,AUC=0.94)[105].Qian等[106]对3个最重要的IgG N-糖链G0(带2个半乳糖基)、G1(带1个半乳糖基)、和G2(无半乳糖基)做相对定量,利用公式根据G0/(G1+G2×2) 计算IgG半乳糖基化程度的相对比率,以此指标与CA125组合,对卵巢癌和良性卵巢肿瘤的检测,ROC分析表明特异性从65.2%(单独的检测CA-125) 提高到84.6%,灵敏度为90%.

对免疫球蛋白位点特异性糖基化特征的研究,不仅有助于进一步阐述Ig糖基化异常在卵巢癌中的生物功能特性,并且能提高检测灵敏度和诊断特异性.

3.2 外泌体和囊泡

外泌体(exosome)和囊泡(vesicles,EV)都是由细胞分泌产生细胞外囊泡状结构,可携带多种生物活性分子.以外泌体和囊泡作为卵巢癌诊断标志物已有广泛的研究,并展现出独特的优势:血浆中含量丰富(108~1013个/ml);极其稳定(在各种冷冻、冷藏和解冻条件下可保存数年);具肿瘤特异性、含量与肿瘤分期和治疗效果相关[107-110].

研究报道,在卵巢癌细胞系的外泌体中蛋白质数量多达2 230种[111],其中半乳凝集素3结合蛋白(galectin-3 binding protein,LGALS3BP),被认为是鉴别正常细胞和卵巢癌来源外泌体和EV的特异性标志物[111-112].因此,目前外泌体和EV糖蛋白的糖组学研究中也多以LGALS3BP为对象.研究显示,复杂型N-糖链是卵巢癌外泌体糖蛋白的优势糖链,并带有较高的唾液酸化,同时也存在携带N-乙酰葡糖胺的平分型N-糖链[113].在卵巢癌细胞OVMz和SKOV3的EVs具有特异性的N-糖链标志,其复杂型N-糖链有α2, 3-连接的唾液酸、岩藻糖、平分型GlcNAc和二乙酰乳糖二胺(lacdiNAc)结构,此外,还检测到含T-抗原的O-糖链[114].由于同样的结构也存在于浆液性和子宫内膜样卵巢癌患者糖蛋白的N-糖链上,因此它被认为是潜在的卵巢癌糖链标志物[111, 115-116].

3.3 补体4结合蛋白

补体4结合蛋白(C4b-binding protein,C4BP)是经典补体激活途径中重要的调节因子,一般由5~7条完全相同的α链组成和1条β链组成,肽链通过偶极性α螺旋区和二硫键之间的相互作用连接于C端结构域[117-118].C4BPB作为特异性的血清生物标志物已应用于人和动物疾病的检测[119-120].Sogawa等[121]使用串联质谱标签(tandem mass tag labelling)和LC-MS/MS对胰腺导管腺癌(PDAC)患者血清做筛选鉴定,发现PDAC患者血清中C4BPA(补体4结合蛋白α-链)水平显著高于健康对照、胰腺炎和其他恶性肿瘤,例如胆管癌患者,鉴别诊断实验ROC曲线AUC为0.86,优于传统标志物CA19-9(AUC=0.84),由此确认C4BPA是PDAC早期检测的血清生物标志物.相对于单纯的C4BPA检测,以C4BA N-糖链结构为靶点,无疑会具有更好的特异性.Mikami等[122]对来自134例EOC和非癌症患者,超过100 000个血清糖蛋白糖链结构做质谱分析,从中鉴定出A2160,补体4结合蛋白完全唾液酸化的α链为EOC候选标志物,糖链分析和多肽测序显示,A1260具有2个N-糖基化结合位点(Asn506和Asn528),2个糖基化位点可结合11个己糖、9个己糖胺(HexNAc)、5个N-乙酰神经氨酸和1个岩藻糖(图 4a),在Asn506位点上可连接4种糖链:A2G2(双天线N-糖链)、A3G3(三天线N-糖链)、A2G2F(含1个岩藻糖的双天线N-糖链)和A3G3F(含1个岩藻糖的三天线N-糖链),Asn528位点只连接2种N-糖链A2G2和A3G3(图 4b).

对血清标本检测特异性的评估实验证实,α链完全唾液酸化的A1260比α糖链部分唾液酸化的A1260对EOC诊断的精度更高,如,Asn 506位携带含3个唾液酸、三天线N-糖链(完全唾液酸化)其AUC值明显高于N-糖链部分唾液酸化的.同样,Asn528位携含2个唾液酸N-糖链(完全唾液酸化)的A1260,其AUC也明显高于仅携带单唾液酸化的,充分表明唾液酸化水平是影响A1260检测特异性的关键因素.

4 问题与展望

缺少特异敏感的早期诊断标志物是卵巢癌病死率长期居高不下的重要原因之一,如何进一步提高和改善检测的敏感度和特异性,减少假阳性是卵巢癌生物标志物研究的重要课题.伴随肿瘤形成和发展过程中产生的各种糖基化修饰异常和糖链结构的改变常具有独特的肿瘤特异性,由此也成为肿瘤生物标志物研究的新靶点.

近年来,借助高通量凝集素芯片、多重质谱分析等糖蛋白组学和糖组学研究技术的迅猛发展和推广普及,卵巢癌生物标志物的研究也从传统的对蛋白质的定性、定量研究,逐步转向于对标志物复杂糖基化修饰体系的研究以及对标志性糖链的结构鉴定和定量分析,开展了许多探索性的尝试和研究.通过对糖基化模式和糖链结构差异性的研究,提高和改善了CA125等经典肿瘤标志物对卵巢癌的鉴别诊断效能,发现和筛选出一批有潜力的卵巢癌糖链标志物,扩大了卵巢癌肿瘤标志物研究的样本来源.利用糖链谱分析技术开展卵巢癌肿瘤标志物的研究已成为普遍的共识和发展趋势.

因糖基化修饰的多样性、糖链结构的复杂性,卵巢癌标志物的糖链谱研究在基础性研究和技术层面仍然面临许多需要解决的问题.例如:如何深化糖链结构的性质、结构与功能的基础性研究;如何建立健康女性血液和体液糖蛋白和糖链结构异质性数据库;如何建立特异性好、简便易行的糖蛋白、糖肽及糖链的分离富集技术和适用于临床检验的快速、高通量的检测方法等,都是研究者当前面临和需要付出艰苦努力去解决的问题.

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中国科学院生物物理研究所和中国生物物理学会共同主办
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文章信息

薛添, 李艳红, 李铮
XUE Tian, LI Yan-Hong, LI Zheng
卵巢癌生物标志物的糖链谱研究进展
Progress in The Study of Glycosylation of Ovarian Cancer Biomarkers
生物化学与生物物理进展, 2017, 44(10): 865-876
Progress in Biochemistry and Biophysics, 2017, 44(10): 865-876
http://dx.doi.org/10.16476/j.pibb.2017.0135

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收稿日期: 2017-04-10
接受日期: 2017-09-04

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