CN111220804B - 基于血清糖蛋白糖型评估乳腺癌化疗效果的标志物及其应用 - Google Patents
基于血清糖蛋白糖型评估乳腺癌化疗效果的标志物及其应用 Download PDFInfo
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Abstract
本发明提供了一种基于血清糖蛋白糖型评估乳腺癌化疗效果的标志物及其应用。通过检测凝集素LCA、GSL‑II、GSL‑I、PHA‑E+L、WFA、RCA120中任一或任意组合所识别糖链的表达水平，能够评估化疗对乳腺癌化疗后患者是否有疗效。具体依据可参考：GSL‑II下调，且下调的Fold‑change值在0.5‑1.9之间；LCA，GSL‑I，RCA120上调，且上调的Fold‑change值在1.68‑2.4，3‑12.5，0.67‑2.61之间；PHA‑E+L、WFA与健康女性对照组无显著性差异，则说明有疗效。
Description
技术领域
本发明涉及一种基于血清糖蛋白糖型检测的乳腺癌化疗效果标志物及其应用。
背景技术
乳腺癌是妇女中最常见的恶性肿瘤，也是女性中癌症死亡的主要原因，在世界范围内，其发病率和死亡率预计在今后5至10年内将显著增加。乳腺癌的发病率之高，严重危害了女性的健康。根据最新几年的统计数据显示，中国女性整体上乳腺癌的发病率和死亡率呈现上升的趋势，同时乳腺癌患者出现了年轻化、死亡率升高的趋势。乳腺癌的治疗通常涉及手术治疗、化学药物治疗、放射性治疗、内分泌治疗和靶向治疗。新辅助化疗已经成为治疗乳腺癌的最佳方案之一，可以提高患者生存质量，改善临床症状，延长患者生存期。目前，临床上对乳腺癌新辅助化疗的疗效评价主要通过临床评价和病理组织学评价。但仍有疗效评估出现偏差以及获得疗效结论较晚、错失调整化疗最佳方案等局限性。因此，早期诊断及评估对于改善预后、延长患者生存期具有重要意义。另一方面，我国人口基数庞大、地区差异明显，采用更为简便、经济的方法进行早期诊断和早期治疗，可避免化疗盲目性。
长期以来，组织活检技术常作为肿瘤诊断的金标准，随着对肿瘤研究的深入发现组织活检技术仍有肿瘤异质性、不适用于所有患者等局限性。但随着液体活检技术的出现，不仅解决了组织活检的局限性问题，同时也缩短了癌症的诊断时间，液体活检就是通过检测血液或者尿液等体液中的某些生物活性分子，从而对癌症等疾病做出诊断。其优势在于能通过非侵入性取样降低活检的危害；由于其具有敏感性高、检测快捷等特点，并且有利于肿瘤的早期发现，已有研究指出，同健康女性血清相比，乳腺癌患者血清中可检测到不同浓度的HER2，VEGF和EGF蛋白等具有乳腺癌诊断潜能的血清标志物。同时，血清中疾病特异性的标志物在肺癌、结直肠癌、胃癌、肝癌、宫颈癌等领域也得到了广泛关注和应用。
蛋白质糖基化在癌症方面的研究日益兴起，越来越多的证据表明，糖基化异常与肿瘤细胞的侵袭和转移有密切关系，这些变化包括糖链在表达水平上的差异和特殊糖基结构的出现。糖链是膜蛋白和分泌蛋白的结构组分，通过糖基化作用影响蛋白质的功能，与细胞通讯、信号转导等密切相关，糖链的改变往往是对机体状态变化更灵敏的应答。因此研究糖复合物糖基化将有望提高标志物的特异性与灵敏度，为癌症早期发现、诊断、预后及化疗疗效评估提供新的方法。
而诸多研究表明，在乳腺癌及其他多种癌症患者血清中可见肿瘤相关性糖基化的改变。乳腺癌患者血清中蛋白质糖基化修饰存在差异，具体表现为增多的MUC型O-糖链、含多聚N-乙酰乳糖胺(poly-LacNAc)结构的糖链、分支型N-糖链，减少的β1-4GlcNAc等分型和拥有LacdiNAc末端结构的复杂型N-糖链，以及糖链岩藻糖基化、唾液酸化修饰增加等；转移性乳腺癌患者化疗前后血清唾液酸转移酶(Serum sialyltransferas，SST)平均水平在进展期乳腺癌患者中显著升高，乳腺癌患者N-糖链的高甘露糖修饰水平与正常人血清中甘露糖修饰水平相比明显增加。
凝集素是一类糖结合蛋白，其可识别不同的糖链结构并以多价形式与这些糖链特异性结合。通过将凝集素探针固定于经环氧化修饰的固体片基上制成凝集素芯片，凭借生物芯片便捷、快速和高通量的优势，人们便可获得临床样本中糖复合物糖基化相关信息，发现与疾病特异性相关的差异性表达的糖链。
发明内容
本发明的目的在于提供一种通过血清中糖蛋白糖链的变化来评估乳腺癌化疗疗效的方案。
本发明给出的方案如下：
第一方面，特定凝集素在构建基于血清糖蛋白糖型检测的乳腺癌化疗疗效评估相关产品方面的用途，所述特定凝集素为LCA、GSL-II、GSL-I、PHA-E+L、WFA、RCA120中任一或任意组合；所述相关产品为凝集素芯片、试剂盒、检测分析系统和/或它们的组合。
用户可根据上述特定凝集素所识别糖链的表达水平来评估乳腺癌患者进行的化疗是否有效果。
进一步的，所述相关产品还提示以下评估依据：
待测样本与健康对照组相比，满足以下条件之任一或任意组合，即表明乳腺癌患者进行的化疗有效果；
GSL-II下调，并且下调的Fold-change值在0.5-1.9之间；
LCA上调，并且上调的Fold-change值在1.68-2.4之间；
GSL-I上调，并且上调的Fold-change值在3-12.5之间；
RCA120上调，并且上调的Fold-change值在0.67-2.61之间；
PHA-E+L与健康对照组无显著性差异，Fold-change值在0.36-0.93，0.66-0.96之间；
WFA与健康对照组无显著性差异，Fold-change值在0.36-0.93，0.66-0.96之间。
这里，“提示评估依据”的具体形式不限，例如：随附的产品说明书中记载上述条件；涉及到软件的，则还可由相应的算法体现该依据。
从中也可以看出，凝集素LCA、GSL-II、GSL-I、PHA-E+L、WFA、RCA120可按照“下调变化”、“上调变化”、“无显著性差异”分为三组。所以，较佳的一种组合方式可以是三组各选一种凝集素。
第二方面，一种基于血清样本用于乳腺癌化疗疗效评估的凝集素芯片，其中包含的凝集素为LCA、GSL-II、GSL-I、PHA-E+L、WFA和RCA120中的任一或任意组合。当然，实际工作中也可以包含其他凝集素，但对于乳腺癌化疗疗效评估来说，只关注上述六种凝集素中的任一或任意组合进行判断。
凝集素芯片的制备和检测为现有常规方法，其步骤一般包括血液采集、血清蛋白处理和荧光标记、凝集素芯片及其数据分析。
第三方面，一种基于血清样本用于乳腺癌化疗疗效评估的试剂盒，其中包含的凝集素为LCA、GSL-II、GSL-I、PHA-E+L、WFA和RCA120中的任一或任意组合。
凝集素芯片产品、试剂盒可进一步给出使用说明，其中包含上述各凝集素在健康对照组的糖蛋白糖链表达水平以及上述评估依据。
第四方面，一种智能终端，包括处理器和程序存储器，所述程序存储器存储的程序被处理器加载时执行以下步骤：
获取待测血清样本的凝集素测试结果，所述凝集素测试结果体现凝集素LCA、GSL-II、GSL-I、PHA-E+L、WFA、RCA120中任一或任意组合对应的糖蛋白糖链表达水平；
获取相应的健康对照组的糖蛋白糖链表达水平以及评估依据(可预先记录在智能终端，也可从外部获取，如通过联网获取等)；
将待测血清样本和健康对照组的糖蛋白糖链表达水平进行比较，输出评估结论，所述评估结论为待测血清样本所属乳腺癌主体进行的化疗是否有效果。
第五方面，一种计算机可读存储介质，存储有计算机程序，所述计算机程序被处理器加载时执行以上各个步骤。
本发明具有如下有益效果：
本发明通过检测经过化疗的乳腺癌患者血清样品中特定糖蛋白糖链结构的表达水平差异，快速确定乳腺癌患者化疗是否有疗效。
附图说明
图1为健康女性(HV)、乳腺癌患者(BC)、乳腺癌化疗后患者(CT)血清糖蛋白糖链荧光信号归一化数据图。
具体实施方式
下面通过给出的具体实施例对本发明做进一步说明，但不作为对本发明的限定。
本发明从37种凝集素(如表1所示)中筛选用于检测女性乳腺癌化疗疗效评估的凝集素探针。
表1缩略语对照表
本发明中使用的试剂材料包括：蛋白酶抑制剂，二甲基亚砜(DMSO)，Cy3，荧光染料，Sephadexg G-25柱，牛血清白蛋白(BSA)，玻璃片基，氧基硅烷试剂(GPTS)，Bradford试剂，Tween-20，以及国产分析纯常用试剂。
采用的设备包括：超速冷冻离心机5804R：德国Eppendorf公司；微量核酸蛋白测定仪：德国Implen公司；芯片点样仪：博奥晶芯SmartArrayer48点样仪；生物芯片扫描仪4000B：美国Axon公司；芯片杂交箱HL-2000：美国UVP公司。
1.研究人群和血液采集
选取的133名女性血液提供者均为无其它疾病，其中包括确诊的乳腺癌患者(BC)18例，乳腺癌化疗后患者(CT)65例(经临床影像学及病理学分析确认有疗效)，健康志愿者(HV)50例，如表2所示，由于血清糖蛋白糖链与提供者年龄具有相关性，因此三组血液提供者年龄均相互匹配。
表2年龄信息统计表
2.血清蛋白处理和荧光标记
收集到的血液，室温或4℃静置半小时，3000rpm离心5分钟分离血清。血清分离后-80℃分装保存。取血清样本100μg，用Cy3荧光染料标记后用Sephadex G-25除盐柱纯化，收集到的荧光标记后的血清蛋白用于凝集素芯片孵育。
3.凝集素芯片及其数据分析
凝集素芯片的制备，Cy3荧光标记的样本蛋白与凝集素芯片的孵育步骤及凝集素芯片数据获取与归一化分析方法为常规技术，可参见专利文献CN201110021447.3等。
4.结果分析
利用凝集素芯片分别对健康女性、乳腺癌女性患者以及化疗后乳腺癌患者血清样本进行检测，通过genepix6.0获取芯片数据并归一化处理后，将未化疗乳腺癌患者(BC)、化疗后乳腺癌患者(CT)同健康女性组(HV)结果进行比较，即每个凝集素对应的归一化后NFI在BC组和CT组分别比HV组得到Fold-change值，将Fold-change>1.5和Fold-change<0.67作为BC组和CT组相较于HV组在血清中上调和下调表达的糖链。
结果发现(见表3和图1)：
LCA识别的α-D-Man，Fucα-1,6GlcNAc，α-D-Glc，GSL-II识别的GlcNAc andagalactosylatedtri/tetra antennary glycans以及GSL-Ⅰ识别的αGalNAc，αGal，anti-Aand B糖链，在未化疗乳腺癌患者(breast cancer，BC)组血清中的表达水平分别是健康志愿者(health volunteer，HV)组的2.4，1.9和12.5倍，均显著上调。经过化疗(chemicaltherapy，CT)后，LCA，GSL-ⅠⅠ和GSL-Ⅰ识别的糖链在血清中的表达水平分别是HV组的1.68，0.5，3倍，明显趋于正常组的表达水平。
PHA-E+L识别的Bisecting GlcNAc，bi-antennary N-glycans，tri-和tetra-antennary complex-type N-glycan；WFA识别的terminating in GalNAcα/β1-3/6Gal，以及RCA120识别的β-Gal，Galβ-1,4GlcNAc(typeⅡ)，Galβ1-3GlcNAc(typeⅠ)糖链，在BC组血清中的表达量分别是HV组的0.36，0.66和0.67倍，均显著下调。经过化疗(chemicaltherapy，CT)后，PHA-E+L，WFA和RCA120识别的糖链在血清中的表达水平分别是HV组的0.93，0.96，2.61倍，明显趋于正常组的表达水平。
表3三组血清糖蛋白糖型的表达比较
a三组之间凝集素归一化后信号值彼此进行比值分析,BC/HV,CT/HV和CT/BC。HV:健康组；BC：乳腺癌患者组；CT：乳腺癌化疗后患者组。
－,无显著性差异；*P<0.05；**P<0.01；***P<0.001；****P<0.0001.
5.凝集素探针组的确定
通过上述结果分析，若以健康女性血清作为对照标准，用于筛选早期乳腺疾病患者血清的凝集素探针表达结果参见表4。
表4用于从乳腺癌化疗女性患者中筛选有化疗疗效的凝集素探针
以健康女性样本为对照组，待测样本的凝集素芯片与对照组对比，当GSL-II下调，并且下调的Fold-change值在0.5-1.9之间，LCA，GSL-I，RCA120上调，并且上调的Fold-change值在1.68-2.4，3-12.5，0.67-2.61区间范围内，PHA-E+L、WFA与对照组无显著性差异并且Fold-change值在0.36-0.93，0.66-0.96区间范围内，则说明其患者的化疗有效果。
Claims (3)
1.特定凝集素在构建基于血清糖蛋白糖型检测的乳腺癌化疗疗效评估相关产品方面的用途，其特征在于：所述特定凝集素为LCA、GSL-II、GSL-I、PHA-E+L、WFA和RCA120的组合；所述相关产品为凝集素芯片、试剂盒、检测分析系统和/或它们的组合；
所述相关产品还提示以下评估依据：
待测样本与健康对照组相比，满足以下条件组合，即表明乳腺癌患者进行的化疗有效果；
GSL-II下调，并且下调的Fold-change值在0.5-1.9之间；
LCA上调，并且上调的Fold-change值在1.68-2.4之间；
GSL-I上调，并且上调的Fold-change值在3-12.5之间；
RCA120上调，并且上调的Fold-change值在0.67-2.61之间；
PHA-E+L与健康对照组无显著性差异，Fold-change值在0.36-0.93，0.66-0.96之间；
WFA与健康对照组无显著性差异，Fold-change值在0.36-0.93，0.66-0.96之间。
2.一种智能终端，包括处理器和程序存储器，其特征在于：所述程序存储器存储的程序被处理器加载时执行以下步骤：
获取待测血清样本的凝集素测试结果，所述凝集素测试结果体现凝集素LCA、GSL-II、GSL-I、PHA-E+L、WFA和RCA120组合对应的糖蛋白糖链表达水平；
评估依据为：
待测样本与健康对照组相比，满足以下条件组合，即表明乳腺癌患者进行的化疗有效果；
GSL-II下调，并且下调的Fold-change值在0.5-1.9之间；
LCA上调，并且上调的Fold-change值在1.68-2.4之间；
GSL-I上调，并且上调的Fold-change值在3-12.5之间；
RCA120上调，并且上调的Fold-change值在0.67-2.61之间；
PHA-E+L与健康对照组无显著性差异，Fold-change值在0.36-0.93，0.66-0.96之间；
WFA与健康对照组无显著性差异，Fold-change值在0.36-0.93，0.66-0.96之间；
获取相应的健康对照组的糖蛋白糖链表达水平以及评估依据；
将待测血清样本和健康对照组的糖蛋白糖链表达水平进行比较，输出评估结论，所述评估结论为待测血清样本所属乳腺癌主体进行的化疗是否有效果。
3.一种计算机可读存储介质，存储有计算机程序，其特征在于：所述计算机程序被处理器加载时执行权利要求2中所列各个步骤。
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