CN111551723A - 特定凝集素在基于唾液糖蛋白糖链结构鉴别胰腺癌方面的应用及相关产品 - Google Patents
特定凝集素在基于唾液糖蛋白糖链结构鉴别胰腺癌方面的应用及相关产品 Download PDFInfo
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Abstract
本发明公开了特定凝集素在基于唾液糖蛋白糖链结构鉴别胰腺癌方面的应用及相关产品。所述特定凝集素分为上调组和下调组；其中：上调组为凝集素ECA、PHA‑E、GSL‑I、LCA、RCA120、ACA、MAL‑I和PHA‑E+L中的任一或任意组合；下调组为凝集素Jacalin、MAL‑II、SJA、MPL、PTL‑II、DSA、PWM和SNA中的任一或任意组合；鉴别依据为：对于被测唾液样本，若所述上调组识别的糖蛋白糖链结构表达显著上调，所述下调组识别的糖蛋白糖链结构表达显著下调，则表明相应主体为胰腺癌患者。
Description
技术领域
本发明涉及一种基于唾液糖蛋白糖链结构鉴别胰腺癌的相关产品。
背景技术
胰腺癌(Pancreatic Cancer,PC)是一种发病隐匿、进展快、疗效不佳、中位生存期短、预后极差的恶性肿瘤，素有“癌中之王”的称号。随着居民生活水平的提高和临床检出率的提升，近年来胰腺癌的发病率呈现逐年增长的趋势，成为威胁居民健康水平的重要因素。2018年全球新发胰腺癌病例约为45.9万，而死亡例数约43.2万,是造成癌症死亡的第七大病因，预测甚至将超越乳腺癌成为恶性肿瘤中的第三大致死原因。由于缺乏有效的筛查指标，且在癌症发生发展过程中几乎没有主观症状，导致了大多数胰腺癌患者确诊时已处于晚期，失去了最佳的治疗机会；另一方面，胰腺癌侵袭性强，转移率高，复发可能性大，手术及放、化疗预后均不理想，共同导致了胰腺癌患者5年生存率不到6％，胰腺癌患者的中位生存期仅为6个月。
许多患者在诊断为胰腺癌时已经是晚期，丧失了最佳的治疗时机。因此，早期诊断胰腺癌成为极为重要的临床问题。已有的研究提出液体活检在早期诊断中的意义，例如一个血清代谢物组合也显示出良好的前景，5种血清代谢物(乙酰精胺、二乙酰精胺、吲哚衍生物和2个溶磷脂胆碱)在早期胰腺癌和对照组的盲法验证中，使得AUC从0.726升至0.892。联合CA19-9、TIMP1及LRG1后AUC更是达到了0.924。在癌前病变中及时发现早期癌也有着不容忽视的意义，采用超声内镜活检下的目标物的质谱分析，发现了一组标志物，包含mucin-5AC和mucin-2，可以准确区分癌前病变/癌变与良性病变，这在验证队列中的精确度为97％(95％CI：89％～99％)。这组标志物效果优于单纯应用癌胚抗原甚至细胞学检查。另外一个组合，包含mucin-5AC和前列腺干细胞抗原，可以准确预测高级别异常增生和癌变，精确度达96％(95％CI：90％～99％)。也有研究提出胰腺癌患者在获得诊断时血浆miRNA会发生改变，但这种改变常发生在疾病晚期，不能用于早期诊断。
糖基化是一种发生在蛋白和脂质上的最为常见的翻译后修饰，在维持正常生命活动中发挥了重要的作用。糖基化影响蛋白的生物活性，参与新生肽链的正确折叠，维持蛋白正确的空间构象，参与维系蛋白质的亚基聚合，参与分子间的识别作用，包括受体与配体的识别结合，细胞与细胞的结合，细胞与细胞外基质的结合。蛋白表面的糖链作为细胞-细胞间、细胞-细胞外基质间相互作用的第一步接触受体，更能准确反映细胞的生理状态。近年来的研究揭露了糖链在生物体内的重要功能以及对疾病的影响，成为了癌症机制研究以及临床辅助诊断领域的研究热点之一。目前许多研究表明，肿瘤细胞糖基化的改变往往发生在肿瘤的早期，某些与肿瘤相关的糖链能在良性病变与癌前病变中出现，使糖链成为强有力的早期诊断标志物。
随着现今科学技术的发展，检测技术的多样性和灵敏度不断提高，近些年来，唾液作为临床样本已被广泛应用于多种疾病诸如艾滋病、自身免疫性疾病、肝硬化、糖尿病、心血管病、龋病等疾病的药物水平监测、病情监控和疗效评价当中。现有公开文献和我们以往的研究工作表明唾液中含有丰富的N-连接糖蛋白和O-连接糖蛋白，唾液蛋白糖基化的改变与疾病的发生发展具有较高的关联性。从变化的唾液糖蛋白糖链中可以找到与疾病相关的生物标志物，基于唾液检测的新技术和新方法也会逐渐成为今后非侵袭性临床诊断发展的一个重要方向。
发明内容
本发明的目的在于提供一种基于唾液糖蛋白糖型鉴别胰腺癌的方案。
本发明给出的方案如下：
第一方面，特定凝集素在构建基于唾液糖蛋白糖链结构鉴别胰腺癌的相关产品方面的用途，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；其特殊之处在于：所述特定凝集素分为上调组和下调组，其中上调组为凝集素ECA、PHA-E、GSL-I、LCA、RCA120、ACA、MAL-I和PHA-E+L中的任一或任意组合，下调组为凝集素Jacalin、MAL-II、SJA、MPL、PTL-II、DSA、PWM和SNA中的任一或任意组合；鉴别依据为：对于被测唾液样本，若所述上调组识别的糖蛋白糖链结构表达显著上调，所述下调组识别的糖蛋白糖链结构表达显著下调，则表明相应主体为胰腺癌患者。
这里，所述相关产品给出“鉴别依据”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。检测分析系统可以是智能终端，也可以是涵盖检测全流程的多个设备组合等。
第二方面，一种基于唾液糖蛋白糖链结构用于鉴别胰腺癌的凝集素芯片，其中包含的凝集素即上述特定凝集素。当然，实际工作中也可以利用更全面的凝集素芯片取样，但对于鉴别胰腺癌来说，只关注上述特定凝集素。
凝集素芯片的制备和检测为现有常规方法，其步骤一般包括唾液采集、唾液蛋白处理和荧光标记、芯片检测及数据分析。
第三方面，一种基于唾液糖蛋白糖链结构用于鉴别胰腺癌的试剂盒，其特殊之处在于：其中包含的凝集素即上述特定凝集素；所述试剂盒的使用说明书给出以下鉴别依据：对于被测唾液样本，若上调组识别的糖蛋白糖链结构表达显著上调，下调组识别的糖蛋白糖链结构表达显著下调，则表明相应主体为胰腺癌患者。
试剂盒的使用说明书中还可直接给出上述特定凝集素在健康对照组的糖蛋白糖链表达水平。
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实现以下步骤：
获取被测唾液样本的凝集素测试结果，所述凝集素测试结果体现上述的特定凝集素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鉴别依据(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)；
将被测唾液样本与健康对照组的糖蛋白糖链表达水平进行比较，输出鉴别结论；鉴别依据为：若上调组识别的糖蛋白糖链结构表达显著上调，下调组识别的糖蛋白糖链结构表达显著下调，则表明相应主体为胰腺癌患者。
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实现以上所列各个步骤。
本发明具有如下有益效果：
本发明通过检测被试者唾液中特定糖蛋白糖链结构的表达水平差异，能够快速、准确鉴别被试者是否为胰腺癌患者。
附图说明
图1为凝集素探针布局图；
图2为聚类分析图；图中，HV：健康志愿者；PC：胰腺癌患者；
图3为胰腺癌中上调的8种凝集素散点图；图中，HV：健康志愿者；PC：胰腺癌患者；*,P<0.05；**,p<0.01；***,P<0.001；
图4为胰腺癌中下调的8种凝集素散点图；图中，HV：健康志愿者；PC：胰腺癌患者；*,P<0.05；**,p<0.01；***,P<0.001；
图5为主成分分析图；图中，PC：胰腺癌患；GC：胃癌患者。
具体实施方式
以下介绍本申请的相关实验及结论，以揭示本申请技术方案的科学性和可行性。应当理解，申请人为此所进行的实验及付出的人力物力不止于此。
1、实验部分
1.1试剂与材料
环氧基硅烷试剂(GPTS)，Bradford试剂，蛋白酶抑制剂，DMSO，Tween-20，盐酸羟胺均购自美国Sigma公司，Cy3、Cy5荧光染料构自美国Amerhsam公司,Sephadex G-25柱购自美国GE Healthcare公司，384孔板构自英国Genetix公司，牛血清白蛋白(BSA)购自德国Calbiochem公司，玻璃片基购自Gold Seal公司，0.2μm滤膜，0.45μm滤膜购自日本Sartorius公司，其他常用试剂购自国产分析纯。37种凝集素(见表1)分别购自Vector公司，Sigma公司。
表1.凝集素对照表
1.2实验仪器
电热鼓风干燥箱：天津泰斯特公司；高压灭菌锅：日本TOMY公司；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；生物芯片扫描仪4000B：美国Axon公司；芯片点样仪：博奥晶芯SmartArrayer48点样仪；芯片杂交箱HL-2000：美国UVP公司。
1.3研究人群和全唾液采集
已确诊的胰腺癌患者26例，对照样本为42例健康志愿者(无其它疾病，一周之内没有服用任何药物)和21例已确诊的胃癌(gastric cancer，GC)患者。饭后两小时，约9点到10点之间，生理盐水漱口三次后迅速采集自然分泌的全唾液。唾液采集至少1ml并立即置于冰上，加入蛋白酶抑制剂(每毫升唾液加入1μL)防止蛋白降解。
表2.健康志愿者，胰腺癌患者以及胃癌患者信息汇总表
1.4唾液蛋白处理和荧光标记
收集到的全唾液经12 000rpm 4℃离心10min后吸取上清弃去不溶沉淀物。上清再经0.22μm孔径的滤膜过滤掉细菌和其他微生物。样本经Cy3荧光染料标记后用Sephadex G-25除盐柱去掉游离荧光。标记好的蛋白准备用于凝集素芯片孵育。
1.5凝集素芯片和数据分析
1.5.1凝集素芯片的制备
将未处理的玻片用无水乙醇清洗三次，每次10min。离心甩干后，将玻片浸泡入250mL10％NaOH溶液中，摇床上轻摇反应，避光过夜。反应后，超声15min，再用超纯水清洗四次，每次2min，无水乙醇清洗两次，每次2min。离心甩干后，再将玻片浸泡到200mL10％GPTS溶液中，摇床上轻摇，避光反应3h。反应后，超声清洗15min，无水乙醇清洗三次，每次10min。离心甩干后，芯片的环氧化修饰完成，并将修饰好的玻片放置于4℃干燥器中保存备用。制备好的凝集素芯片的点样设计如图1,每张芯片共分为4个矩阵，每个矩阵规格为12*10，每个样品点重复三次。
1.5.2凝集素芯片的孵育及数据分析
(1)凝集素芯片的封闭
将点制好的凝集素芯片从4℃干燥器中取出，回温。首先用PBST、PBS各清洗玻片一次，每次3min，离心甩干。将凝集素芯片与600μl封闭缓冲液在芯片杂交盒中孵育，25℃旋转反应1h。封闭结束后用PBST、PBS各清洗玻片两次，每次3min，甩干。用Genepix4000B芯片扫描仪扫描封闭后芯片，检查封闭效果。
(2)唾液样本的凝集素芯片检测
将荧光标记的唾液蛋白3μg与孵育缓冲液混匀，配置成600μl上样体系。并均匀加载在盖玻片上，盖上封闭后的凝集素芯片，于芯片杂交仪中25℃避光旋转孵育3h。孵育结束后用PBST、PBS各清洗玻片两次，每次5min，离心甩干。
(3)数据的扫描与分析
用Genepix4000B芯片扫描仪扫描芯片，光电倍增管(PMT)功率设为100％，先对整张芯片进行预扫描，然后选定点样区域，进行精确扫描，调节明暗度和对比度，达到最佳视觉效果。用GenePix3.0软件从芯片扫描结果图中获取荧光信号强度值和背景信号值。筛选出大于2倍背景标准偏差的荧光信号值，选取中值进行归一化分析，获得归一化后的荧光信号强度(Normalized Fluorescent Intensities,NFIs)。
2、结果部分
(1)胰腺癌患者唾液中的糖蛋白糖链结构的变化
首先利用凝集素芯片分别对13例胰腺癌患者和21例健康志愿者混合唾液进行检测，获取芯片数据并归一化处理后，比较胰腺癌患者与健康志愿者在唾液糖蛋白糖型上的差异。结果如表3a和3b所示，有8种凝集素(ECA,PHA-E,GSL-I,LCA,RCA120,ACA,MAL-I,PHA-E+L)识别的糖蛋白糖链结构在胰腺癌患者的唾液中表达显著上调；有8种凝集素(Jacalin,MAL-II,SJA,MPL,PTL-II,DSA,PWM,SNA)识别的糖蛋白糖链结构在胰腺癌患者的唾液中表达显著下调。
表3a.相较于健康志愿者，胰腺癌患者唾液中表达显著上调的糖链结构
表3b.相较于健康志愿者，胰腺癌患者唾液中表达显著下调的糖链结构
(2)胰腺癌患者唾液中的糖蛋白糖链结构变化的个例验证
利用凝集素芯片再分别对另外的13例胰腺癌患者和另外的21例健康志愿者个例唾液样本进行检测，获取芯片数据并归一化处理后，首先将健康志愿者组芯片结果与胰腺癌患者组芯片结果进行聚类分析，在总体水平观测健康志愿者与胰腺癌患者在唾液糖蛋白糖链水平上的差异。结果如图2所示，层次聚类分析能够将健康志愿者与胰腺癌患者进行一定程度的区分。如图3所示，总计有8种凝集素(ECA,PHA-E,GSL-I,LCA,RCA120,ACA,MAL-I,PHA-E+L)的NFIs在胰腺癌患者唾液糖蛋白中显著上调。如图4所示，总计有8种凝集素(Jacalin,MAL-II,SJA,MPL,PTL-II,DSA,PWM,SNA)的NFIs在胰腺癌患者唾液糖蛋白中显著下调。胰腺癌患者个例唾液中的糖蛋白糖链结构变化与胰腺癌患者混合唾液中的糖蛋白糖链结构变化具有一致性。
(3)胰腺癌患者唾液糖蛋白糖型与胃癌患者同样存在显著的差异
胃癌和胰腺癌同属于消化道恶性肿瘤，且胃癌的发病率要显著高于胰腺癌。因此基于21例已公开的胃癌患者唾液糖蛋白糖型数据，对这两种癌症进行了比较。结果如图5所示，主成分分析能够胰腺癌患者以及胃癌患者进行明显的区分，表明了胰腺癌患者与胃癌患者的唾液糖蛋白糖型存在显著的差异。
Claims (5)
1.特定凝集素在构建基于唾液糖蛋白糖链结构鉴别胰腺癌的相关产品方面的用途，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；其特征在于：所述特定凝集素分为上调组和下调组；其中：
上调组为凝集素ECA、PHA-E、GSL-I、LCA、RCA120、ACA、MAL-I和PHA-E+L中的任一或任意组合；
下调组为凝集素Jacalin、MAL-II、SJA、MPL、PTL-II、DSA、PWM和SNA中的任一或任意组合；
鉴别依据为：对于被测唾液样本，若所述上调组识别的糖蛋白糖链结构表达显著上调，所述下调组识别的糖蛋白糖链结构表达显著下调，则表明相应主体为胰腺癌患者。
2.一种基于唾液糖蛋白糖链结构用于鉴别胰腺癌的凝集素芯片，其特征在于：其中包含的凝集素为权利要求1中所述的特定凝集素。
3.一种基于唾液糖蛋白糖链结构用于鉴别胰腺癌的试剂盒，其特征在于：其中包含的凝集素为权利要求1中所述的特定凝集素；所述试剂盒的使用说明书给出以下鉴别依据：对于被测唾液样本，若上调组识别的糖蛋白糖链结构表达显著上调，下调组识别的糖蛋白糖链结构表达显著下调，则表明相应主体为胰腺癌患者。
4.一种智能终端，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实现以下步骤：
获取被测唾液样本的凝集素测试结果，所述凝集素测试结果体现权利要求1中所述的特定凝集素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鉴别依据；
将被测唾液样本与健康对照组的糖蛋白糖链表达水平进行比较，输出鉴别结论；鉴别依据为：若上调组识别的糖蛋白糖链结构表达显著上调，下调组识别的糖蛋白糖链结构表达显著下调，则表明相应主体为胰腺癌患者。
5.一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实现权利要求4中所列各个步骤。
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