CN112630431A - 人凝血酶敏感蛋白-1在制备预测肝内胆管癌化疗效果的试剂盒中的应用 - Google Patents
人凝血酶敏感蛋白-1在制备预测肝内胆管癌化疗效果的试剂盒中的应用 Download PDFInfo
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Abstract
本发明涉及医学生物检测技术领域,具体是人凝血酶敏感蛋白‑1在制备预测肝内胆管癌化疗效果的试剂盒中的应用。本发明优点在于:本发明利用蛋白芯片筛选出中晚期肝内胆管癌(ICC)化疗耐药组和敏感组病人组织中存在的表达差异的蛋白,而且该蛋白可以在血清中检测到,发现凝血酶敏感蛋白‑1在两组病人中差异最为明显而且在敏感组病人中表达水平非常高。本发明提供了人凝血酶敏感蛋白‑1作为肝内胆管癌病人化疗敏感与否的血清标志物中的应用,其表达水平高低和病人的生存预后密切相关。本发明为血清学诊断提供了新的临床手段。
Description
技术领域
本发明涉及医学生物检测技术领域,具体地说,是人凝血酶敏感蛋白-1在制备预测肝内胆管癌化疗效果的试剂盒中的应用。
背景技术
蛋白质作为生命功能的执行者,在生物体中发挥着重要的功能,其中分泌到细胞膜外的蛋白质称为分泌性蛋白或分泌蛋白。分泌蛋白占人类蛋白质组的十分之一,它扮演着细胞信息传递的重要角色,且以旁分泌、自分泌、内分泌等途径发挥作用,而细胞生长、分化,组织器官生理功能的正常发挥及炎症、肿瘤等疾病的发生、发展和转归都依赖细胞间这种信息传递。分泌蛋白研究的一个重要方向便是作为一些疾病诊断的血清标志物进行应用。
1999年,国外学者首次用酶联免疫吸附试验(ELISA)分析头颈部鳞癌细胞原代培养上清液中的14种细胞因子,并用患者血清和病变组织标本进行分析验证,发现这些因子在炎症反应和血管形成方面起重要作用,其中白细胞介素-6、血管内皮生长因子(VEGF)等可作为肿瘤诊断、治疗的标志物。之后我国学者通过癌细胞分泌蛋白质组学用来筛选恶性肿瘤分子标志物,并在鼻咽癌中取得初步成功。这些都反映了分泌蛋白在疾病的诊断中发挥着巨大的潜能。由于分泌蛋白在血清中可以检测,通过酶联免疫吸附试验(ELISA)等方法简单易行、便于检测。有助于临床的推广临床应用。
凝血酶敏感蛋白-1(Thrombospondin-1;以下简称TSP1)是一种多功能的细胞外基质糖蛋白,参与多种重要的生物过程,在细胞黏附、迁移、增殖、凋亡、血管生成、肿瘤细胞转移、炎症和血栓形成中起重要作用。但是,TSP1在血清学诊断标志物中的应用研究还非常少。而且,其作为肝内胆管癌(intrahepatic cholangiocarcinoma,ICC)化疗敏感性预测的标志物,更是没有这样的研究所涉及。
因此,我们通过酶联免疫吸附试验(ELISA)来检测血清中TSP1的表达以预测肝内胆管癌病人化疗的敏感性的研究也是首次,之前并未有文献报道。
发明内容
本发明的目的在于提供凝血酶敏感蛋白-1(TSP1)的新用途,具体是TSP1可以作为血清学诊断标志物,来指导肝内胆管癌患者在以吉西他滨为主的化疗方案治疗时,能够提前预判出病人对于吉西他滨为主方案的治疗效果。
本发明的另一个目的在于提供一种检测血清中TSP1分泌蛋白的试剂盒,及其该试剂盒在肝内胆管癌化疗病人血清学诊断中的应用。
在前期的研究中,本发明利用蛋白芯片筛选了化疗敏感和耐药病人组织中表达差异显著的分泌蛋白,结果发现TSP1在化疗敏感的肝内胆管癌病人中高表达并可以调节指示化疗的敏感性。而且TSP1在不同病人身上的表达水平存在差异,而且组织中和血清中清TSP1具有正相关性,可以通过检测病人血清中TSP1的含量来判断病人以吉西他滨为主要化疗方案的治疗效果。
进一步地,本发明建立了人血清中TSP1的检测方法和提供了血清中TSP1检测的试剂盒,通过对肝内胆管癌病人血清的检测,我们首先发现在血清中能够检测到TSP1,并且56例ICC病人血清TSP1的差异和化疗效果以及生存时间相关。
因此,本发明认为,TSP1具备作为指导肝内胆管癌病人化疗敏感性的血清学标志物的可能性。
本发明的第一方面,提供人凝血酶敏感蛋白-1在制备预测肝内胆管癌化疗敏感性标志物中的应用。
本发明的第二方面,提供人凝血酶敏感蛋白-1在制备预测肝内胆管癌化疗效果的试剂或试剂盒中的应用。
进一步的,所述的试剂或试剂盒,是通过酶联免疫吸附剂测定(Elisa)技术等方法检测血清中TSP1的表达量。
本发明的第三方面,提供人凝血酶敏感蛋白-1在制备肝内胆管癌化疗预后分型的试剂或试剂盒中的应用。
进一步的,所述的试剂或试剂盒根据血清中TSP1的表达量高低,将肝内胆管癌患者分为化疗敏感型和化疗耐药型,高于试剂盒检测出的83.625ng/ml为敏感型,低于此阈值的为耐药型(本试剂盒检测的浓度为病人血清中TSP1稀释后的浓度,病人血清TSP1的实际浓度需按试剂盒稀释比例进行换算)。
进一步的,所述的化疗为以吉西他滨为主的化疗治疗方案。
本发明的第四方面,提供人凝血酶敏感蛋白-1在制备预测肝内胆管癌化疗后生存期的试剂或试剂盒中的应用。
进一步的,化疗病人血清中TSP1的水平和生存相关:TSP1血清含量高者,化疗病人总生存期更长;TSP1血清含量高者,化疗病人无进展生存期更长。
本发明的第五方面,提供一种预测肝内胆管癌化疗效果的试剂盒,所述的试剂盒通过酶联免疫吸附剂测定(Elisa)技术等方法检测血清中TSP1的表达量;根据血清中TSP1的表达量高低,将肝内胆管癌患者分为化疗敏感型和化疗耐药型。
进一步的,所述的试剂盒包括以下试剂:
本发明优点在于:
本发明提供了TSP-1在作为肝内胆管癌病人化疗敏感与否的血清标志物中的应用,TSP-1的表达水平高低和病人的生存预后密切相关。本发明通过对肝内胆管癌病人血清TSP-1的检测,发现在血清中能够检测到TSP-1,并且ICC化疗敏感组病人血清中的TSP1含量明显高于化疗耐药组病人,TSP1血清含量高者,化疗病人总生存期和无进展生存期更长。本发明为血清学诊断提供了新的临床手段。
附图说明
图1为TSP1的筛选以及来源;
图2为56例子化疗癌患者TSP1表达水平存在差异,且组织和血清水平存在正相关性;A图是肝内胆管癌(ICC)患者中TSP1水平的验证;B图是ICC病人血清中水平的验证;C图是TSP1表达水平在ICC病人组织和血清中的相关性分析
图3为化疗病人血清中TSP1的水平和生存相关;A:TSP1血清含量高者,化疗病人总生存期(OS)更长;B:TSP1血清含量高者,化疗病人无进展生存期(PFS)更长。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
实施例1:TSP1的筛选、鉴定
前期我们利用高通量普通转录组筛选了吉西他滨耐药的人源肿瘤异种移植模型(Patient-Derived tumor Xenograft,PDX),进一步利用这些模型中的耐药模型以及敏感模型中表达水平差异的基因,从筛到的基因中选出了变化最显著的10个基因(见图1),而且通过进一步筛选,发现在这些基因中只有TSP1其编码的蛋白能够在人血清中能检测,因此有作为血清标志物的潜力,便将TSP1作为研究的基因。
实施例2:血清样本的采集、处理以及TSP1的检测
(1)血清样本收集:收集肝内胆管癌病人化疗前的血清。
(2)血清样本处理:采集的外周血样品3000rpm 4℃离心15min,取上清分装于EP管中,-80℃保存。
(3)检测血清中TSP1含量:
准备试剂与收集血样:
1)10×标本稀释液用蒸馏水作1:10倍稀释(示例:1ml浓稀释液+9ml蒸馏水)。
2)收集标本:血清、血浆(EDTA、柠檬酸盐、肝素抗凝)、细胞培养上清液、组织匀浆等尽早检测,2-8℃保存48小时;更长时间须冷冻(-20℃或-70℃)保存,避免反复冻融。血清至少100倍稀释。人唾液可不稀释。血浆或细胞上清或乳汁至少2倍稀释。
3)标准品液配制:取8个1.5ml离心管,第一管加标本稀释液900ul,第二至第八管加入标本稀释液500ul。在第一管中加入5000ng/ml的标准品溶液100ul置于漩涡混合器上混匀后用加样器吸出500ul,移至第二管。如此反复作对倍稀释,从第七管中吸出500ul弃去。第八管为空白对照。
4)洗涤液:用重蒸水1:20稀释(示例:1ml浓缩洗涤液加入19ml的重蒸水)
检测程序:
1)加样:每孔各加入标准品或待测样品100ul,将反应板充分混匀后置37℃40分钟。
2)洗板:用洗涤液将反应板充分洗涤4-6次,向滤纸上印干。
3)每孔加入蒸馏水和第一抗体工作液各50ul(空白除外)。将反应板充分混匀后置37℃20分钟。
4)洗板:同前。
5)每孔加酶标抗体工作液100ul。将反应板置37℃10分钟。
6)洗板:同前。
7)每孔加入底物工作液100ul,置37℃暗处反应15分钟。
8)每孔加入100ul终止液混匀。
9)30分钟内用酶标仪在450nm处测吸光值。
实施例3:不同病人之间血清中TSP1的表达存在差异
通过实施例2中的检测方法对于经历过手术的肝内胆管癌病人TSP1组织以及血清中TSP1的表达水平进行了检测,发现TSP1在不同ICC病人中组织(图2A)以及血清(图2B)的水平存在差异,而且血清中TSP1的表达水平和组织中呈现正相关关系(图2C),这体现了TSP1有作为血清标志物的基本条件和临床转化的潜力。
实施例4:TSP1表达水平的高低可以指导病人的化疗效果
按照实施例2相同的方法,我们目前对于以吉西他滨为基础化疗方案的肝内胆管癌病人化疗前的血清进行了检测,对于不同病人的血清中TSP1的表达水平通过ROC曲线确定的阈值分为高表达组(>=83.625ng/ml)以及低表达组(<83.625ng/ml),通过对于这些病人的随访,发现TSP1的表达水平和病人的化疗敏感性相关(表1);而且TSP1表达高组别的病人相比较于化疗敏感组别较低的病人,其OS(总生存期)以及PFS(无进展生存期)均时间更长(图3),显示病人的生存获益。
这一结果提示:TSP1可作为预测晚期肝内胆管癌病人化疗敏感与否的血清学标志物清学进行使用,可以填补预测肝内胆管癌化疗效果的血清标志物这一空白。
表1.化疗病人血清中TSP1表达水平和临床指标相关性
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (9)
1.人凝血酶敏感蛋白-1在制备预测肝内胆管癌化疗敏感性标志物中的应用。
2.人凝血酶敏感蛋白-1在制备预测肝内胆管癌化疗效果的试剂或试剂盒中的应用。
3.根据权利要求2所述的人凝血酶敏感蛋白-1在制备预测肝内胆管癌化疗效果的试剂或试剂盒中的应用,其特征在于,所述的试剂或试剂盒,是通过酶联免疫吸附剂测定技术检测血清中TSP1的表达量。
4.人凝血酶敏感蛋白-1在制备肝内胆管癌化疗预后分型的试剂或试剂盒中的应用。
5.根据权利要求4所述的人凝血酶敏感蛋白-1在制备肝内胆管癌化疗预后分型的试剂或试剂盒中的应用,其特征在于,所述的试剂或试剂盒根据血清中TSP1的表达量高低,将肝内胆管癌患者分为化疗敏感型和化疗耐药型。
6.根据权利要求4所述的人凝血酶敏感蛋白-1在制备肝内胆管癌化疗预后分型的试剂或试剂盒中的应用,其特征在于,所述的化疗为以吉西他滨为主的化疗治疗方案。
7.人凝血酶敏感蛋白-1在制备预测肝内胆管癌化疗后生存期的试剂或试剂盒中的应用。
8.根据权利要求7所述的人凝血酶敏感蛋白-1在制备预测肝内胆管癌化疗后生存期的试剂或试剂盒中的应用,其特征在于,TSP1血清含量高者,化疗病人总生存期更长;TSP1血清含量高者,化疗病人无进展生存期更长。
9.一种预测肝内胆管癌化疗效果的试剂盒,其特征在于,所述的试剂盒,通过酶联免疫吸附剂测定技术检测血清中TSP1的表达量;根据血清中TSP1的表达量高低,将肝内胆管癌患者分为化疗敏感型和化疗耐药型。
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