CN110656178A - 胃癌预后诊断标志物trem1的应用及检测试剂盒 - Google Patents
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Abstract
本发明公开了一种胃癌预后诊断标志物TREM1在胃癌预后检测及评估中的应用,涉及生物医学领域,所述应用是通过检测所述标志物TREM1的相对表达水平来评估胃癌患者预后情况。本发明还公开了包括所述标志物TREM1的引物与内参的检测试剂盒。本发明提供的胃癌预后诊断标志物TERM1具有高特异性和敏感性,可通过快速、简便的方法检测其相对表达量、进而对胃癌患者的预后进行准确的判断和评估,从分子水平实现对胃癌危险等级的分级分层,迅速、准确地预测胃癌预后,对不同预后程度的患者进行分类治疗,从而指导胃癌病人的个体化精准治疗,节约医疗成本,具有极高的临床应用价值。
Description
技术领域
本发明涉及生物医学领域,尤其涉及一种胃癌预后诊断标志物TREM1的应用及检测试剂盒。
背景技术
胃癌是一种全球范围内常见的恶性肿瘤,其发病率和死亡率均居全球恶性肿瘤前列。据国际癌症研究机构数据显示,2012年全球胃癌新发病例约95.1万例,其中死亡病例约72.3万例,位于恶性肿瘤发病率第5位、死亡率第3位。同时,胃癌病人在治疗后往往还面临着较差的预后,这也严重威胁着人类健康。中国的胃癌发病数和死亡数分别占全球胃癌发病和死亡的42.6%和45.0%,属于胃癌高发国家。近年来,胃癌临床诊断和治疗方面取得很大的进步,多种治疗和诊断方法也在不断出现,但由于癌症的异质性及个体差异,原发性胃癌切除后易产生局部复发或转移,部分患者术后还会发生原位复发、异位转移和化疗耐药的反应。因此及时判断胃癌患者的病情,了解患者预后生存情况,有助于对不同患者采取个性化治疗方案。所以,寻找简便有效的生物标志物用于胃癌患者的预后诊断对于临床治疗有着十分重要的指导意义。
髓样细胞表达触发受体1(trigger receptors expressed on myeloid cells 1,TREM1)是最早由Bouchon于2000年发现的主要表达于中性粒细胞、CD14+单核/巨噬细胞表面TREM免疫球蛋白超家族激活型受体成员。目前研究表明细菌脂多糖(LPS)及其他微生物等能上调TREM1在细胞表面的表达及其可溶性亚型(sTREM1)的胞外分泌,并通过激活相关炎症细胞上调炎症前化学增活素-白介素-8(interleukin 8,IL-8)、单核细胞趋化蛋白-1(monocyte chemotactic protein 1,MCP-1)、肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)等的分泌,协同Toll样受体(Toll-like receptors,TLR)作用等,在炎症反应发生和级联放大中具有重要作用。TREM1作为肿瘤微环境中炎症、免疫反应中的重要因子,在肿瘤发生、发展中起着重要的桥梁、枢纽作用。
流行病学研究发现可通过抑制癌前病变患者或肿瘤易感者机体慢性炎症反应,降低其罹患肿瘤或肿瘤复发的风险。近年来研究认为,TREM1高表达与炎症相关性肿瘤的发生有着密切关系,其介导的慢性炎症反应会使机体细胞高度增生,进而提高变异几率,最终引发肿瘤。TREM1作为一种新型的炎症激发受体,影响肿瘤发生、转移及预后。TREM1及与其相应的可溶形式sTREM1在患者肿瘤组织、血液和其他体液中的监测也是疾病良恶性鉴别的重要依据,具有重要临床研究价值。
目前已出现一些关于胃癌的预后诊断标志物的相关专利,中国发明专利CN109852696A公开说明了DLC3在制备胃癌预后检测的试剂盒中的用途;中国发明专利CN102998459A公开了通过检测BCL6B基因的抑制表达,来对个体的胃癌进行确诊和个体的胃癌预后进行判断的方法。但TREM1作为胃癌预后诊断标志物的应用尚未见报道。寻找更多高特异性和敏感性的分子标志物对胃癌辅助诊断和预后分析具有重要意义。
因此,本领域的技术人员致力于开发一种具有高特异性和敏感性的胃癌预后诊断标志物TERM1,并能够通过对其相对表达量的检测从分子水平实现对胃癌危险等级的分级分层,迅速、准确地预测胃癌预后,对不同预后程度的患者进行分类治疗,从而指导胃癌病人的个体化精准治疗。
发明内容
有鉴于现有技术的上述缺陷,本发明所要解决的技术问题是如何开发一种具有高特异性和敏感性的胃癌预后诊断标志物TERM1,并能够通过对其相对表达量的检测从分子水平实现对胃癌危险等级的分级分层,迅速、准确地预测胃癌预后,对不同预后程度的患者进行分类治疗,从而指导胃癌病人的个体化精准治疗。
为实现上述目的,本发明提供了一种胃癌预后诊断标志物TREM1在胃癌预后检测及评估中的应用,所述应用是通过检测所述标志物TREM1的相对表达水平来评估胃癌患者预后情况。
进一步地,所述标志物TREM1的相对表达水平的检测方法可以采用qPCR法,具体包括以下步骤:
步骤1、获取胃癌组织样本和对照组织样本,研磨,提取总RNA样品;
步骤2、将所述步骤1制得的所述总RNA样品进行反转录;
步骤3、将所述步骤2得到的反转录产物进行qPCR反应,并计算所述标志物TREM1的相对表达量。
进一步地,所述步骤2中需要先将所述总RNA样品中的基因组DNA去除。
进一步地,所述qPCR反应中需要所述标志物TREM1的引物和内参,其中,所述标志物TREM1的引物可选择DNA序列为5’-TGTGATCTACCAGCCTCCCA-3’的正向引物和DNA序列为5’-GGGGTCCCTGAAAAACCCTT-3’的反向引物;所述内参选择GAPDH时,所述内参的正向引物的DNA序列为5’-CCTCATCTGGGATGCTGAAT-3’,所述内参的反向引物的DNA序列为5’-GCTGCGGGCTCAATTTATAG-3’。
进一步地,所述步骤1中的所述对照组织样本可选择胃癌旁组织。
进一步地,所述步骤1中所述研磨需要的器具需经过液氮预冷,且在所述研磨的过程中需要不断补充液氮保持超低温状态。
进一步地,所述标志物TREM1的相对表达量的计算方法为:TREM1表达量的倍数变化=2-(ΔCt(胃癌组织样本)-ΔCt(对照组织样本)),其中ΔCt(胃癌组织样本)=Ct(胃癌组织样本中的TREM1)-Ct(胃癌组织样本中的内参),ΔCt(对照组织样本)=Ct(对照组织样本中的TREM1)-Ct(对照组织样本中的内参)。
进一步地,所述标志物TREM1的相对表达水平还可采用抗TREM1的特异性抗体进行检测。
本发明还提供了一种胃癌预后诊断标志物TREM1检测试剂盒,所述试剂盒包括所述标志物TREM1的引物和内参。
进一步地,所述试剂盒可用于检测所述标志物TREM1的相对表达量。
与现有技术相比,本发明至少具备以下有益技术效果:
(1)本发明提供的肿瘤微环境相关因子TREM1作为胃癌预后诊断标志物具有极高的特异性和敏感性,其在胃癌肿瘤中呈特异性高表达现象,且表达量高低指示患者预后生存期;
(2)本发明通过对标志物TREM1的相对表达量的检测,可以快速、方便、准确地判断胃癌病人的预后状况,为患者治疗方案的确定提供指导;
(3)本发明提供的标志物TREM1及其应用可从分子水平实现胃癌危险度的分级分期,将不同预后程度的患者进行分类治疗,不仅指导了个体化精准治疗,而且节约了医疗成本,具有较高的临床应用价值。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例的胃癌组之样本和对照组织样本中TREM1的表达量差异数据图;
图2是本发明的一个较佳实施例的胃癌患者TREM1表达量与生存期绘制生存曲线数据图。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例,使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现,本发明的保护范围并非仅限于文中提到的实施例。
(一)利用qPCR法对收集的人胃癌组织及癌旁正常组织进行表达量检测
1、RNA提取
1)胃癌组织及胃癌旁组织研磨
将研磨所需的研钵、研磨杵、剪刀、手术刀、镊子、勺子用超纯水清洗干净,于60℃烘箱烘烤3h后包好锡箔纸,后置于180℃烘箱烘烤过夜;进行组织研磨前上述器材均需要经过液氮预冷;取出保存于液氮的临床样品,切取黄豆大小的组织块放入预冷好的研钵中,研磨,同时不断的少量多次地补充液氮,保持研钵超低温状态,直至研磨至粉状,这个过程一般需要10min-15min;研磨充分后,加入1mL预冷的TRIzoI继续进行研磨,将组织粉末和TRIzol充分混匀;待TRIzol完全融化后转移至做好标记的1.7mL DNase/RNase Free的离心管中,进行RNA抽提,若不马上使用则冻于-80℃超低温冰箱内。
2)总RNA的抽提与质检
采用TRIzol说明书中分离RNA的方法进行总RNA的提取,具体步骤如下:
将装有经过TRIzol裂解的组织的离心管置于室温平衡5min;按每毫升TRIzol加0.2mL氯仿的比例加入氯仿,盖好管盖,上下剧烈震荡15s,之后静置5min,4℃条件下12000×g离心15min;离心过后可看见明显的分层现象,小心吸取上清的无色水相,转移至新的1.7mL DNase/RNase Free离心管中,按0.5mL异丙醇/mL TRIzol的比例加入异丙醇,上下颠倒混匀,在室温条件下静置10min,4℃条件下12000×g离心10min,可见管底有白色总RNA沉淀;小心弃去上清,按每毫升TRIzol加1mL 75%乙醇的比例加入75%乙醇洗涤沉淀,4℃条件下7500×g离心5min;吸去上清,空气干燥5min-10min,加入适量的DNase/RNase-Free无菌水溶解RNA沉淀,之后若不立即使用就保存于-80℃。
提取完成后,使用Nanodrop 2000超微量分光光度计对总RNA进行定量,之后用琼脂糖凝胶电泳检测RNA的完整性,具体步骤如下:
取2μL提取好的总RNA经过适当的稀释后用Nanodrop 2000定量,使用DNase/RNase-Free无菌水作为校准时的溶液。质量较好的总RNA A260/280的比值应该在2.0-2.2之间。配制0.8%的琼脂糖凝胶,RNA上样量500-800ng,电泳缓冲液为0.5×TBE,120V恒压电泳20min,之后在凝胶成像仪上观察。完整的RNA样品在电泳图中应该呈现出3条清晰的条带,由上至下分别代表了28S rRNA、18S rRNA和5S rRNA,其中28S rRNA的条带亮度应是18SrRNA的两倍。
2、总RNA的反转录
使用Takara公司的PrimeScriptTMRT reagent试剂盒进行反转录,包括去除基因组DNA和反转录两个过程,具体步骤如下所示:
1)去除总RNA样品中的基因组DNA,体系配方如下所示:
5×gDNA Eraser Buffer | 2μL |
gDNA Eraser | 1μL |
Total RNA | 1μg |
RNase free water | up to 10μL |
Total volume | 10μL |
置于PCR仪中进行反应,42℃反应2min。
2)反转录反应
将总RNA样品均分至两个PCR管中,在冰上配置mRNA反转录体系,体系配方如下所示:
去除gDNA后的总RNA样品 | 5μL |
PrimeScript RT Enzyme Mix | 0.5μL |
5×PrimeScript Buffer | 2μL |
Oligo dT Primer(50μM) | 0.5μL |
Random 6mers(100μM) | 0.5μL |
RNase Free dH<sub>2</sub>O | 1.5μL |
Total volume | 10μL |
轻柔混匀后立即置于PCR仪中进行反转录反应,反应条件为:45℃,15min;85℃,5sec;4℃,5min。
3、qPCR反应
取0.5μL反转录产物,加至19.5μL RNase free water中,稀释40倍。配置qPCR反应体系,体系如下所示:
2×SsoAdvanced SYBR Green Supermix | 5μL |
cDNA(稀释后) | 1μL |
正向引物(10μM) | 1μL |
反向引物(10μM) | 1μL |
Total volume | 10μL |
充分混匀后瞬离,轻轻弹击管壁以除去气泡,再次离心。之后置于StepOne PlusReal-Time PCR System中,设定程序并进行qPCR反应,反应程序如下:95℃,10s;95℃、3sec,60℃、30sec,共40个循环。
4、qPCR数据分析
qPCR反应完成之后对获得的数据进行相对表达量分析,具体计算过程如下:
ΔCt(胃癌组织样本)=Ct(胃癌组织样本中的TREM1)-Ct(胃癌组织样本中的内参基因GAPDH)
ΔCt(对照组织样本)=Ct(对照组织样本中的TREM1)-Ct(对照组织样本中的内参基因GAPDH)
ΔΔCt=ΔCt(胃癌组织样本)-ΔCt(对照组织样本)
TREM1表达量的倍数变化=2(-ΔΔCt)
结果如图1所示,胃癌组织样本中TREM1的表达量显著高于对照组织正常样本,其差异具有统计学差异(p<0.01)。
(二)结合胃癌患者临床生存信息分析癌症组织中TREM1低表达的临床意义
利用R语言软件(更新版本3.5.2),对上述实验中的qPCR表达值作为参考标准,依据基因表达的中位值,将407例胃癌患者分为TREM1高表达组和TREM1低表达组,并结合其生存期资料,绘制Kaplan-Meier生存曲线,分析结果如图2所示。TREM1低表达胃癌患者的生存期明显长于TREM1高表达胃癌患者的生存期,其差异具有统计学意义(p<0.05),这表明TREM1在胃癌预后评估中具有重要意义,可作为胃癌患者预后标志物。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术无需创造性劳动就可以根据本发明的构思做出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
Claims (10)
1.一种胃癌预后诊断标志物TREM1在胃癌预后检测及评估中的应用,其特征在于,所述应用是通过检测所述标志物TREM1的相对表达水平来评估胃癌患者预后情况。
2.如权利要求1所述的胃癌预后诊断标志物TREM1在胃癌预后检测及评估中的应用,其特征在于,所述标志物TREM1的相对表达水平的检测方法可以采用qPCR法,具体包括以下步骤:
步骤1、获取胃癌组织样本和对照组织样本,研磨,提取总RNA样品;
步骤2、将所述步骤1制得的所述总RNA样品进行反转录;
步骤3、将所述步骤2得到的反转录产物进行qPCR反应,并计算所述标志物TREM1的相对表达量。
3.如权利要求2所述的胃癌预后诊断标志物TREM1在胃癌预后检测及评估中的应用,其特征在于,所述步骤2中需要先将所述总RNA样品中的基因组DNA去除。
4.如权利要求2所述的胃癌预后诊断标志物TREM1在胃癌预后检测及评估中的应用,其特征在于,所述qPCR反应中需要所述标志物TREM1的引物和内参,其中,所述标志物TREM1的引物可选择DNA序列为5’-TGTGATCTACCAGCCTCCCA-3’的正向引物和DNA序列为5’-GGGGTCCCTGAAAAACCCTT-3’的反向引物;所述内参选择GAPDH时,所述内参的正向引物的DNA序列为5’-CCTCATCTGGGATGCTGAAT-3’,所述内参的反向引物的DNA序列为5’-GCTGCGGGCTCAATTTATAG-3’。
5.如权利要求2所述的胃癌预后诊断标志物TREM1在胃癌预后检测及评估中的应用,其特征在于,所述步骤1中的所述对照组织样本可选择胃癌旁组织。
6.如权利要求2所述的胃癌预后诊断标志物TREM1在胃癌预后检测及评估中的应用,其特征在于,所述步骤1中所述研磨需要的器具需经过液氮预冷,且在所述研磨的过程中需要不断补充液氮保持超低温状态。
7.如权利要求2所述的胃癌预后诊断标志物TREM1在胃癌预后检测及评估中的应用,其特征在于,所述标志物TREM1的相对表达量的计算方法为:TREM1表达量的倍数变化=2-(ΔCt(胃癌组织样本)-ΔCt(对照组织样本)),其中ΔCt(胃癌组织样本)=Ct(胃癌组织样本中的TREM1)-Ct(胃癌组织样本中的内参),ΔCt(对照组织样本)=Ct(对照组织样本中的TREM1)-Ct(对照组织样本中的内参)。
8.如权利要求1所述的胃癌预后诊断标志物TREM1在胃癌预后检测及评估中的应用,其特征在于,所述标志物TREM1的相对表达水平还可采用抗TREM1的特异性抗体进行检测。
9.一种胃癌预后诊断标志物TREM1检测试剂盒,其特征在于,所述试剂盒包括所述标志物TREM1的引物和内参。
10.如权利要求9所述的胃癌预后诊断标志物TREM1检测试剂盒,其特征在于,所述试剂盒可用于检测所述标志物TREM1的相对表达量。
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