CN116334220A - 一种外泌体来源的lncRNA在制备甲状腺癌诊断和/或预后的产品中的应用 - Google Patents
一种外泌体来源的lncRNA在制备甲状腺癌诊断和/或预后的产品中的应用 Download PDFInfo
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Abstract
本发明公开了一种外泌体来源的lncRNA在制备甲状腺癌诊断和/或预后的产品中的应用,所述lncRNA为LINC02555,其序列为SEQ ID NO:1。使用该产品检测受试者时,其具有快速、高效的特点,对甲状腺癌的诊断和/或预后评估具有重要价值。
Description
技术领域
本发明属于体外诊断或生物医药领域,具体涉及一种外泌体来源的lncRNA在制备甲状腺癌诊断和/或预后的产品中的应用。
背景技术
甲状腺癌起源于甲状腺滤泡上皮,是全球范围内十分常见的内分泌系统恶性肿瘤,且其发病率呈逐年上升趋势,跃居癌症新发病率第九位,约每20个人中就有1例甲状腺癌。甲状腺癌病因复杂,大部分甲状腺癌起病隐匿,临床表现典型性不足、影像学特征又易与甲状腺良性病变混淆。
甲状腺结节是指由放射线照射等各种原因导致甲状腺内出现一个或多个组织结构异常的团块。甲状腺结节临床极为常见,其多为良性。B超检查是目前检查甲状腺结节的首选手段,根据结节影像学特点可初步判断其性质。但B超检查不能确诊甲状腺癌,当甲状腺癌病变较小时,与结节性甲状腺肿难以区分,给临床和影像诊断带来一定困难。良性甲状腺结节和甲状腺癌的临床处理及预后差异很大,如不能及时准确地鉴别甲状腺结节的良恶性,对甲状腺癌病人就意味着延迟诊断或误诊,从而耽误治疗。
若B超检查结果高度怀疑恶变,则需进行甲状腺细针穿刺与细胞学检查,即FNAC检查,这是目前鉴别甲状腺结节良恶性最可靠的诊断方法。该法不受甲状腺结节大小限制,在超声引导下对结节进行穿刺检查,可重复操作。但该法为有创性检查,同时操作较复杂,病人也需要承受较多心理与生理上的压力,降低了人群普查的依从性。且由于设备的局限及每个医生的穿刺技术水平不一,目前该技术仍未在全国普及。总结分析FNAC出现假阴性的原因主要如下:(1)针吸穿刺和涂片制作的技术原因,包括,结节太小且部位较深,或结节较大但只有部分癌变,导致未采集到有效细胞成分;结节与周围组织分界不清,无法确定肿瘤范围,穿刺可能未真正刺入结节;抽吸组织少,或血液过多致细胞量过少;涂片制片质量差。(2)FNAC对某些病理组织学类型的甲状腺恶性肿瘤诊断较困难。如滤泡型乳头状癌、甲状腺髓样癌及增生结节的鉴别仍较为困难。(3)诊断标准难以掌握,容易造成误诊和漏诊。因此,本领域迫切需要开发一种高灵敏度、特异度的无创甲状腺癌检测方法,以便进行及时、有针对性地治疗,提高患者生存质量与生存率,降低医疗成本,节约资源。
外泌体(Exosome)是一类直径约为40-150nm,具有完整膜结构的细胞外囊泡,其所包含蛋白质、核酸和脂类物质等。现有研究表明外泌体可作为细胞间信息传递的重要媒介,参与到人体内多种病理、生理过程。由于外泌体内部携带的物质被磷脂双分子层包裹,其对于血液环境中的蛋白酶与RNA酶降解有一定的抵御能力,其作为诊断检测靶标具有天然的优势。发现甲状腺癌血浆外泌体来源的分子标志物,进一步建立非侵入性、易操作性、高灵敏性和高特异性的甲状腺癌诊断或检测方法,将有助于甲状腺癌早发现、早诊断和早治疗。
发明内容
为解决现有技术中缺乏非侵入性、易操作性、高灵敏性和高特异性的甲状腺癌诊断或检测方法的问题,本发明提供了一种外泌体来源的lncRNA在制备甲状腺癌诊断和/或预后的产品中的应用,其中所述lncRNA为LINC02555。
在一些实施方案中,所述LINC02555序列为SEQ ID NO:1。
本发明进一步提供了一种试剂盒在制备甲状腺癌诊断和/或预后的产品中的应用,所述包含LINC02555和/或检测LINC02555的试剂;所述LINC02555序列为SEQ ID NO:1。
在一些实施方案中,所述检测LINC02555的试剂包括检测LINC02555的引物对,所述引物对包括检测LINC02555的上游引物和检测LINC02555的下游引物。
在一些具体的实施方案中,所述上游引物为SEQ ID NO:2,所述下游引物为SEQ IDNO:3。
在一些实施方案中,所述试剂盒还包括检测内标的引物对,和/或,检测内标的试剂。
在一些实施方案中,所述诊断的样品源自体液,例如血液。
本发明进一步还提供了一种用于甲状腺癌诊断和/或预后评估的系统,所述系统包括以下模块:
输入模块,其用于输入待测样本数据,所述待测样本数据包括LINC02555检测数值;
分析模块,其用于分析待测样本数据得到分析结果;其中,当所述待测样本数据达到判断条件时,输出分析结果为“阳性”;当所述待测样本数据不满足判断条件时,输出分析结果为“阴性”;
判断模块,所述判断模块根据所述待测样本数据判断样本来源的受试者是否有患甲状腺癌风险,并输出判断结果;其中,当LINC02555的分析结果为“阳性”时,输出判断结果为“有患甲状腺癌风险/预后欠佳”;当分析结果为“阴性”时,输出判断结果为“无患甲状腺癌风险/预后佳”。
在一些具体的实施方案中,所述判断条件为当待测样本中的LINC02555的表达量大于来自正常人的样本中的1.55倍时,判断待测样本数据达到判断条件,输出分析结果为“阳性”;当待测样本中的LINC02555的表达量小于来自正常人的样本中的1.55倍时,判断待测样本数据不满足判断条件,输出分析结果为“阴性”。
本发明进一步提供了一种可读介质,所述可读介质存储了程序,所述程序被处理器执行时可实现前述的用于甲状腺癌诊断和/或预后评估的系统的功能。
本发明提供了一种用于甲状腺癌诊断和/或预后评估的装置,其包括:
(1)前述的可读介质;
(2)处理器,用于执行程序以实现所述甲状腺癌诊断和/或预后评估的系统的功能。
在一些实施方案中,所述装置还包括输出装置,用于输出判断结果。
本发明还提供了前述的系统、前述的可读介质或前述的装置在甲状腺癌诊断和/或预后评估中的应用。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
本发明公开了一种外泌体来源的lncRNA在甲状腺癌诊断和/或预后的产品中的应用,使用该产品检测受试者时,其具有快速、高效的特点,对甲状腺癌的诊断和/或预后评估具有重要价值。
附图说明
图1为LINC02555在健康对照与THCA患者血浆外泌体中的差异表达的情况。
图2为LINC02555临床应用价值的评估。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1基于高通量测序筛选与甲状腺癌相关的外泌体RNA分子标志物
取来自于5例早期甲状腺癌患者和5例对照组健康受试者的血液10mL,分离血浆后利用QIAGEN ExoEasy试剂盒进行外泌体提取,采用QIAGEN miRNeasy mini kit提取RNA,并进行微量RNA建库测序。对测序结果进行生物信息学分析,比较早期甲状腺癌患者中相比于对照组具有差异表达的长链RNA,发现LINC02555在两组中具有显著差异表达。
实施例2基于荧光定量PCR平台的lncRNA检测体系
反转录,按照HiFiScript cDNA第一链合成试剂盒(购自江苏康为世纪生物科技股份有限公司)说明书指示操作,进行反转录合成cDNA。
定量PCR扩增检验,反应体系为:
反应程序为:95℃预变性10min,然后以95℃变性15s、55℃退火30s、72℃延伸30s,共进行38个循环。ABI 7500荧光定量PCR仪器选定熔解曲线程序,在爬坡过程中连续收集样品荧光信号以获得熔解曲线。Real-Time PCR采用2-ΔΔCt法进行相对定量分析。所述LINC02555的上游引物为具体如SEQ ID NO:2所示的核苷酸序列,下游引物具体为如SEQ IDNO:3所示的核苷酸序列。
实施例3利用RNA分子标志物进行甲状腺癌诊断检测
(1)样本采集,收集包括经医院确诊的甲状腺癌患者(40例)、甲状腺结节良性病人和健康人对照样本(合计40例)的血液10mL,分离血浆。
(2)外泌体RNA提取,采用QIAGEN ExoEasy试剂盒进行血浆外泌体分离,分离后的外泌体用QIAGEN miRNeasy mini kit提取外泌体中的RNA,并利用Agilent 2100检测RNA浓度和质量,记录RNA浓度。
(3)荧光定量PCR,采用实施例2中基于荧光定量PCR平台的lncRNA检测体系,对步骤2中提取的血浆外泌体,检测目标RNA。
(4)结果分析
对40例甲状腺癌病人和40例对照样本(健康人和良性病变)的血浆外泌体进行荧光定量PCR检测,结果如图1所示,LINC02555在甲状腺癌患者血浆外泌体中高表达,与高通量测序结果一致。进一步采用ROC曲线评估LINC02555的临床应用价值,具体用AUC(AreaUnder the Curve,ROC曲线下方面积)来表示,AUC值在0.5和1.0之间,AUC越接近于1,表明特异性与灵敏性越好,临床应用价值越大。
本发明依据LINC02555在甲状腺癌中的表达水平,进行ROC曲线分析,如图2所示,ROC曲线分析其AUC数值为0.929。
基于上述发现,推测外泌体LINC02555可为甲状腺癌早期诊断提供新的思路与手段。
Claims (10)
1.一种外泌体来源的lncRNA在制备甲状腺癌诊断和/或预后的产品中的应用,其特征在于,所述lncRNA为LINC02555。
2.如权利要求1所述的应用,其特征在于,所述LINC02555序列如SEQ ID NO:1所示。
3.一种试剂盒在制备甲状腺癌诊断和/或预后的产品中的应用,其特征在于,所述试剂盒包含LINC02555和/或检测LINC02555的试剂;所述LINC02555序列如SEQ ID NO:1所示。
4.如权利要求3所述的应用,其特征在于,所述检测LINC02555的试剂包括检测LINC02555的引物对,所述引物对包括上游引物和下游引物;
较佳地,所述上游引物的核苷酸序列如SEQ ID NO:2所示,所述下游引物的核苷酸序列如SEQ ID NO:3所示。
5.如权利要求3所述的应用,其特征在于,所述试剂盒还包括检测内标的引物对,和/或,检测内标的试剂。
6.如权利要求1-5任一项所述的应用,其特征在于,所述诊断使用的样品源自体液,例如血液。
7.一种用于甲状腺癌诊断和/或预后评估的系统,其特征在于,所述系统包括以下模块:
输入模块,其用于输入待测样本数据,所述待测样本数据包括LINC02555检测数值;
分析模块,其用于分析待测样本数据得到分析结果;其中,当所述待测样本数据达到判断条件时,输出分析结果为“阳性”;当所述待测样本数据不满足判断条件时,输出分析结果为“阴性”;
判断模块,所述判断模块根据所述待测样本数据判断样本来源的受试者是否有患甲状腺癌风险,并输出判断结果;其中,当LINC02555的分析结果为“阳性”时,输出判断结果为“有患甲状腺癌风险/预后欠佳”;当分析结果为“阴性”时,输出判断结果为“无患甲状腺癌风险/预后佳”;
较佳地,所述判断条件为当待测样本中的LINC02555的表达量大于来自正常人的样本中的1.55倍时,判断待测样本数据达到判断条件,输出分析结果为“阳性”;当待测样本中的LINC02555的表达量小于来自正常人的样本中的1.55倍时,判断待测样本数据不满足判断条件,输出分析结果为“阴性”。
8.一种可读介质,其特征在于,所述可读介质存储了程序,所述程序被处理器执行时可实现如权利要求7所述的系统的功能。
9.一种用于甲状腺癌诊断和/或预后评估的装置,其特征在于,所述装置包括:
(1)如权利要求8所述的可读介质;
(2)处理器,用于执行程序以实现所述甲状腺癌诊断和/或预后评估的系统的功能;
优选地,所述装置还包括输出装置,用于输出判断结果。
10.一种如权利要求7所述的系统、权利要求8所述的可读介质或权利要求9所述的装置在甲状腺癌诊断和/或预后评估中的应用。
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