CN108753980B - 一种甲状腺微小乳头状癌的转移性筛查的筛查试剂盒 - Google Patents
一种甲状腺微小乳头状癌的转移性筛查的筛查试剂盒 Download PDFInfo
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Abstract
本发明公开了一种甲状腺微小乳头状癌的转移性筛查试剂盒以及用途。本发明有效地确定检测对象是否患有转移性甲状腺微小乳头状癌，检测阳性者建议早期手术，若阴性者则可继续观察，避免了甲状腺微小癌的过度治疗和治疗不足，临床应用前景优良。
Description
技术领域
本发明涉及甲状腺微小乳头状癌的检测方法。
背景技术
甲状腺癌发病率在全球范围内快速增长，是年增长率最快的实体恶性肿瘤，预计到2019年，甲状腺癌会成为排名第三的女性常见恶性肿瘤。其中大多数新发病例都是甲状腺微小乳头状癌(PTMC)，即肿瘤直径≤1cm的甲状腺乳头状癌。根据国家的不同，PTMC在新发甲状腺癌中的比例在40％-50％间浮动。目前国内外甲状腺微小乳头状癌患者是否存在过度诊断和治疗是甲状腺外科最热门也是最具有争议的话题之一，但目前国内外尚无方法在手术前精准地判断PTMC的侵袭性及预后，也无法提前预判哪些PTMC会对病人造成较大的危害需及时治疗。
转录组学(RNA-seq)是为了全面准确的鉴定肿瘤中的转录本调控、可变剪接、基因融合等，解析频发基因融合在肿瘤发生、发展过程中的重要作用。通过转录组学这种基于基因表达谱的分子标签，不仅可以辨别细胞的表型归属，还可以用于疾病的诊断。截止目前，经检索还没有甲状腺乳头状癌(PTC)和甲状腺微小乳头状癌(PTMC)(肿瘤直径≤1cm)相关的转录组学的研究及文献报道。转录组学对样本量要求比较低，少量组织即可检测，尤其适合PTMC的研究。
因此，若能找到提前准确预判PTMC高转移性和较差预后的指标，针对侵袭性高的PTMC及时手术治疗以达到最佳预后，对于较为惰性的PTMC，选择观察即可。这样就可以避免PTMC的过度治疗，既可以减轻病人的心理负担，也可以减轻国家社会的经济负担，还可以更合理的利用昂贵且有限的医疗资源。
发明内容
为了解决上述问题，本发明提供了一种确定检测对象(甲状腺微小乳头状癌)是否处于异常状态(高转移性或较差预后)的方法。
本发明发现了一种用于检测甲状腺微小乳头状癌标志物发展进程及预后判断的生物标志物。
本发明提供了一种甲状腺微小乳头状癌的转移性筛查试剂盒，它包括任选的用于检测甲状腺微小乳头状癌组织中Trpc5的表达水平的试剂。
Trpc5：transient receptor potential cation channel subfamily C member5[Homo sapiens(human)]，瞬时受体电位通道5，Ensembl:ENSG00000072315MIM:300334；Vega:OTTHUMG00000022212。
优选地，所述检测甲状腺微小乳头状癌组织中Trpc5表达水平的试剂是PCR检测用试剂。
优选地，所述PCR检测用试剂包含SEQ ID NO.1～2所示引物对。
优选地，所述检测甲状腺微小乳头状癌组织中Trpc5表达水平的试剂是Western-blot检测方法用试剂。
本发明还提供了检测甲状腺微小乳头状癌组织中Trpc5的表达水平的试剂在制备甲状腺微小乳头状癌的转移性筛查试剂中的用途。
优选地，所述检测甲状腺微小乳头状癌组织中Trpc5表达水平的试剂是PCR检测用试剂。
优选地，所述PCR检测用试剂包含SEQ ID NO.1～2所示物对。
优选地，所述检测甲状腺微小乳头状癌组织中Trpc5表达水平的试剂是Western-blot检测方法用试剂。
本发明试剂盒通过检测待检甲状腺微小乳头状癌患者肿瘤组织中Trpc5的表达水平，将高转移性患者筛查出来，针对转移性高的PTMC及时手术治疗以达到最佳预后，对于较为惰性的PTMC，选择观察即可。这样就可以避免PTMC的过度治疗，既可以减轻病人的心理负担，也可以减轻国家社会的经济负担，还可以更合理的利用昂贵且有限的医疗资源。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1Trpc5表达水平检测结果图，a为荧光实时定量PCR检测Trpc5在组织中RNA表达水平检测结果，b为免疫组化的结果。
具体实施方式
实施例1Trpc5的表达水平与甲状腺微小乳头状癌状态的关系
1.病理收集
收集四川大学华西医院2016年12月起至2018年5月进行首次手术的PTMC病例，术前均签署标本留取知情同意书，符合上述条件的均常规收集手术切除的病理组织标本，术中甲状腺癌灶所在腺叶切下15分钟以内取癌灶和正常组织分管装液氮，并详细登记病人资料做好记录，几个月后进行病理检测，确定是淋巴结高转移还是无转移，严格挑选纳入无转移及高转移组的肿瘤组织进行Trpc5的表达水平检测，无转移35例，高转移26例。
样本纳入的标准如下：
①甲状腺左叶或右叶微小乳头状癌(因峡部较薄，故TMC很容易侵出被膜，故峡部癌排除)，彩超提示结节最大径在5-10mm之间；
②非妊娠期或哺乳期女性(因女性发病率明显高于男性，另外为避免性激素等因素的干扰，故转录组测序只纳入了女性病人，男性病人用于后期功能验证)；
②病例均为首次手术。
2.Trpc5的表达水平检测
2.1荧光实时定量PCR检测Trpc5在组织中RNA表达水平检测
取出液氮中保存的组织，冰上放置5分钟使其软化，用组织匀浆器粉碎，称重，每100mg组织加入1ml TRIZOL。每1ml的TRIZOL试剂裂解的样品中加入0.2ml的氯仿，盖紧管盖。手动剧烈振荡管体15秒后，15到30℃孵育2到3分钟。4℃下12000rpm离心15分钟。离心后混合液体将分为下层的红色酚氯仿相，中间层以及无色水相上层。RNA全部被分配于水相中。水相上层的体积大约是匀浆时加入的TRIZOL试剂的60％。将水相上层转移到一干净无RNA酶的离心管中。加等体积异丙醇混合以沉淀其中的RNA，混匀后15到30℃孵育10分钟后，于4℃下12000rpm离心10分钟。此时离心前不可见的RNA沉淀将在管底部和侧壁上形成胶状沉淀块。移去上清液，每1mlTRIZOL试剂裂解的样品中加入至少1ml的75％乙醇(75％乙醇用DEPCH2O配制)，清洗RNA沉淀。混匀后，4℃下7000rpm离心5分钟。小心吸去大部分乙醇溶液，使RNA沉淀在室温空气中干燥5-10分钟。溶解RNA时，先加入无RNA酶的水40μl用枪反复吹打几次，使其完全溶解，获得的RNA溶液保存于-80℃待用。使用Agilent 2100Bioanalyzer(Agilent RNA 6000Nano Kit)检测total RNA的浓度。
样品cDNA合成：采用货号为Femantas K1631的试剂盒合成，逆转录buffer 4μl，随机引物0.2μl，逆转录酶0.5μl，DEPC水13.3μl，RNA模版2μl，总体积20μl。轻弹管底将溶液混合，6000rpm短暂离心。混合液先70℃干浴3分钟，然后37℃水浴60分钟。取出后立即95℃干浴3分钟，得到逆转录终溶液即为cDNA溶液，保存于-80℃待用。
荧光实时定量PCR：反应体系：2×PCR酶Mix10ul,上游引物F 0.5ul，下游引物R0.5ul,加水至总体积20ul。反应步骤：45个PCR循环(94℃ 20秒；60℃5秒)。完成反应后记录Ct值，计算表达水平。采用的试剂盒为bio-rad ssofast evagreen,货号172-5204AP。
Trpc5引物序列如下：
Forward：TCCTGTTTCCCATGCTGTCT；Reverse：GCCCCTGTACATGAAGGTCT。
2.2Western方法(biorad 1620177，Trpc5抗体是购自Novus，货号为nbp2-12919)检测2例高转移组织和3例无转移肿瘤组织中Trpc5的蛋白表达水平。
3 Trpc5的表达水平与甲状腺微小乳头状癌状态的关系
3.1荧光实时定量PCR检测目的基因在组织中RNA表达水平
实验结果如图1和表1所示：
表1组织中Trpc5与甲状腺微小乳头状癌状态的相关性
| Trpc5的表达平均水平 | |
| 甲状腺微小乳头状癌非转移组织 | 0.15copy/ng cDNA |
| 甲状腺微小乳头状癌高转移组织 | 14.45copy/ng cDNA |
如表1和图1a图所示，甲状腺微小乳头状癌患者中，非转移患者的Trpc5的表达水平低，而高转移患者的表达水平显著高。
在实际检测中，若发现待检样本的Trpc5表达水平大于14.45copy/ng cDNA，则建议进行手术，若为0.15copy/ng cDNA～14.45copy/ng cDNA，则建议密切随访复查，监测Trpc5表达水平，必要时手术，若为小于0.15copy/ng cDNA，则建议观察。
3.2Western方法检测2例高转移组织和3例无转移肿瘤组织中Trpc5的蛋白表达水平。
如图1b图所示，甲状腺微小乳头状癌患者中，非转移患者的Trpc5的蛋白表达平均水平低，而高转移患者Trpc5的蛋白表达水平显著高，二者差异显著。
由以上结果可以看出，甲状腺微小乳头状癌非转移患者相比，甲状腺微小乳头状癌高转移患者的Trpc5的RNA表达水平和蛋白表达水平均显著升高，说明甲状腺微小乳头状癌的转移性与肿瘤组织中Trpc5的表达水平呈正相关，Trpc5的高表达会显著提高甲状腺微小乳头状癌高转移性的可能性。
因此，可以通过检测待检甲状腺微小乳头状癌患者肿瘤组织中Trpc5的表达水平，将高转移性患者筛查出来。
实施例2本发明检测Trpc5的试剂盒的组成及其使用方法
一、PCR检测试剂盒
1、试剂盒的组成
检测试剂盒(50人份)：
| 组分 | 体积 |
| 上游引物 | 0.5ul(10μM) |
| 下游引物 | 0.5ul(10μM) |
| 2×PCR酶Mix | 10μL |
| dd water | 至总体积20μL |
Trpc5引物序列如下：
Forward：TCCTGTTTCCCATGCTGTCT
Reverse：GCCCCTGTACATGAAGGTCT。
2、试剂盒的使用方法
取出液氮中保存的组织，冰上放置5分钟使其软化，用组织匀浆器粉碎，称重，每100mg组织加入1ml TRIZOL。每1ml的TRIZOL试剂裂解的样品中加入0.2ml的氯仿，盖紧管盖。手动剧烈振荡管体15秒后，15到30℃孵育2到3分钟。4℃下12000rpm离心15分钟。离心后混合液体将分为下层的红色酚氯仿相，中间层以及无色水相上层。RNA全部被分配于水相中。水相上层的体积大约是匀浆时加入的TRIZOL试剂的60％。将水相上层转移到一干净无RNA酶的离心管中。加等体积异丙醇混合以沉淀其中的RNA，混匀后15到30℃孵育10分钟后，于4℃下12000rpm离心10分钟。此时离心前不可见的RNA沉淀将在管底部和侧壁上形成胶状沉淀块。移去上清液，每1mlTRIZOL试剂裂解的样品中加入至少1ml的75％乙醇(75％乙醇用DEPCH2O配制)，清洗RNA沉淀。混匀后，4℃下7000rpm离心5分钟。小心吸去大部分乙醇溶液，使RNA沉淀在室温空气中干燥5-10分钟。溶解RNA时，先加入无RNA酶的水40μl用枪反复吹打几次，使其完全溶解，获得的RNA溶液保存于-80℃待用。使用Agilent 2100Bioanalyzer(Agilent RNA 6000Nano Kit)检测total RNA的浓度。
样品cDNA合成：采用货号为Femantas K1631的试剂盒合成，逆转录buffer 4μl，随机引物0.2μl，逆转录酶0.5μl，DEPC水13.3μl，RNA模版2μl，总体积20μl。轻弹管底将溶液混合，6000rpm短暂离心。混合液先70℃干浴3分钟，然后37℃水浴60分钟。取出后立即95℃干浴3分钟，得到逆转录终溶液即为cDNA溶液，保存于-80℃待用。
PCR扩增：选择Trpc5特异性引物进行PCR反应。荧光实时定量PCR：完成下列反应体系：2×PCR酶Mix10ul,上游引物F 0.5ul，下游引物R 0.5ul,加水至总体积20ul。反应步骤：45个PCR循环(94℃20秒；60℃5秒)。完成反应后记录Ct值。
综上，本发明试剂盒通过检测待检甲状腺微小乳头状癌患者肿瘤组织中Trpc5的表达水平，将高转移性患者筛查出来，可用于临床甲状腺微小乳头状癌的辅助诊断，为患者采取相关的治疗措施或者决策提供有效的依据，临床应用前景良好。
序列表
<110> 四川大学华西医院
<120> 一种甲状腺微小乳头状癌的转移性筛查的筛查试剂盒
<130> GY026-18P1354
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Trpc5-F（Artificial Sequence）
<400> 1
tcctgtttcc catgctgtct 20
<210> 2
<211> 20
<212> DNA
<213> Trpc5（Artificial Sequence）
<400> 2
gcccctgtac atgaaggtct 20
Claims (2)
1.检测甲状腺微小乳头状癌组织中Trpc5的表达水平的试剂在制备甲状腺微小乳头状癌的转移性筛查试剂中的用途；
所述检测甲状腺微小乳头状癌组织中Trpc5表达水平的试剂是PCR检测用试剂，所述PCR检测用试剂包括检测Trpc5表达的引物对。
2.根据权利要求1所述的用途，其特征在于：所述PCR检测用试剂包含序列如SEQ IDNO.1~2所示的引物对。
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