CN107937521B - 用于检测肾上腺皮质癌的试剂盒 - Google Patents
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Abstract
本发明涉及检测领域，具体涉及一种肾上腺皮质癌检测试剂盒。该试剂盒特异性检测STX10的表达量，进而判断是否换有肾上腺皮质癌。因此，本发明的这一发现将为肾上腺皮质癌的诊断提供一条全新的途径。而且本发明的诊断准确性也高，具有较好的应用前景。
Description
技术领域
本发明涉及检测技术领域，具体涉及一种用于检测肾上腺皮质癌的试剂盒。
背景技术
肾上腺皮质癌(adrenocortical carcinoma，ACC)是一罕见的恶性肿瘤，其年发病率约为1-2/100万，约占所有恶性肿瘤的0.12％。从婴幼儿到80岁以上的老年人均可患此病，平均发病年龄为44岁。在年龄分布上肾上腺皮质癌有两个高发年龄段：一是小于5岁的幼儿，二是40-69岁年龄段的成年人。肾上腺皮质癌发病率无明显性别差异，男女发病比例约为1:1.3。患者多数为单侧肾上腺发病，双侧同时发病的仅占2-6％。
肾上腺皮质癌50％为功能性，即发生了内分泌功能改变，表现为皮质醇增多症、原发性醛固酮增多症、肾上腺嗜铬细胞瘤等，其中皮质醇增多症常见，醛固酮增多症较少出现，而其余患者均为无功能性。
目前肾上腺皮质癌诊断主要有四种方式：激素水平检测：功能性肾上腺皮质癌患者中，约有30％的患者会出现血浆皮质醇升高，20％出现雄激素水平升高，10％发生雌激素水平升高，1-2％有血浆醛固酮水平升高。因此可通过相关激素水平的检测来辅助诊断肾上腺皮质癌。影像学诊断：目前普遍认为，CT检查是诊断肾上腺皮质癌的首选影像学诊断手段。其中肿瘤大小是诊断肾上腺皮质癌最重要的指标，其恶性的可能性与大小成一定的正相关，但并不是绝对可靠。大于4cm的肾上腺皮质肿瘤，CT诊断为肾上腺皮质癌的敏感性为96％，特异性为51％；大于6cm者，CT诊断肾上腺皮质癌的敏感性为90％，特异性为78％；大于8cm者，敏感性为77％，特异性为93％。CT表现的特点有：瘤体大、边缘不规则、密度不均匀、出血、坏死等，均为肾上腺皮质癌的恶性征象。中央坏死和钙化在肾上腺皮质癌中很常见，约20％的病例出现钙化。MRI可多平面成像，对肾上腺皮质癌的定位，观察肿瘤与邻近器官的关系以及静脉受累情况优于CT，对评价肾上腺皮质癌的静脉浸润很有价值。瘤体较大尤其是右侧的肾上腺皮质癌患者应行MRI检查有无下腔静脉瘤栓，因为9-19％的病例可能合并下腔静脉转移。病理诊断：Weiss等提出肾上腺皮质癌的9项病理特征，分别为：核异型性大；核分裂数>6/50HPF；异常核分裂；具有嗜酸性胞质的瘤细胞占全部细胞75％以上；瘤细胞呈弥漫性分布≥33％；坏死；静脉侵犯；窦隙状结构浸润；包膜浸润。以上9项中只要具备3项即可作出肾上腺皮质癌的诊断；同时，核分裂像、不典型分裂、静脉侵犯是最重要的3项诊断依据。细针抽吸活组织检查(FNA)已应用于诊断肾上腺肿物的性质，但也有一定风险性、有争议：其缺点是假阴性率高，肿瘤穿刺道种植出血，如果肿瘤为嗜铬细胞瘤可致严重的血液动力学改变。只有当高度怀疑肾上腺皮质癌远处转移或肾上腺转移癌、与临近器官肿瘤鉴别困难、无法手术切除，同时排除嗜铬细胞瘤时才考虑应用细针抽吸活组织检查。除此之外，均需首选手术切除活检。
在CN103417986A中公开了通过体外实验研究MTDH对肾上腺皮质癌细胞增殖的影响，降低MTDH的表达量可抑制肾上腺皮质癌细胞系H295R增殖。同时MTDH在原发性醛固酮增多症腺瘤中表达量明显升高。因此，MTDH基因5对于肾上腺皮质癌细胞的增殖有调控作用，可用于制备筛选肾上腺皮质癌或肾癌的药物。其不涉及对癌的诊断。而浆膜融合蛋白质STX10，在细胞的发育过程中起着较强的作用，目前该基因与肾上腺皮质癌的关系尚不清楚。
而现有技术的肾上腺皮质癌检测都比较复杂，而且准确性方面也需要改进，因此，在本领域具有迫切的研发需求。
发明内容
本发明目的是，提供STX10在制备肾上腺皮质癌临床诊断试剂或试剂盒中的应用。
所述的STX10在制备肾上腺皮质癌临床诊断试剂或试剂盒中的应用，是以STX10为靶标，设计特异性的荧光定量PCR引物，建立检测STX10的定量方法及配套的试剂或试剂盒。
所述的检测STX10的定量的方法及配套的试剂或试剂盒，具体是体外检测STX10的表达是否异常。
本发明另外一个显著的进步在于：改进现有STX10基因mRNA荧光定量RT-PCR技术，使之具有特异、灵敏、稳定、操作简便等的特点，提出一种质量控制系列方法的组合，这一系列质控方法的组合消除了STX10基因mRNA荧光定量RT-PCR检测的假阳性和假阴性，提高了该检测方法的准确性和在临床实验诊断的实用性。
本发明另外提供一宗荧光定量PCR的引物对，其上游引物:GCCCGCGGGCTGTACCAGCG(SEQ ID NO:1)；下游引物:TCCTGCAGGTCCCCGGCTGG(SEQ ID NO:2)；扩增大小200bp。本发明另外提供内参基因GAPDH的SYBRGreen荧光定量RT-PCR检测引物，上游引物：CTCCTCCTGTTCGACAGTCAGC(SEQ ID NO:3)；下游引物：CCCAATACGACCAAATCCGTT(SEQ ID NO:4)。
本发明另外提供一种用于肾上腺皮质癌诊断的的检测试剂盒，其特征在于，所述试剂盒包含外周血总RNA提取试剂、逆转录PCR的反应液、实时荧光定量RT-PCR反应液，所述实时荧光定量RT-PCR反应液包含目的基因标志物及内参基因GAPDH的检测引物，所述引物序列如SEQ ID NO:1-4所示的序列。
鉴于到目前为止，尚未有关STX10与肾上腺皮质癌的任何相关性报道。因此，本发明的这一发现将为肾上腺皮质癌的诊断提供一条全新的途径。而且本发明的诊断准确性也高，具有较好的应用前景。
附图说明
图1为实时荧光定量PCR扩增曲线图，左边为癌组织，右边为非癌组织。
图2是STX10在健康人与癌患者中表达情况图。
具体实施方式
本发明提供的具体实施方式作详细说明，但本发明的实施并不仅限于此。
下列实施例中未注明具体条件的实验方法，通常按照本领域常规的所述常规条件，或试剂制造厂商所建议的条件实施。
实施例1 STX10在肾上腺皮质癌组织芯片中的免疫组织化学分析
在前期细胞学研究的基础上，为证实STX10在肾上腺皮质癌癌组织和癌旁组织之间的表达差异，随机选取30对肾上腺皮质癌的癌组织和对应的癌旁组织样本进行免疫组织化学研究，其中选取的样本来自苏州大学第一附属医院。
免疫组织化学研究的实验过程及结果判定：
实验过程：
1、人类肾上腺皮质癌组织芯片(制备方式参考文献：“应用组织芯片技术研究A103和Inhibinα在肾上腺皮质腺瘤中的表达”，也可以采用商业购买方式)用含10％的山羊血清的PBS室温封闭20分钟；
2、加入鼠抗人STX10抗体(购自深圳市豪地华拓生物科技有限公司，货号：PL0402920 1:200稀释)，4度过夜，PBS洗3次，每次5分钟；
3、加入辣根过氧化物酶标记的山羊抗鼠的抗体(购自SIGMA公司，1:1000稀释)，室温孵育30分钟，PBS洗3次，每次4分钟；
4、二氨基联苯胺(DAB)(购自Sigma公司)显色；
5、苏木精-伊红(购自Sigma公司)染色20秒；
6、组织切片依次使用：50％乙醇、70％乙醇、80％乙醇、90％乙醇、100％乙醇、二甲苯/乙醇(1:1)、二甲苯，进行脱水透明处理；
7、使用中性树脂封片，于显微镜观察。
免疫组化结果为STX10在肾上腺皮质癌癌组织中呈阳性，在癌旁组织中呈弱阳性。
根据染色强度和阳性率对组织芯片进行打分评估，染色强度打分标准为：阴性为0分、弱阳性为1分、中等阳性为2分、强阳性为3分；阳性率打分标准为：低于5％为0分，5％_30％为1分，31-60％为2分，60％以上为3分。将染色强度的得分与阳性率的得分相加，获得组织切片的综合得分，得分结果为：癌组织的平均综合得分为6；癌旁组织的平均综合得分为3.23，两者具有极显著差异，P值为0.0001。统计发现，在超过96％的样本对中，STX10在肾上腺皮质癌组织中显著上调表达。
由上述可知，STX10在肾上腺皮质癌组织和癌旁组织中存在明显的差异表达，显然与肾上腺皮质癌的发生发展有着密切的相关性，因此它的表达量可以用于检测肾上腺皮质癌。相应的，特异性的扩增STX10的引物，由于其能够用于检测STX10的表达量，因而可以用于检测肾上腺皮质癌，或者用于制备检测肾上腺皮质癌的试剂或试剂盒，这对本领域人员来说是显而易见的。
实施例2实时荧光定量PCR用于检测STX10的表达量
1、核酸抽提(癌组织和正常组织2对):
使用Invitrogen Trizol RNA提取试剂盒。按照说明书进行操作，最后用30μLDEPC水溶解，之后立即进行逆转录。
2、逆转录:
使用QIAGENReverse Transcription试剂盒。按照说明书进行操作。
3、SYBR Green I实时荧光定量PCR对逆转录产物扩增
5μL逆转录产物与5μL DEPC水[作为PCR无模板对照(PCR-NTC)]分别加入FastStart Universal SYBR Green Master(Rox)10μL、DEPC水5μL、PCR上下游引物各1uL(SEQ ID NO：1-4)；然后用实时荧光定量PCR检测。检测参数为95℃1Omin；95℃15s,62℃lmin，45个循环；绘制熔解曲线。结果分析:反应结束后打开Analysis窗口，选择样本孔,点击Results自动分析结果。通过结果发现(图1)：Real-Time PCR反应后溶解曲线显示阴性对照样品无扩增峰；肿瘤组织和癌旁组织样品溶解曲线均仅有单峰，表明扩增产物单一，上述样品的Ct值分别为：
肿瘤组织样品1 Ct值27.4075
癌旁组织样品1 Ct值31.2932
肿瘤组织样品2 Ct值27.5523
癌旁组织样品2 Ct值31.3855
肿瘤组织中STX10的mRNA表达显著高于对应的癌旁组织。
实施例3 STX10表达对肾上腺皮质癌的辅助诊断
将优化的试剂盒用于肾上腺皮质癌的辅助诊断。其中肾上腺皮质癌标本来自苏州大学第一附属医院经病理确诊的30例女性癌患者，平均年龄55岁。健康人标本来自同院同期30名女性健康体检者，年龄54岁。
用SYBR Green I实时荧光定量PCR检测癌患者/健康人STX10的相对表达量为分别为20.83±18.18和9.33±4.44(见图2)。两组表达量经检验，之间差异有统计学意义(P<0.01)。以STX10的相对表达量16.48作为临界值时，本方法对肾上腺皮质癌诊断的敏感度为90.0％(27/30)，特异度为95.0％(57/60)。具有较好的辅助诊断效果。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
序列表
<110> 苏州立豪生物科技有限公司
<120> 用于检测肾上腺皮质癌的试剂盒
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
gcccgcgggc tgtaccagcg 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
tcctgcaggt ccccggctgg 20
<210> 3
<211> 22
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
ctcctcctgt tcgacagtca gc 22
<210> 4
<211> 21
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
cccaatacga ccaaatccgt t 21
Claims (4)
1.STX10在制备用于检测肾上腺皮质癌的试剂中的用途。
2.如权利要求1所述的用途，其特征在于，根据STX10设计的引物对SEQ ID NO：1-2用于制备检测试剂。
3.权利要求1-2任一所述的用途，其特征在于：所述试剂为试剂盒或基因芯片。
4.SEQ ID NO：1-4所示的引物组在制备检测肾上腺皮质癌的试剂盒中的用途。
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| CN1852974A (zh) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | 用于治疗和诊断癌症的组合物和方法 |
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