CN107267642A - Bambi作为鼻咽癌标志物的应用 - Google Patents
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Abstract
本发明公开了BAMBI作为鼻咽癌标志物的应用。申请人及其整个团队经过3年的实验研究，总共对38例鼻咽癌患者、12例慢性鼻咽炎以及49例健康志愿者的进行组织和血清的研究，研究表明：鼻咽癌患者鼻咽组织BAMBI表达量明显增高，而鼻咽炎(鼻咽炎为普通的良性疾病)表达量相对较低，p＝0.0038，两者之间的差异极显著。鼻咽癌患者血清BAMBI明显增高，经统计学分析，t＝2.639，p＝0.012。无论在鼻咽癌组织及鼻咽癌血清中，BAMBI均呈上调表达，因此BAMBI的检测可作为鼻咽癌分子筛查指标。
Description
技术领域
本发明属于检测领域，具体涉及BAMBI作为鼻咽癌标志物的应用。
背景技术
骨形成蛋白和激活素的跨膜抑制剂(BMP and activin membrane—boundinhibitor，BAMBI)是一种跨膜糖蛋白，BAMBI基因是由Onichtchouk等(Onichtchouk D，Chen YG，Dosch R，el a1.Silencing of TGF-beta signalling by the pseudoreceptorBAMBI.Nature，1999，401(6752)：480-485)于1999年发现的、定位在10号染色体p12.3-pl1.2区域，编码产物为一个由260个氨基酸组成的跨膜的糖蛋白，分子量29108kD，又称作伪受体(Pseudoreceptor)，与骨形态发生蛋白(bone morphogenetic protein，BMP)、激活素(activin)等配体的I型受体胞外区都具有相似的结构，BAMBI与TGF-βI型受体竞争结合配体，其表达上调可更多的阻断TGF-β信号，其另一个作用相当于使I型和Ⅱ型受体表达下调或功能丧失，正因为如此，BAMBI因其结构上的特点而广泛参与了TGF-β信号的调控，而TGF-β信号在正常情况下对上皮细胞的增殖起抑制作用，而其表达下调或功能丧失都可能引起癌变，且伴随肿瘤的生长过程。
近年来国内外学者研究了BAMBI与肿瘤的关系，发现在人类某引些癌有有异常高表达，如结肠癌高表达(Sekiya T,Adachi S,Kohu K,et a1.Identification of BMP andactivin membrane-bound inhibitor(BAMB I),an inhibitor of transforming growthfactor-beta signaling,as a target of the beta-catenin pathway in colorectaltumor cells.J Biol Chem.2004,279(8):6840-6846)，非小细胞肺癌高表达(苗参,赵蕾,高建,王辉,崔泽实.BAMBI在非小细胞肺癌中的分布和mRNA表达.中国癌症杂志,2009,12(3):203-207、Sebastian Marwitz,Sofia Depner,Dmytro Dvornikov,etal.Downregulation of the TGFβ Pseudoreceptor BAMBI in Non–Small Cell LungCancer Enhances TGFβ Signaling and Invasion.Cancer Res,2016,76(13):3785–801)，胃癌高表达(Yining Zhang,Zhaojin Yu,Qinghuan Xiao,et al.Expression of BAMBIand its combination with Smad7 correlates with tumor invasion and poorprognosis in gastric cancer.Tumor Biol.2014,35:7047–7056)胰腺癌呈现高表达(王平,郎海波,刘心娟,等.骨形成蛋白和激活素的跨膜抑制剂在胰腺癌中的表达及其意义.中华胰腺病杂志,2015,15(2):78-80)。
上述研究结果显示BAMBI可能与某些癌症相关，但上述研究一般是在研究病变组织标本或培养的癌细胞中发现的，并不是通过检测患者的血清标本发现。事实上，血清标本对癌症的诊断更有意义，因为血清标本易于取材，并且无痛苦，基本上无创伤，所以患者易于接受。而组织取材有一定创伤，患者相对难以接受。然而，血清检查与组织标本的检查存在着差别，有时血清标本检查的结果与组织标本或培养的肿瘤细胞检验结果不一致，限制了BAMBI基因作为标志物用于血清学的基因检测及后续诊断。
鼻咽癌的诊断金标准为病理诊断，对于鼻咽癌检测的标志物多集中于EBV的相关检测。但是，EBV与鼻咽癌的关系并非确切的因果关系，只能是说存在相关性，研究学者发现淋巴瘤、胃癌、肝癌等也与EBV相关，使其用于鼻咽癌的筛查受到一定限制。所以目前鼻咽癌的筛查是通过检测EB病毒抗体(阳性或阴性)而确定鼻咽癌高危人群，再进一步进行鼻咽镜检查并活检而诊断鼻咽癌，起着筛查鼻咽癌作用。EBV的血清检查仍然是鼻咽癌主要的筛查方法。其它确定为鼻咽癌的分子标志物，基本很少用于临床应用。
发明内容
本发明的目的在于提供BAMBI作为鼻咽癌标志物的应用。
本发明所采取的技术方案是：
BAMBI在鼻咽癌辅助诊断或鼻咽癌治疗/预后评估中的应用。
BAMBI在制备鼻咽癌辅助诊断或鼻咽癌治疗/预后评估试剂或试剂盒中的应用。
用于检测BAMBI的试剂在制备鼻咽癌辅助诊断或鼻咽癌治疗/预后评估试剂或试剂盒中的应用。
优选的，用于检测BAMBI的试剂包括用于扩增BAMBI的特异性引物对。
优选的，该特异性引物对序列如下所示：
F：5'-CCAAAGGTGAAATTCGATGCTA3-'(SEQ ID NO：3)；
R：5'-GGTATGGTGGTGCCAGAGTG3-'(SEQ ID NO：4)。
一种用于鼻咽癌辅助诊断或预后评估的试剂盒，该试剂盒中含有能够定量检测BAMBI表达水平的试剂。
优选的，该试剂包括用于扩增BAMBI的特异性引物对。
优选的，该特异性引物对序列如下所示：
F：5'-CCAAAGGTGAAATTCGATGCTA3-'(SEQ ID NO：3)；
R：5'-GGTATGGTGGTGCCAGAGTG3-'(SEQ ID NO：4)。
本发明的有益效果是：
申请人及其整个团队经过3年的实验研究，总共对38例鼻咽癌患者、12例慢性鼻咽炎以及49例健康志愿者的进行组织和血清的研究，研究表明：鼻咽癌患者鼻咽组织BAMBI表达量明显增高，而鼻咽炎(鼻咽炎为普通的良性疾病)表达量相对较低，p＝0.0038，两者之间的差异极显著。鼻咽癌患者血清BAMBI明显增高，经统计学分析，t＝2.639，p＝0.012。无论在鼻咽癌组织及鼻咽癌血清中，BAMBI均呈上调表达，因此BAMBI的检测可作为鼻咽癌分子筛查指标。
本发明公开的鼻咽癌标志物为人骨形成蛋白和激活素的跨膜抑制剂BAMBI。通过定量检测血清中的BAMBI基因的表达量，根据BAMBI基因的表达量，来判断鼻咽癌的情况。也即，仅利用血清标本就可以对鼻咽癌进行筛查，血清标本易于取材，并且无痛苦，基本上无创伤，因此，患者易于接受。而鼻咽癌组织取材有一定创伤和痛苦，患者相对难以接受，而且取材还受到其它因素(如鼻腔疾病使用活检钳难以到达鼻咽部等)限制。
本发明公开的鼻咽癌标志物BAMBI效果优于现有技术中常用的鼻咽癌标志物EBV。本发明的标志物是在明确了鼻咽癌血清及鼻咽组织BAMBI高表达的因果关系基础上得到，可以直接通过检测血清中BAMBI含量而发挥诊断鼻咽癌的作用。
附图说明
图1为BAMBI扩增曲线；
图2为BAMBI溶解曲线。
具体实施方式
下面结合具体实验，进一步阐述本发明内容。下列实验中未注明具体条件的实验方法，通常按照常规条件，如《分子克隆实验指南》(第三版)中所述的条件，或按照制造厂商所建议的条件。
实验1、鼻咽癌鼻咽组织BAMBI表达
(一)材料与方法：
1.临床资料：鼻咽癌患者17例，年龄从22岁到64岁，平均年龄为46.47±11.17岁，男11例，女6例，临床分期：Ⅱ期6例，Ⅲ期6例，Ⅳ期5例，病理分类：分化型非角化性癌10例，未分化型癌7例。慢性鼻咽炎患者12例，年龄从20岁到55岁，平均年龄为34.17±11.25岁，男8例，女4例。
2.RT-PCR检测鼻咽组织BAMBI
取-80℃保存的待检鼻咽组织100mg，使用BioPulverizerTM冰冻粉碎组织，加1ml的RNA抽提试剂Trizol(Invitrogen)，使用Mini-Bead-Beater-16匀浆后抽提RNA。
将总体积20ul多聚A聚合酶和逆转录酶混合后加1ug总RNA进行反转录：37℃反应60min，65℃反应20min，生成cDNA后再进行20倍稀释，取2ul做为模板。采用两步法行Real-time PCR检测。
引物的设计应用软件为Primer5，各个目的基因及引物序列见表1。
表1 PCR引物序列
反应条件为：95℃预变性3min，95℃变性30s，560退火20s，72℃延伸20s，40个循环，β-actin为内参，检测各模板的Ct值(C代表cycle，T代表threshold)。Ct值为每个反应管内荧光信号到达设定的阈值时所经历的循环数，△Ct越小表示其起始拷贝数越多，表达越高。以Folds＝2-△△Ct以表示目的miRNA在实验组与对照组中表达的相对倍比关系。
3.分析、计算、统计
各样品的目的基因(BAMBI)和管家基因(β-actin)分别进行Realtime PCR反应。根据绘制的梯度稀释DNA标准曲线，各样品目的基因和管家基因的浓度结果直接由机器生成。每个样品的目的基因浓度除以其管家基因的浓度，即为此样品此基因的校正后的相对含量。采用RQ＝2-△△CT进行计算相对表达量，荧光定量PCR结果采用t检验进行数据分析。
(二)结果
1.BAMBI扩增曲线见图1。
2.BAMBI溶解曲线见图2。
3.BAMBI在鼻咽癌鼻咽组织中的表达结果见表2。
表2 BAMBI在鼻咽癌和鼻咽组织中的表达
| 样本量 | BAMBI相对表达量 | |
| 鼻咽癌 | 17 | 0.00371±0.00327 |
| 鼻咽炎 | 12 | 0.00052±0.00023 |
从表2结果可知：鼻咽癌患者鼻咽组织BAMBI表达量明显增高，而鼻咽炎(普通的良性疾病)表达量相对较低，p＝0.0038，两者之间的差异极差异。
也即利用检测的BAMBI表达量，可以区分鼻咽癌和普通的良性疾病-鼻咽炎。但是，BAMBI的表达量在鼻咽癌分期中无差异。
实验2、鼻咽癌血清BAMBI表达
(一)材料与方法：
1.临床资料：鼻咽癌患者21例，年龄从30岁到68岁，平均年龄为49.7±12.3岁，男15例，女6例，临床分期：Ⅱ期6例，Ⅲ期11例，Ⅳ期4例，病理分类：分化型非角化性癌16例，未分化型癌5例。健康志愿者49例，年龄从20岁到66岁，平均年龄为48.3±12.5岁，男20例，女10例。
2.研究对象于上午8-9点抽取静脉血，静置30分钟后离心取血清，保存于-80度待测。
3.试剂及仪器：人BAMBI ELISA试剂盒：E-14444(上海恒远生物科技有限公司)，酶标仪：芬兰(Labsystems Multiskan MS)，仪器型号：352型，洗板机：芬兰(ThermoLabsystems)：仪器型号：AC8，离心机：微量高速离心机(国产)：仪器型号：TG16W。培养箱：隔水式恒温培养箱(国产)：仪器型号：GNP-9080型。
4.采用ELISA法检测血清BAMBI含量，方法如下：
常规设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μL；样本孔中加入待测样本50μL；空白孔不加。除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶(HRP)标记的检测抗体100μL，用封板膜封住反应孔，37℃水浴锅或恒温箱温育60min。弃去液体，吸水纸上拍干，每孔加满洗涤液(350μL)，静置1min，甩去洗涤液，吸水纸上拍干，如此重复洗板5次(也可用洗板机洗板)。每孔加入底物A、B各50μL，37℃避光孵育15min。每孔加入终止液50μL，15min内，在450nm波长处测定各孔的OD值。
(二)结果
鼻咽癌血清BAMBI表达见表3。
表3 BAMBI在鼻咽癌和鼻咽血清中的表达量
| 样本量 | BAMBI(pg/ml) | |
| 鼻咽癌 | 21 | 480.95±130.06 |
| 鼻咽炎 | 49 | 393.07±121.96 |
表3结果显示与鼻咽炎患者相比较，鼻咽癌患者血清BAMBI明显增高，经统计学分析，t＝2.639，p＝0.012。
综上研究显示，无论在鼻咽组织及鼻咽癌血清中，BAMBI均呈上调表达，因此BAMBI的检测可作为鼻咽癌分子筛查指标。
SEQUENCE LISTING
<110> 周小军
<120> BAMBI作为鼻咽癌标志物的应用
<130>
<160> 4
<170> PatentIn version 3.5
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<211> 20
<212> DNA
<213> 人工序列
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gtggccgagg actttgattg 20
<210> 2
<211> 23
<212> DNA
<213> 人工序列
<400> 2
cctgtaacaa cgcatctcat att 23
<210> 3
<211> 22
<212> DNA
<213> 人工序列
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ccaaaggtga aattcgatgc ta 22
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<400> 4
ggtatggtgg tgccagagtg 20
Claims (8)
1.BAMBI在鼻咽癌辅助诊断或鼻咽癌治疗/预后评估中的应用。
2.BAMBI在制备鼻咽癌辅助诊断或鼻咽癌治疗/预后评估试剂或试剂盒中的应用。
3.用于检测BAMBI的试剂在制备鼻咽癌辅助诊断或鼻咽癌治疗/预后评估试剂或试剂盒中的应用。
4.根据权利要求3所述的应用，其特征在于：用于检测BAMBI的试剂包括用于扩增BAMBI的特异性引物对。
5.根据权利要求4所述的应用，其特征在于：该特异性引物对序列如下所示：
F：5'-CCAAAGGTGAAATTCGATGCTA3-'；
R：5'-GGTATGGTGGTGCCAGAGTG3-'。
6.一种用于鼻咽癌辅助诊断或预后评估的试剂盒，其特征在于：该试剂盒中含有能够定量检测BAMBI表达水平的试剂。
7.根据权利要求6所述的试剂盒，其特征在于：该试剂包括用于扩增BAMBI的特异性引物对。
8.根据权利要求7所述的试剂盒，其特征在于：该特异性引物对序列如下所示：
F：5'-CCAAAGGTGAAATTCGATGCTA3-'；
R：5'-GGTATGGTGGTGCCAGAGTG3-'。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108490178A (zh) * | 2018-02-13 | 2018-09-04 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | 用于npc诊断及预后预测的标志物及其应用 |
| CN109628590A (zh) * | 2018-11-21 | 2019-04-16 | 南方医科大学 | 锌指蛋白znf146的用途 |
| CN116694768A (zh) * | 2023-06-27 | 2023-09-05 | 清远市人民医院 | 人类白细胞抗原hla-b*5801基因在鼻咽癌易感性筛查中的应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1482256A (zh) * | 2002-09-13 | 2004-03-17 | 中南大学 | 鼻咽癌分子标志物—brd7试剂盒 |
| US20070065446A1 (en) * | 2003-05-28 | 2007-03-22 | Scimedia Ltd. | Anti-bambi antibody and diagnostic or remedy for colon cancer and liver cancer |
| US20100125042A1 (en) * | 2008-05-15 | 2010-05-20 | The Regents Of The University Of California | Peripheral gene expression biomarkers for autism |
-
2017
- 2017-07-28 CN CN201710629354.6A patent/CN107267642B/zh not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1482256A (zh) * | 2002-09-13 | 2004-03-17 | 中南大学 | 鼻咽癌分子标志物—brd7试剂盒 |
| US20070065446A1 (en) * | 2003-05-28 | 2007-03-22 | Scimedia Ltd. | Anti-bambi antibody and diagnostic or remedy for colon cancer and liver cancer |
| US20100125042A1 (en) * | 2008-05-15 | 2010-05-20 | The Regents Of The University Of California | Peripheral gene expression biomarkers for autism |
Non-Patent Citations (3)
| Title |
|---|
| LIU KAI等: "Knockdown of BAMBI inhibits beta-catenin and transforming growth factor beta to suppress metastasis a gastric cancer cells", 《MOLECULAR MEDICINE REPORTS》 * |
| SEBASTIAN MARWITZ等: "Downregulation of the TGFb Pseudoreceptor BAMBI in Non–Small Cell Lung Cancer Enhances TGFb Signaling and Invasion", 《MOLECULAR AND CELLULAR PATHOBIOLOGY》 * |
| 关旭等: "Smad7、BAMBI在肿瘤中的作用及意义", 《中华结直肠疾病电子杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108490178A (zh) * | 2018-02-13 | 2018-09-04 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | 用于npc诊断及预后预测的标志物及其应用 |
| CN109628590A (zh) * | 2018-11-21 | 2019-04-16 | 南方医科大学 | 锌指蛋白znf146的用途 |
| CN116694768A (zh) * | 2023-06-27 | 2023-09-05 | 清远市人民医院 | 人类白细胞抗原hla-b*5801基因在鼻咽癌易感性筛查中的应用 |
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