CN111893112A - 胃癌肿瘤标志物及其应用 - Google Patents
胃癌肿瘤标志物及其应用 Download PDFInfo
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- CN111893112A CN111893112A CN202010807781.0A CN202010807781A CN111893112A CN 111893112 A CN111893112 A CN 111893112A CN 202010807781 A CN202010807781 A CN 202010807781A CN 111893112 A CN111893112 A CN 111893112A
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- gastric cancer
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Abstract
本发明提供了一种胃癌肿瘤标志物，胃癌肿瘤标志物的氨基酸序列如SEQ ID NO:2所示。较佳地，胃癌肿瘤标志物的核苷酸序列如SEQ ID NO:1所示。还提供了一对上述的胃癌肿瘤标志物的应用。还提供了一对上述的胃癌肿瘤标志物的检测引物对及其应用。还提供了一种上述的胃癌肿瘤标志物的抑制剂及其应用。本发明的胃癌肿瘤标志物能够使得胃癌诊断简便准确快速，提高胃癌早期诊断水平，适于大规模推广应用。
Description
技术领域
本发明涉及生物医药技术领域，更具体地，涉及肿瘤标志物技术领域，特别是指一种胃癌肿瘤标志物及其应用。
背景技术
胃癌(gastric carcinoma)是起源于胃黏膜上皮的恶性肿瘤，在我国各种恶性肿瘤中发病率居首位，胃癌发病有明显的地域性差别，在我国的西北与东部沿海地区胃癌发病率比南方地区明显为高。好发年龄在50岁以上，男女发病率之比为2：1。由于饮食结构的改变、工作压力增大以及幽门螺杆菌的感染等原因，使得胃癌呈现年轻化倾向。胃癌可发生于胃的任何部位，其中半数以上发生于胃窦部，胃大弯、胃小弯及前后壁均可受累。绝大多数胃癌属于腺癌，早期无明显症状，或出现上腹不适、嗳气等非特异性症状，常与胃炎、胃溃疡等胃慢性疾病症状相似，易被忽略，因此，目前我国胃癌的早期诊断率仍较低。胃癌的预后与胃癌的病理分期、部位、组织类型、生物学行为以及治疗措施有关。
胃癌肿瘤标记物，临床上最为常见的是CEA，就是癌胚抗原的测定。也包括其他相关的肿瘤标记物，包括系列CA199、CA125以及相关的糖类抗原。在胃癌患者肿瘤标记物当中，常见的标记物指标会明显升高，在正常人群当中，肿瘤标记物的浓度很低，经常表现为阴性。当患有胃癌肿瘤比较明显或发展到中晚期时，肿瘤标记物的测值会出现免疫性的反应，表现为明显的升高。胃癌的患者也会产生相应的胃肠道症状，出现反酸以及腹部胀痛、消瘦、乏力，甚至呕血、黑便等症状，结合肿瘤标记物，基本能够初步怀疑或定诊胃癌的可能性。因此，发现灵敏度和特异性高的新的胃癌肿瘤标志物将是提高胃癌早期诊断水平的关键。
因此，希望提供一种胃癌肿瘤标志物，其能够使得胃癌诊断简便准确快速，提高胃癌早期诊断水平。
发明内容
为了克服上述现有技术中的缺点，本发明的一个目的在于提供一种胃癌肿瘤标志物，其能够使得胃癌诊断简便准确快速，提高胃癌早期诊断水平，适于大规模推广应用。
本发明的另一目的在于提供一对胃癌肿瘤标志物的检测引物对，其能够简便快速准确地检测胃癌肿瘤标志物，提高胃癌早期诊断水平，适于大规模推广应用。
本发明的另一目的在于提供一种胃癌肿瘤标志物的抑制剂，其能够抑制胃癌肿瘤标志物的表达，进而抑制胃癌肿瘤细胞的生长，适于大规模推广应用。
本发明的另一目的在于提供一种胃癌肿瘤标志物在作为胃癌诊断试剂中的应用，其能够使得胃癌诊断简便准确快速，提高胃癌早期诊断水平，适于大规模推广应用。
本发明的另一目的在于提供一种胃癌肿瘤标志物的检测引物对在作为胃癌诊断试剂中的应用，其能够简便快速准确地检测胃癌肿瘤标志物，提高胃癌早期诊断水平，适于大规模推广应用。
本发明的另一目的在于提供一种胃癌肿瘤标志物的抑制剂在作为胃癌治疗药物中的应用，其能够抑制胃癌肿瘤标志物的表达，进而抑制胃癌肿瘤细胞的生长，适于大规模推广应用。
为达到以上目的，在本发明的第一方面，提供了一种胃癌肿瘤标志物，其特点是，所述胃癌肿瘤标志物的氨基酸序列如SEQ ID NO:2所示。
较佳地，所述胃癌肿瘤标志物的核苷酸序列如SEQ ID NO:1所示。
在本发明的第二方面，提供了一对上述的胃癌肿瘤标志物的检测引物对，其特点是，所述的胃癌肿瘤标志物的检测引物对具有如SEQ ID NO:3所示的核苷酸序列和SEQ IDNO:4所示的核苷酸序列。
在本发明的第三方面，提供了一种上述的胃癌肿瘤标志物的抑制剂，其特点是，所述的胃癌肿瘤标志物的抑制剂具有如SEQ ID NO:5所示的核苷酸序列。
在本发明的第四方面，提供了一种上述的胃癌肿瘤标志物的抑制剂，其特点是，所述的胃癌肿瘤标志物的抑制剂具有如SEQ ID NO:6所示的核苷酸序列。
在本发明的第五方面，提供了上述的胃癌肿瘤标志物在作为胃癌诊断试剂中的应用。
在本发明的第六方面，提供了上述的胃癌肿瘤标志物的检测引物对在作为胃癌诊断试剂中的应用。
在本发明的第七方面，提供了上述的胃癌肿瘤标志物的抑制剂在作为胃癌治疗药物中的应用。
本发明的有益效果在于：
a.本发明的胃癌肿瘤标志物的氨基酸序列如SEQ ID NO:2所示，其能够使得胃癌诊断简便准确快速，提高胃癌早期诊断水平，适于大规模推广应用。
b.本发明的上述的胃癌肿瘤标志物的检测引物对具有如SEQ ID NO:3所示的核苷酸序列和SEQ ID NO:4所示的核苷酸序列，其能够简便快速准确地检测胃癌肿瘤标志物，提高胃癌早期诊断水平，适于大规模推广应用。
c.本发明的上述的胃癌肿瘤标志物的抑制剂具有如SEQ ID NO:5所示的核苷酸序列，或者具有如SEQ ID NO:6所示的核苷酸序列，其能够抑制胃癌肿瘤标志物的表达，进而抑制胃癌肿瘤细胞的生长，适于大规模推广应用。
d.本发明的上述的胃癌肿瘤标志物在作为胃癌诊断试剂中的应用，其能够使得胃癌诊断简便准确快速，提高胃癌早期诊断水平，适于大规模推广应用。
e.本发明的上述的胃癌肿瘤标志物的检测引物对在作为胃癌诊断试剂中的应用，其能够简便快速准确地检测胃癌肿瘤标志物，提高胃癌早期诊断水平，适于大规模推广应用。
f.本发明的上述的胃癌肿瘤标志物的抑制剂在作为胃癌治疗药物中的应用，其能够抑制胃癌肿瘤标志物的表达，进而抑制胃癌肿瘤细胞的生长，适于大规模推广应用。
本发明的这些和其它目的、特点和优势，通过下述的详细说明，附图和权利要求得以充分体现，并可通过所附权利要求中特地指出的手段、装置和它们的组合得以实现。
附图说明
图1是采用定量RT-PCR检测AGR3基因在8例胃癌病人癌组织和癌旁组织中的表达情况示意图，其中，“Non-Cancer”指癌旁组织，“Cancer”指胃癌组织。
图2是采用定量RT-PCR检测AGR3基因在胃癌细胞株中的表达情况示意图，其中：****，p<0.0001。
图3是采用定量RT-PCR检测AGR3基因在敲减和过表达AGR3基因的胃癌细胞系AGS和NCI-N87中的表达情况示意图，其中：****，p<0.0001。
图4是敲减和过表达AGR3基因的胃癌细胞系AGS和NCI-N87的细胞增殖能力变化示意图。
具体实施方式
为了使得胃癌诊断简便准确快速，提高胃癌早期诊断水平，本发明提供了一种胃癌肿瘤标志物，所述胃癌肿瘤标志物的氨基酸序列如SEQ ID NO:2所示。
所述胃癌肿瘤标志物的核苷酸序列可以根据需要确定，较佳地，所述胃癌肿瘤标志物的核苷酸序列如SEQ ID NO:1所示。该基因即为AGR3基因，anterior gradient 3,protein disulphide isomerase family member[Homo sapiens(human)]。
本发明还提供了一对上述的胃癌肿瘤标志物的检测引物对，所述的胃癌肿瘤标志物的检测引物对具有如SEQ ID NO:3所示的核苷酸序列和SEQ ID NO:4所示的核苷酸序列。
本发明还提供了一种上述的胃癌肿瘤标志物的抑制剂，所述的胃癌肿瘤标志物的抑制剂具有如SEQ ID NO:5所示的核苷酸序列。
本发明还提供了一种上述的胃癌肿瘤标志物的抑制剂，所述的胃癌肿瘤标志物的抑制剂具有如SEQ ID NO:6所示的核苷酸序列。
本发明还提供了上述的胃癌肿瘤标志物在作为胃癌诊断试剂中的应用。
本发明还提供了上述的胃癌肿瘤标志物的检测引物对在作为胃癌诊断试剂中的应用。
本发明还提供了上述的胃癌肿瘤标志物的抑制剂在作为胃癌治疗药物中的应用。
为了能够更清楚地理解本发明的技术内容，特举以下实施例详细说明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:ColdSpring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1定量RT-PCR检测AGR3基因在胃癌组织中的表达情况
1、组织分离：实验用组织来源于原发性胃癌的手术病人。手术切除的胃脏一经离体，迅速切取病灶及周围5公分外癌旁组织，放入液氮中(-80℃)保存。癌和癌旁的诊断均以病理诊断为最终依据。
2、核糖核酸(RNA)的抽提：用Trizol试剂盒(Invitrogen)提取总RNA，提取方法参照Trizol试剂盒说明书。当准备使用RNA时，可以使用下列方法沉淀RNA：加入NaAc至0.3M，12,000×g离心5分钟。
3、cDNA的合成：
(1)冰浴离心管里面加入模板RNA4μL(0.5μg/μL)，20pmol/μL的随机引物2μL，去离子水5μL，混匀，离心3-5秒；
(2)70℃水浴5分钟，冰浴30秒；
(3)加入5×M-MLV逆转录酶反应液4μL，RNase抑制剂1μL，dNTP 2μL(100mM)，混匀；
(4)37℃水浴5分钟，加入1μL M-MLV反转录酶(200酶活性单位/μL)，混匀；
(5)37℃水浴1小时；
(6)70℃，10分钟结束反应，产物置冰上进行下一步PCR实验，余下的-70℃保存。
4、定量RT-PCR的引物设计及扩增
根据SEQ ID NO:1所示的AGR3基因序列设计引物对，分别如SEQ ID NO:3所示的核苷酸序列和SEQ ID NO:4所示的核苷酸序列。
另外，以GAPDH作为内对照，其扩增引物对分别如SEQ ID NO:7所示的核苷酸序列和SEQ ID NO:8所示的核苷酸序列。
实验仪器试剂：Thermal Cycler DiceTM Real Time System TP800,Takara；Premix Ex TaqTM,Takara。
PCR的反应体系：总体积20μL，SYBR Premix Ex Taq 10μL，上下游引物(10μmol/L)各0.4μL，cDNA1μL，ddH2O 8.2μL，混匀试剂，离心后放入PCR仪中进行扩增反应。PCR条件为:94℃4min；94℃30s，60℃退火20s，40个循环。琼脂糖凝胶电泳检测PCR扩增产物，结果见图1。
5、实验结果：由图1结果可见，AGR3基因在胃癌组织中的表达水平明显高于癌旁组织中的表达水平，经t检验，p值小于0.05(有统计学意义)。
实施例2定量RT-PCR检测AGR3基因在胃癌细胞株中的表达情况
用定量RT-PCR检测AGR3基因在人胃黏膜上皮细胞株GES-1和人胃癌细胞株BGC823、HGC27、AGS、NCI-N87和SNU-1中的表达。其中所述细胞株来源于中国科学院上海细胞库。
按照实施例1所述方法提取不同胃癌细胞株RNA，反转录合成cDNA，以GAPDH作为内对照，以合成的不同胃癌细胞株cDNA为模板，按照实施例1所述方法进行PCR反应，琼脂糖凝胶电泳检测PCR扩增产物，结果见图2。
由图2结果可见，AGR3基因在AGS、NCI-N87和SNU-1中高表达，在GES-1、BGC823和HGC27细胞仅有较弱的表达。
实施例3胃癌细胞系AGS和NCI-N87中敲减和过表达AGR3后AGR3的表达和细胞的增殖能力变化
体外化学合成短发卡核糖核酸(shRNA)具有快速、简单和特异性强等特点，在抗肿瘤治疗等方面有广泛的应用前景。利用shRNA研究AGR3基因对AGS和NCI-N87细胞生长增殖的影响。
针对AGR3基因mRNA设计合成两条shRNA序列，分别是AGR3(s1)和AGR3(s2)，分别如SEQ ID NO:5所示的核苷酸序列和SEQ ID NO:6所示的核苷酸序列；设置无关序列NC作为阴性对照，如SEQ ID NO:9所示的核苷酸序列；另外体外化学合成AGR3基因全序列，如SEQ IDNO:1所示；设置空载体(vector)作为阴性对照。
利用脂质体(Lipofectamine 2000)转染试剂分别将上述2种shRNA、NC序列、vector、AGR3基因按终浓度50nmol/L转染进AGS和NCI-N87细胞(由中国科学院上海细胞库提供)中，用含10％胎牛血清的DMEM培养。按照实施例1所述方法提取不同胃癌细胞株RNA，反转录合成cDNA，以GAPDH作为内对照，以合成的不同胃癌细胞株cDNA为模板，按照实施例1所述方法进行定量RT-PCR实验检测AGR3基因在胃癌细胞株AGS和NCI-N87中的表达，结果见图3。
在96孔板中每孔接种3×103胃癌细胞系AGS和NCI-N87细胞(无血清培养液，由中国科学院上海细胞库提供)，待细胞密度达到40％左右时，利用脂质体(Lipofectamine2000)转染试剂分别将上述2种shRNA、NC序列、vector、AGR3基因按终浓度50nmol/L转染进AGS和NCI-N87细胞中，4h后换成含10％胎牛血清的DMEM。以24h为1个检测单位培养5d，每天检测1次该细胞密度。每孔待测细胞液中加10μL CCK-8，37℃孵育1h。利用酶标仪在450nm波长下检测其吸光度以代表细胞存活能力，绘制生长曲线，结果见图4。
由图3～图4结果可见，与对照组(NC)相比较，特异性干扰AGR3基因表达的短发卡核糖核酸(shRNA)：AGR3(s1)和AGR3(s2)能够有效抑制AGR3基因的mRNA水平，同时能够抑制胃癌细胞系AGS和NCI-N87细胞的生长，胃癌细胞的存活率明显低于对照组的细胞。结果表明人AGR3基因的表达下调能在一定程度上抑制胃癌细胞生长。与对照组(vector)相比较，过量表达AGR3基因能够有效促进AGR3基因的mRNA水平，同时能够促进胃癌细胞系AGS和NCI-N87细胞的生长，胃癌细胞的存活率明显高于对照组的细胞。结果表明人AGR3基因的表达上调能在一定程度上促进胃癌细胞生长。
因此，AGR3基因及其表达产物作为诊断胃癌的标志物，使胃癌诊断更加准确、快速，本发明的AGR3基因及其表达产物作为制备治疗胃癌药物的靶基因为治疗胃癌提供了新的治疗靶点和治疗途径。
综上所述，本发明的胃癌肿瘤标志物能够使得胃癌诊断简便准确快速，提高胃癌早期诊断水平，适于大规模推广应用。
在此说明书中，本发明已参照其特定的实施例作了描述。但是，很显然仍可以作出各种修改和变换而不背离本发明的精神和范围。因此，说明书和附图应被认为是说明性的而非限制性的。
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atgatgctac actcagcttt gggtctctgc ctcttactcg tcacagtttc ttccaacctt 60
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gacatcactt gggtacaaac ttatgaagaa ggtctctttt atgctcaaaa aagtaagaag 180
ccattaatgg ttattcatca cctggaggat tgtcaatact ctcaagcact aaagaaagta 240
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Claims (9)
1.一种胃癌肿瘤标志物，其特征在于，所述胃癌肿瘤标志物的氨基酸序列如SEQ IDNO:2所示。
2.根据权利要求1所述的胃癌肿瘤标志物，其特征在于，所述胃癌肿瘤标志物的核苷酸序列如SEQ ID NO:1所示。
3.一对根据权利要求1或权利要求2所述的胃癌肿瘤标志物的检测引物对，其特征在于，所述的胃癌肿瘤标志物的检测引物对具有如SEQ ID NO:3所示的核苷酸序列和SEQ IDNO:4所示的核苷酸序列。
4.一种根据权利要求1或权利要求2所述的胃癌肿瘤标志物的抑制剂，其特征在于，所述的胃癌肿瘤标志物的抑制剂具有如SEQ ID NO:5所示的核苷酸序列。
5.一种根据权利要求1或权利要求2所述的胃癌肿瘤标志物的抑制剂，其特征在于，所述的胃癌肿瘤标志物的抑制剂具有如SEQ ID NO:6所示的核苷酸序列。
6.根据权利要求1或权利要求2所述的胃癌肿瘤标志物在作为胃癌诊断试剂中的应用。
7.根据权利要求3所述的胃癌肿瘤标志物的检测引物对在作为胃癌诊断试剂中的应用。
8.根据权利要求4所述的胃癌肿瘤标志物的抑制剂在作为胃癌治疗药物中的应用。
9.根据权利要求5所述的胃癌肿瘤标志物的抑制剂在作为胃癌治疗药物中的应用。
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