CN104142397A - 胸腺素alpha原在制备乳腺癌诊断标记物中的应用 - Google Patents

胸腺素alpha原在制备乳腺癌诊断标记物中的应用 Download PDF

Info

Publication number
CN104142397A
CN104142397A CN201410383719.8A CN201410383719A CN104142397A CN 104142397 A CN104142397 A CN 104142397A CN 201410383719 A CN201410383719 A CN 201410383719A CN 104142397 A CN104142397 A CN 104142397A
Authority
CN
China
Prior art keywords
breast cancer
urine
former
alpha
prothymosin alpha
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201410383719.8A
Other languages
English (en)
Inventor
周克夫
崔若辰
梁贤明
王世媛
陈越月
段莹
陈艳芳
张长弓
陈雪飞
孙雨涵
胡鑫炎
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xiamen University
Original Assignee
Xiamen University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xiamen University filed Critical Xiamen University
Priority to CN201410383719.8A priority Critical patent/CN104142397A/zh
Publication of CN104142397A publication Critical patent/CN104142397A/zh
Pending legal-status Critical Current

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57415Specifically defined cancers of breast
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575Hormones
    • G01N2333/5759Thymosin or related peptides
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/70Mechanisms involved in disease identification
    • G01N2800/7023(Hyper)proliferation
    • G01N2800/7028Cancer

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

胸腺素alpha原在制备乳腺癌诊断标记物中的应用,涉及肿瘤标记物。采用斑点杂交法和ELISA方法检测乳腺癌患者尿液中胸腺素alpha原蛋白的水平,探讨胸腺素alpha原与乳腺癌生物学行为的关系。采用免疫检测方法,只需要检测尿液中的胸腺素alpha原水平,并能快速地证明乳腺癌病人尿液中的胸腺素alpha原水平明显高于正常人,对乳腺癌进行初步判断,为进一步诊断提供依据。所需尿液样品容易获取,不需要有任何创伤,不需要昂贵的仪器设备,方便操作,容易推广应用。

Description

胸腺素alpha原在制备乳腺癌诊断标记物中的应用
技术领域
本发明涉及肿瘤标记物,尤其是涉及胸腺素alpha原在制备乳腺癌诊断标记物中的应用。
背景技术
乳腺癌是女性恶性肿瘤中常见的一种,全球乳腺癌发病率自20世纪70年代末开始一直呈上升趋势[Yang L,Parkin DM,Ferlay J,et a1.Estimates of cancer incidence in Chinafor 2000 and projections for 2005[J].Cancer Epidemiol Biomarkers Prev,2005,14(1):243-250]。8名妇女中就会有一人患乳腺癌,中国不是乳腺癌的高发国家,但不宜乐观,今年我国乳腺癌发病率的增长速度高出发达国家1~2个百分点。据国家癌症中心和卫生部疾病预防控制局2012年公布的的乳腺癌发病数据显示:全国肿瘤登记地区乳腺癌发病率位居女性恶性肿瘤的第1位[全国肿瘤防治研究办公室,全国肿瘤登记中心,卫生部疾病防御控制局.全国第三次死因回顾抽样调查[M].北京:人名卫生出版社,2012:120-131]。
目前,乳腺癌已成为当前社会的重大公共卫生问题,究其原因,1:早期乳腺癌往往不具备典型的症状和体征,不易引起重视;2:在乳腺门诊,医生了解病史后首先会进行体检,检查双侧乳腺,结合影像学检查,包括乳腺学检查,包括乳腺X线摄影(乳腺钼靶照相)[Armstrong K,Handorf EA,Chen J,et al.Breast cancer risk prediction and mammographybiopsy decisions:a model-based study[J].Am J Prev Med,2013,44:15-22]、彩超,必要时还要进行乳腺磁共振检查[王鹏.保乳手术治疗早期乳腺癌86例疗效观察[J].当代军医,2012,18(9):107-108],以上检测手段费用高,时间长,更加频繁的辐射使乳腺癌的风险显著增加。
为了补充乳腺癌筛选方法,我们尝试使用肿瘤标记物水平来诊断乳腺癌。
肿瘤标志物是由肿瘤组织产生的存在于肿瘤组织,或分泌至血液,或其他体液,或因肿瘤组织刺激,由宿主细胞产生而含量明显高于正常参考值的一类物质。随着医学的发展,近年来,许多学者致力于发现肿瘤特征性的标记物,为恶性肿瘤的早期诊断提供新的方向。
胸腺素alpha原是一种在体内广泛表达的强酸性蛋白质,其功能与细胞免疫调节、细胞增殖和细胞凋亡有关[Wu C L,Shiau A L,Lin C C.Prothymosin alpha promotes cell proliferation inNIH3T3 cells[J].Life Sci,1997,61:2091-2099],作为一种典型的肿瘤相关蛋白,在细胞增殖和凋亡调控方面的深入研究有助于发现细胞癌变的发生机理,曾在多种癌症中检测到了PTMA水平的升高[郑金来,刘佃辛,高兰兴.胸腺素α原研究进展[J].国外医学免疫学分册,1998,21(4):210-214],这种在癌细胞中高水平的PTMA蛋白产物不仅维持细胞处于高度增殖状态,还保护细胞避免进入凋亡途径[EvatafievaαG,Belov C A,Rubtsov Y P,et al.Apoptosis-related fragmentation,translocation and properties of humanprothymosinalpha[J].Exp Cell Res,2003,284(2):211-223],有报导指出,乳腺癌患者体内β族胸腺素含量增高[兰海燕等.胸腺素β4和β10及β15在乳腺癌中的表达及意义[J].中国肿瘤临床与康复,2009,16(1):22-24],可作为术后复发标记物,但乳腺癌体内胸腺素alpha原表达情况却鲜见报道[徐雅莉等.中国女性乳腺癌发病相关危险因素病例对照研究[J].协和医学杂志,2011,2:7-14]。
发明内容
本发明的目的在于提供胸腺素alpha原在制备乳腺癌诊断标记物中的应用。
本发明检测了正常人、乳腺癌尿液中胸腺素alpha原蛋白的水平,以证明和评价胸腺素alpha原作为肿瘤诊断新标记物的可行性提供依据,其结果为广大女性提供一种更方便快捷的诊断方法,并为进一步研究胸腺素alpha原在肿瘤发生和治疗中的生物学功能提供依据。本发明为此提供了以下免疫学实验:
1)DOT-BLOT法检测胸腺素alpha原在样本中的水平;
2)ELISA法检测胸腺素alpha原在样本中的水平。
本发明采用斑点杂交法和ELISA方法检测乳腺癌患者尿液中胸腺素alpha原蛋白的水平,探讨胸腺素alpha原与乳腺癌生物学行为的关系。
本发明所述胸腺素alpha原作为乳腺癌诊断标记物,尤其是通过检测人体尿液中的胸腺素alpha原水平来进行乳腺癌诊断,迄今尚未见有相关报道。
本发明通过人体尿液中的胸腺素alpha原水平检测,对乳腺癌的诊断提供依据,为乳腺癌的治疗和预后判断提供科学依据,同时也为乳腺癌的治疗方法的筛选及疗效判断提供依据。此外,通过本发明的实施,还为胸腺素alpha原在参与肿瘤细胞的迁移、生长过程在其中发挥的重要调控功能,阐明胸腺素alpha原与肿瘤发生发展的相关性,为今后乳腺癌新靶点的治疗提供一种新的治疗途径。
本发明的优点与积极效果在于,采用免疫检测方法,只需要检测尿液中的胸腺素alpha原水平,并能快速地证明乳腺癌病人尿液中的胸腺素alpha原水平明显高于正常人,对乳腺癌进行初步判断,为进一步诊断提供依据。本发明所需尿液样品容易获取,不需要有任何创伤,不需要昂贵的仪器设备,方便操作,容易推广应用。
附图说明
图1为胸腺素alpha原在乳腺癌病人与正常人尿液中的斑点杂交法检测结果。
图2为乳腺癌病人与正常人尿液ELISA检测后OD450值统计结果。在图2中,横坐标:正常,代表健康人;病人,代表乳腺癌病人;纵坐标:OD值,代表吸光度;P=0.003代表统计结果,**代表极显著。
具体实施方式
以下实施例仅为帮助所属领域技术人员更好地理解本发明,但不以任何方式限制本发明。
实施例1 DOT-BLOT法定性检测胸腺素alpha原在肿瘤人与正常人尿液中的水平。
用制备的胸腺素alpha原多克隆抗体,采用斑点杂交方法,以肿瘤病人尿液为检测对象,检测其中胸腺素alpha原水平,以正常健康人尿液为对照(尿液样品均来自厦门大学附属中山医院,由主管技师梁贤明提供)。
1)将尿液按表1滴(2μL)于NC膜上,待干燥之后用封闭液37℃封闭2h;
2)兔抗胸腺素alpha原(1∶1000)孵育,4℃过夜;
3)用PBST洗膜3次,摇床震荡,每次5min;
4)HRP标记羊抗兔二抗(1∶1000)室温孵育1h;
5)用PBST洗膜,3次,每次10min,然后用DAB显色。
采用实施例1中所述的方法,检测胸腺素alpha原在尿液中的表达水平(参见图1和表2),其结果表明正常人检出率为9%,而乳腺癌病人的检出率为73%,肿瘤病人中阳性率远远高于正常人,而IL-2、IFN-γ、PBS空白对照则没有阳性反应。可以证明,本实验作为乳腺癌诊断具有明显的特异性,可以用于乳腺癌的诊断。
实施例2 ELISA法定量检测胸腺素alpha原在肿瘤人与正常人尿液中的水平。
用制备的胸腺素alpha原单克隆抗体,采用间接ELISA方法,以实施例1中尿液为检测对象,正常健康人尿液和PBS为对照,检测其中胸腺素alpha原水平。
1)包被:抗原按照表1用Coating buffer稀释成2X,200μL/well包板,避光,4℃过夜。
2)PBST洗板3次。
3)Blocking buffer(5%脱脂奶粉)按200μL/Well加入,25℃封闭2h。
4)PBST洗板3次,最后1次甩干但不扣干。
5)小鼠抗胸腺素alpha原单克隆抗体(1∶1000)200μL/well,25℃避光2h。
6)PBST洗板3次,最后1次甩干但不扣干。
7)HRP-羊抗鼠二抗稀释(1∶1000)200μL/well快速上样,25℃避光孵育1h。
8)PBST洗板3次,最后1次甩干但不扣干。
9)加入100μl/孔的TMB,室温、避光显色1h。
10)加入100μL/孔的2NH2SO4终止显色并测OD450值。
表1乳腺癌病人与正常人尿液编号
627 613 593 585 600 635 570 616
597 584 604 588 617 630 596 639
577 579 569 636 567 595 568 601
589 586 598 587 635 574 638 571
389 568 127 296 796 585 248 Pbs
978 754 216 208 556 513 435 426
IL-2 IFN-γ IL-2 IFN-γ PBS PBS PBS PBS
125 753 213 109 530 622 383 724
230 363 913 931 366 056 405 388
686 833 326 291 388 098 533 432
815 525 768 192 466 151 333 493
315 244 180 742 413 PBS PBS PBS
注:加下划线为乳腺癌病人,不加下划线为正常健康人,IL-2、IFN-γ为白细胞介素-2,γ干扰素作为其他细胞因子对照,PBS-磷酸缓冲液为空白对照。
表2乳腺癌病人与正常人尿液经过斑点杂交后阳性率情况表
采用实施例2中所述的方法,检测胸腺素alpha原在尿液中的水平(表3),检测其OD结果显示,胸腺素alpha原在人尿液均有不同程度的表达,但正常人Mean=0.8122,病人Mean=1.0134,经过统计软件GraphPad Prism5分析证明胸腺素alpha原在病人尿液中的含量高于正常人,P值为0.0030(图2),P<0.01差异性极显著,说明本实验在证明胸腺素alpha原呈肿瘤性相关性表达的可行性,其结果与实施例1相一致。
表3乳腺癌病人与正常人尿液ELISA检测后OD450值
0.705 0.798 0.511 0.658 1.633 0.913 0.554 0.952
0.983 0.826 0.758 0.597 1.076 0.841 0.848 0.935
0.954 0.657 0.705 0.823 0.725 0.787 0.73 1.101
0.927 0.908 0.864 0.891 1.247 0.779 0.747 1.766
0.601 0.8 0.951 1.237 0.564 0.691 0.886 0.768
1.102 0.943 0.893 1.351 1.998 1.609 1.251 1.081
1.068 0.581 0.889 1.084 2.088 0.659 0.824 0.791
1.041 0.852 1.047 0.934 1.012 1.686 0.843 1.082
1.013 0.776 1.008 1.911 0.871 0.938 0.709 1.026
0.908 1.366 1.551 1.014 1.038 0.772 1.295 0.763
0.98 0.873 0.866 0.664 0.789 0.791 0.744 0.857
注:加下划线为乳腺癌病人,不加下划线为正常健康人。
两种免疫学实验能够特异性检测到尿液中微量的胸腺素alpha原,同样的方法不能检测出如白细胞介素-2,γ干扰素等其它细胞因子。说明该方法有明显的特异性。通过比较乳腺癌病人和正常人尿液中胸腺素alpha原水平,结果证明,乳腺癌病人尿液样品中的胸腺素alpha原水平比正常健康人明显高,斑点杂交显色反应,病人阳性率显著高于正常人,而ELISA结果也同样证明病人OD值比正常人高,差异极显著。这些结果证明通过检测尿液中的胸腺素alpha原水平可以对乳腺癌病人进行初步判断,其判断结果为乳腺癌的筛查和进一步采用开展更深入的检查提供依据。由于本发明不需要高端仪器设备,操作简单,容易推广,适合基层农村等地对广大妇女的初筛诊断。

Claims (2)

1.胸腺素alpha原在制备乳腺癌诊断标记物中的应用。
2.如权利要求1所述应用,其特征在于采用检测尿液中的胸腺素alpha原水平。
CN201410383719.8A 2014-08-06 2014-08-06 胸腺素alpha原在制备乳腺癌诊断标记物中的应用 Pending CN104142397A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410383719.8A CN104142397A (zh) 2014-08-06 2014-08-06 胸腺素alpha原在制备乳腺癌诊断标记物中的应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410383719.8A CN104142397A (zh) 2014-08-06 2014-08-06 胸腺素alpha原在制备乳腺癌诊断标记物中的应用

Publications (1)

Publication Number Publication Date
CN104142397A true CN104142397A (zh) 2014-11-12

Family

ID=51851630

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410383719.8A Pending CN104142397A (zh) 2014-08-06 2014-08-06 胸腺素alpha原在制备乳腺癌诊断标记物中的应用

Country Status (1)

Country Link
CN (1) CN104142397A (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106568954A (zh) * 2016-10-26 2017-04-19 北京师范大学 乳腺癌的尿液蛋白标志物及其在诊断和预后中的用途
CN107167612A (zh) * 2017-06-20 2017-09-15 深圳市人民医院 与唐氏综合征相关的母血标志物及应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6294349B1 (en) * 1999-03-01 2001-09-25 University Of Mississippi Medical Ctr. Method of diagnosing and monitoring malignant breast carcinomas
US20110223616A1 (en) * 2010-03-12 2011-09-15 The Curators Of The University Of Missouri HuR-Associated Biomarkers
CN102295701A (zh) * 2011-07-18 2011-12-28 厦门大学 抗胸腺素α原多克隆抗体及其制备方法与应用
CN102393458A (zh) * 2011-08-24 2012-03-28 绍兴市人民医院 一种早期乳腺癌血清诊断试剂及其检测方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6294349B1 (en) * 1999-03-01 2001-09-25 University Of Mississippi Medical Ctr. Method of diagnosing and monitoring malignant breast carcinomas
US20110223616A1 (en) * 2010-03-12 2011-09-15 The Curators Of The University Of Missouri HuR-Associated Biomarkers
CN102295701A (zh) * 2011-07-18 2011-12-28 厦门大学 抗胸腺素α原多克隆抗体及其制备方法与应用
CN102393458A (zh) * 2011-08-24 2012-03-28 绍兴市人民医院 一种早期乳腺癌血清诊断试剂及其检测方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
C MAGDALENA ET AL.: "Tumour prothymosin alpha content, a potential prognostic marker for primary breast cancer", 《BRITISH JOURNAL OF CANCER》 *
王梅等: "胸腺素α原与肿瘤关系的研究进展", 《癌症》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106568954A (zh) * 2016-10-26 2017-04-19 北京师范大学 乳腺癌的尿液蛋白标志物及其在诊断和预后中的用途
CN106568954B (zh) * 2016-10-26 2018-11-06 北京师范大学 乳腺癌的尿液蛋白标志物及其在诊断和预后中的用途
CN107167612A (zh) * 2017-06-20 2017-09-15 深圳市人民医院 与唐氏综合征相关的母血标志物及应用

Similar Documents

Publication Publication Date Title
CN102822676B (zh) 用于预测三阴性乳腺癌对疗法的应答的方法
Chern et al. Effects of obesity in rectal cancer surgery
Atak et al. Strategies to treat idiopathic granulomatous mastitis: retrospective analysis of 40 patients
Chen et al. Prognostic value of a positive-to-negative change in hormone receptor status after neoadjuvant chemotherapy in patients with hormone receptor–positive breast cancer
CN106489074B (zh) 作为转移性乳腺癌的生物标志物的s100p和透明质酸
Albergotti et al. Association of pretreatment body mass index and survival in human papillomavirus positive oropharyngeal squamous cell carcinoma
CN104142397A (zh) 胸腺素alpha原在制备乳腺癌诊断标记物中的应用
Chen et al. Lymph node counts and ratio in axillary dissections following neoadjuvant chemotherapy for breast cancer: a better alternative to traditional pN staging
Sarkar et al. Correlation between cyclin D1 expression and standard clinicopathological variables in invasive breast cancer in Eastern India
Yu et al. Nucleus DJ-1/Park7 acts as a favorable prognostic factor and involves mucin secretion in invasive breast carcinoma in Chinese population
Colón et al. Prevalence of steroid receptors and HER 2/neu in breast cancer biopsies of women living in Puerto Rico
Sana et al. Incidental neuroendocrine tumor of the appendiceal base less than20 mm in diameter: is appendectomy enough?
Agaimy et al. Loss of DOG-1 expression associated with shift from spindled to epithelioid morphology in gastric gastrointestinal stromal tumors with KIT and platelet-derived growth factor receptor α mutations
Yau et al. Ductal carcinoma in situ of breast: detection and treatment pattern in Hong Kong
Najam-ul-Haq et al. Clinicopathological Variants of Breast Cancer in a Tertiary Care Hospital in Pakistan
Mehedinţeanu et al. Expression of M3 muscarinic acetylcholine receptors in gastric cancer
Masoud et al. Clinicopathological characteristics and androgen receptor expression of triple negative breast cancer: A retrospective study in upper Egypt
RU2618408C2 (ru) Способ прогнозирования эффективности лечения больных с рецидивами рака шейки матки
Getzler et al. Semaphorin-3A levels in urine demonstrate promising sensitivity for detection of upper-tract urothelial carcinoma-a preliminary case series
Szep et al. Annular erythema as a cutaneous sign of recurrent ductal breast carcinoma, misdiagnosed as erythema chronicum migrans
Zhuang et al. Correlations of special AT-rich sequence binding protein 2 and chitinase-3-like protein-1 with sensitivity to paclitaxel chemotherapy for gastric cancer
Gameiro Improving Feline Mammary Carcinoma Treatment Through Her2-Related Immunochemotherapy Agents and Biomarkers
Rashed et al. Value of CA 15-3 in Female Patients with Breast Cancer
Zhao et al. Molecular typing of breast cancer in Northern Henan Province
Mok et al. FRI0257 Urinary angiostatin, cxcl4 and vcam-1 as biomarkers for lupus nephritis

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20141112