摘要
恶性肿瘤是严重威胁人类健康的疾病，且在世界范围内发病率逐年上升。但由于目前诊断技术及水平有限，很多癌症患者确诊时已经是晚期，失去了许多治疗的机会。因此寻找一种敏感、快捷、方便、无创的检测方法是目前国内外学者关注的课题。经血清学肿瘤标志物的蛋白定量检测来筛查各种类型肿瘤国内外已有报道，在检测各种类型肿瘤患者中的价值已得到充分肯定。MUC1作为一种新的肿瘤标志物近年来得到了广泛的研究。多种腺癌患者包括乳腺癌、肺癌等外周血中MUC1蛋白水平较正常人升高。近年研究表明，恶性血液病组织及细胞中MUC1水平也较正常人升高，但血清中MUC1粘蛋白的表达水平国内外报道较少。
目前，检测外周血MUC1蛋白的方法最常用的为酶联免疫吸附试验（ELISA），但现有的试剂盒多采用单克隆抗体，仅仅是针对某单一抗原表位，而肿瘤患者血清中的黏蛋白是含有多种抗原表位的，这样势必会漏检很多阳性病例，针对目前商业化试剂盒的局限性，本研究通过制备抗人MUC1多克隆抗体，与GP1.4单克隆抗体进行组合，建立了双抗体间接夹心ELISA方法，通过健康人、乳腺癌、肺癌患者外周血MUC1的水平的测定对该试剂盒进行分析评价，并对恶性血液病血清MUC1黏蛋白的表达水平进行了初步检测。
研究方法和结果：
1. MUC1-GST融合蛋白的制备培养表达MUC1-GST融合蛋白的pGEX-MUC1/BL21细菌，菌体通过超声破碎提取上清液，Glutathione Sepharose4B亲和层初步纯化，非变性聚丙烯酰胺凝胶电泳进一步纯化，获得较纯的MUC1-GST融合蛋白。
2.抗体制备及效价检测重组MUC1-GST融合蛋白免疫家兔，获得高效价家兔抗人MUC1多克隆抗体，效价为320000。
3.抗体纯化将家兔血清进行饱和硫酸铵沉淀、Protein A纯化及抗GST抗体的吸收，获得了纯化的家兔抗人MUC1多克隆抗体。
4. MUC1标准品的制备经过亲和层析纯化的MUC1-GST融合蛋白进行凝血酶裂解，获得MUC1多肽标准品。
5.双抗体间接夹心ELISA法的建立通过抗人MUC1多克隆抗体与GP1.4单克隆抗体的不同组合筛选，成功建立了双抗体间接夹心ELISA方法，其包被抗体为GP1.4单克隆抗体，检测抗体为家兔抗人MUC1多克隆抗体。酶标抗体为HRP标记的山羊抗兔IgG抗体。
6.双抗体间接夹心ELISA法最佳工作条件的确定通过棋盘滴定法选择包被抗体、检测抗体和HRP标记的山羊抗兔IgG抗体的最佳工作浓度，包被抗体，检测抗体最佳工作浓度浓度分别为：1ug/ml，15μg/ml；酶标抗体最适稀释比例为1:5000；MUC1标准品的稀释浓度为：0.5ng/ml、1ng/ml、2ng/ml、4ng/ml、8ng/ml、16ng/ml、32ng/ml，64ng/ml。该试剂盒的灵敏度为0.5ng/ml。
7.外周血MUC1蛋白水平的检测通过建立的双抗体间接夹心ELISA法，对收集的225例血清样本（13例乳腺癌，60例肺癌、48例急性髓系白血病、26例急性淋巴细胞白血病、24例淋巴瘤、17例多发性骨髓瘤，20例健康人，7例肺良性疾病，10例良性血液患者）进行检测，结果表明乳腺癌、肺癌患者外周血MUC1蛋白平均水平明显高于健康对照组(P<0.01)，恶性血液病外周血MUC1蛋白水平与健康对照组无显著性差异。进一步绘制ROC曲线分析得出以1.92ng/ml为乳腺癌患者与健康对照组临界值，乳腺癌患者检测的敏感度为100%，正常人的特异度为100%。以1.88ng/ml为肺癌患者与健康人的临界值，肺癌的敏感度为63.3%，健康人的特异度为100%。以1.98ng/ml为肺癌患者与肺良性疾病患者临界值，肺癌的敏感度为61.7%，肺良性疾病的特异度为100%。
8.双抗体间接夹心ELISA法与CA15-3试剂盒比较对于乳腺癌，健康对照者同一病例样本用酶联免疫法CA15-3诊断试剂盒进行对比检测，通过绘制ROC曲线对比显示，双抗体间接夹心ELISA法对乳腺癌的诊断准确度明显高于CA15-3试剂盒。
结论：
1.本研究建立了灵敏度高特异性强的双抗体间接夹心ELISA方法
2.对乳腺癌、肺癌诊断的灵敏度及特异度有很大程度的提高，有望开发为临床辅助诊断的常规试剂盒。
3.对恶性血液病患者外周血MUC1水平的检测尚待进一步探讨。
Malignant tumors are such diseases that seriously threaten human health, and theincidence rate is increasing year by year worldwide. However, due to the limited diagnostictechnology, many patients with cancers are diagnosed in the late stage, and the properopportunity for treatment was missed. As a result, a sensitive, fast, convenient, andnon-invasive detection method attracts much concern to researchers. The protein quantitativedetection of serum tumor markers to screen various types of tumors has been reported athome and abroad, and the value in the detection of various types of tumors has been affirmed.MUC1, a novel tumor marker, has been widely studied in recent years. Patients with manykinds of adenocarcinomas, such as breast cancer and lung cancer, were detected an evaluatedsoluble MUC1protein level in peripheral blood than normal control. Recent studies haveshown that cells of hematologic malignancies express MUC1protein levels higher thannormal control. However, the expression level of soluble muc1mucin in peripheral bloodhas not been reported.
At present, enzyme-linked immunosorbent assay (ELISA) is the most common methodof detecting the soluble MUC1protein in peripheral blood. Monoclonal antibodies are usedto prepare the commercial ELISA kits. Although monoclonal antibodies can greatly improvethe specificity of antigen recognition, they only bind to certain epitopes. Differentindividuals with cancers have different antigen epitopes, and therefore, the detection ratio isdecreased. Taking the limitations of commercial kit into account, in the present study, weprepared a new kit of double antibody indirect sandwich ELISA by combined anti-humanpolyclonal antibodies with GP1.4monoclonal antibodies. The kit was evaluated by detectingMUC1in serum of health subject and patients with breast cancer and lung cancer, and thenwe attempted to analyze the expression level of MUC1mucin in peripheral blood of patientswith hematologic malignancies.
Methods and Results:
1. Preparation of MUC1-GST fusion protein Frist,culture large numbers ofpGEX-MUC1/BL21which express target proteinMUC1-GST. Then,the bacteria werebroken by sonicate and removed supernatant containing fusion protein.Pure MUC1-GSTfusion protein was obtained after Glutathione Sepharose4B affinity layer andNative-Polyacrylamide Gel Electrophoresis.
2. Antibody preparation and titer assay MUC1-GST fusion protein was used toimmunize rabbit in order to prepare polyclonal antibodies. We detected the titers of rabbitanti-MUC1polyclonal antibody.the antibody titer was320000.
3. Antibody purification Purified rabbit anti-MUC1Polyclonal antibodies wereobtained after precipitated by saturated ammonium sulfate, purified by protein A andabsorbed by anti-GST antibody.
4. Preparation of standard sample MUC1-GST fusion protein was digested withthrombin to obtain MUC1standard.
5. Establishment of double antibodies indirect sandwich ELISA A double antibodyindirect sandwich ELISA method was successfully established after screening differentcombinations of anti-human MUC1polyclonal antibody and GP1.4monoclonal antibody.The coated antibody was GP1.4monoclonal antibody; rabbit anti-human polyclonal MUC1antibodies were the detected antibodies, and the enzyme-labeled antibody was HRPAffiniPure Goat Anti-Rabbit IgG.
6. Determination of the optimum working conditions for double antibodies indirectsandwich ELISA The antibody concentration of coated antibody, detection antibody andHRP-labeled antibody was chose by checkerboard titration method. The optimum workingconcentration of coated antibody and detection antibody were1ug/ml and15μg/ml,respectively. The optimum dilution ratio of HRP AffiniPure Goat Anti-Rabbit IgG was1:5000. The MUC1standard sample was diluted as follow:0.5ng/ml,1ng/ml,2ng/ml,4ng/ml,8ng/ml,16ng/ml,32ng/ml, and64ng/ml. The sensitivity of the kit was0.5ng/ml.
7. Detection of MUC1protein levels in peripheral blood After establishment of thedouble antibody sandwich ELISA,225patients were enrolled for detection of MUC1proteinof peripheral blood, including13cases of breast cancer,60cases of lung cancer,48cases ofacute myeloid leukemia,26cases of acute lymphoblastic leukemia,24cases oflymphatictumor,17cases of multiple myeloma,20cases of healthy individuals,7cases oflung benign disease, and10cases of benign hematological diseases. The resultsdemonstrated that patients with breast cancer and lung cancer had significantly higher MUC1protein levels in peripheral blood compared to the healthy control (P<0.01). However,no significant difference of MUC1protein levels was observed between patients withhematologic malignancies and healthy controls (P>0.05). Cutoff value of MUC1protein wasdetermined by ROC curve analysis.1.92ng/ml was the cutoff value for patients with breastcancer and healthy control, and the sensitivity and specificity were100%and100%,respectively. For patients with lung cancer,1.88ng/ml was the cutoff value, and thesensitivity and specificity were63.3%and100%, respectively. For the patients of lungcancer and benign lung diseases,1.98ng/ml was the cutoff value with a sensitivity of61.7%and a specificity of100%.
8. Comparing between double-antibody sandwich ELISA and CA15-3kit The sameblood sample from breast cancer and health subject were assayed by ELISA and CA15-3.After drawing ROC curves, the results showed that double-antibody indirect sandwichELISA had a significantly higher accuracy in diagnosis of breast cancer compared withCA15-3kit.
Conclusions
1. In this study, we develop a double antibody sandwich ELISA method which has abetter sensitivity and specificity.
2. The sensitivity and specificity are greatly improved for diagnosis of lung and breastcancer using the present ELISA kit which is expected to develop conventional kit for clinicaldiagnosis.
3. The detection of MUC1protein level in peripheral blood of patients withhematologic malignancies remains to be further explored.
引文
[1] Satoh S, Hinoda Y, Hayashi T, et al. Enhancement of metastatic properties ofpancreatic cancer cell by MUC1gene encoding an anti-adhesion molecule[J]. Int JCancer,2000,88(4):507-518.
[2] Gong J, Apostolopoulos V, Chen D, et al. Selection and characterization ofMUC1-specific CD8+T cells from MUC1transgenic mice immunized withdendritic-carcinoma fusion cells[J]. Immunology,2000,101(31):316-324.
[3] Xu Y, Zhang L, Hu GX. Potential application of alternatively glycosylated serumMUC1and MUC5AC in gastric cancer diagnosis[J]. Biologicals,2009,37(1):18-25.
[4]张天泽,徐光炜肿瘤学[M].天津:天津科学技术出版社,1996:547-553.
[5] Velaiutham S, Taib NA, Ng KL, et al. Does the pre-operative value of serum CA15-3correlate with survival in breast cancer[J]. Asian pac J Cancer Prev,2008,9(3):445-448.
[6]王纪文.肺癌相关血清肿瘤标志物的临床应用研究[D].中国医学科学院肿瘤研究所,2011.
[7]夏同礼(主编).肿瘤特检诊断[M].北京:人民卫生出版社,2002:99-117.
[8]刘鲁明(主编).肿瘤科疾病临床诊断与鉴别诊断[M].北京：科学技术出版社,2005:46-63.
[9] Chaturvedi P, Singh A.P, Batra S.K. Structure, evolution, and biology of the MUC4mucin[J].FASEB J,2008，22(4)：966-981.
[10] Hattrup C.L, Gendler S.J. Structure and function of the cell surface (tethered) mucins,Annu[J]. Annu Rev. Physiol,2008，70：431-457.
[11] Andrianifahanana M, Moniaux N, Schmied B.M, et al. Mucin (MUC) gene expressionin human pancreatic adenocarcinoma and chronic pancreatitis: apotential role of MUC4as a tumor marker of diagnostic significance[J], Clin. Cancer Res,2001，7(12):4033-4040.
[12] Senapati S, Das S, Batra S.K. Mucin-interacting proteins: from function totherapeutics[J]. Trends Biochem Sci,2010,35(4)：236-245.
[13] Fowler J, Vinall L, Swallow D. Polymorphism of the human muc genes[J].Front.Biosci,2001,6: D1207-D1215.
[14] Diaz L.K, Wiley E.L, Morrow. M. Expression of epithelial mucins Muc1, Muc2, andMuc3in ductal carcinoma in situ of the breast[J]. Breast J.2001,7(1):40-45.
[15] Meseguer A, Pellicer A, simon C.MUC1and endometrial receptivity [J]. Mol.Hum.Report,1998,4(12):1089-1098.
[16]康美,熊汉鹏. MUC1基因, CA15-3与肺癌的临床研究进展[J].江西医药,2008,43(2):166-169.
[17] Kruger W, Kroger N, Zander AR. MUC1expression in hemopoietic tissues.[J]. JHematotheer. Stm. Cell Res.2000,9(4):409-410.
[18] Perez-Vilar J, Hill RL.The structure and assembly of secreted mucins[J].J Biol Chem,1999,274(45):31751-31754.
[19]马吉春.重组MUC1融合蛋白的制备及检测MUC1双抗体夹心ELISA方法的建立
[D].吉林大学,2007.
[20] Ligtenberg MJ, Kruijshaar L, Buijs F, et al. Cell-associated episialin is a complexcontaining two proteins derived from a common precursor[J]. J Biol Chem,1992,267(9):6171-6177.
[21] Levitin F, Stern O, Weiss M, et al. The MUC1SEA module is a self-cleavingdomain[J]. J Biol Chem,2005,280(39):33374-33386.
[22] Macao B, Johansson DG, Hansson GC, et al. Autoproteolysis coupled to proteinfolding in the SEA domain of the membrane-bound MUC1mucin[J]. Nature Struct MolBiol2006,13(1):71-76.
[23] Siddiqui J, Abe M, Hayes D, et al. Isolation and sequencing of a cDNA coding forthe human DF3breast carcinomaassociated antigen[J]. Proc Natl Acad SciUSA,1988,85(7):2320-2323.
[24] Gendler S, Taylor-Papadimitriou J, Duhig T,et al. A highly immunogenic region of ahuman polymorphic epithelial mucin expressed by carcinomas is made up of tandemrepeats[J]. J Biol Chem,1988,263(26):12820-12823.
[25]魏玲,易艳萍,刘萱,等.MUC1及其C端活性片段突变体具有抑制肿瘤的活性[J].生物化学与物理进展,2007,34(4):375-381.
[26] Merlo GR, Siddiqui J, Cropp CS, et al. Frequent alteration of the DF3tumor-associatedantigen gene in primary human breast carcinomas[J]. Cancer Res,1989,49(24):6966-6971.
[27] Baruch A, Hartmann M, Yoeli M, et al. The breast cancer-associated MUC1genegenerates both a receptor and its cognate binding protein[J].Cancer Res,1999,59(7):1552-1561.
[28] Rottger S, White J, Wandall HH, et al. Localization of three human polypeptideGalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylationthroughout the Golgi apparatus[J]. J Cell Sci,1998,111(Pt1):45-60.
[29] Gum JR, Byrd JC, Hicks JW, et al. Molecular cloning of human intestinal mucincDNAs. J.Biol. Chem.1989;264(11):6480-6487
[30]张立新，李春海. MUC1粘蛋白的免疫生物学作用及其在肿瘤生物学治疗中的应[J].中国肿瘤生物治疗杂志,2000,7(3):165-170.
[31] Hartmann M, Baruch A, Ron I, et al. MUC1isoform specific monoclonal antibody6E6/2detects preferential expression of the novel MUC1/Y protein in breast andovarian cancer[J]. Int. J Cancer,1999,82(2):256-267.
[32] Swallow DM, Gendler S, Griffith B, et al. The human tumor-associated epithelialmucins are code for by an expressed hypervariable gene locus PEM[J]. Nature.1987,328(6125):82-84.
[33] Bramwell ME, Wiseman G, Shotton DM. Electron-microscopic studies of the CAantigen, epitectin[J]. J Cell Sci1986,86:249-261.
[34] Hilkens J, Ligtenberg MJL, Vos HL, et al The structure of cell-associated mucin-likemolecules and their adhesion modulating property[J]. Trends Biochem Sci1992,17:359-63.
[35] Von Mensdorff-Pouilly S, Snijdewint FG, Verstraeten AA, et al. Human MUC1mucin:a multifaceted glycoprptein[J]. Int J Biol Markers,2000,15(4):343-356.
[36]周人杰,肖颖彬.粘蛋白MUC1与非小细胞肺癌研究进展[J].免疫学杂志,2011,27(8):729-733.
[37] Jerome KR, Domenech N, Finn OJ. Tumor-specific cytotoxic T cell clones frompatients with breast and pancreatic adenocarcinoma recognize EBV-immortalized Bcells transfected with polymorphic epithelial mucin complementary DNA[J]. JImmunol.1993,151(3):1654-1662.
[38] Engelmann K, Shen H, Finn OJ. MCF7side population cells with characteristics ofcancer stem/progenitor cells express the tumor antigen MUC1[J]. Cancer Res,2008,68(7):2419-26.
[39]许崇艳. MUC1多肽致敏树突状细胞诱导CTLs体外杀伤白血病细胞的实验研究
[D].中国协和医科大学,2007.
[40] Rochlitz C. Figlin R. Phase I immunotherapy with a modified vacciniavirus(MVA)expressing human MUC1as antigen-specific immunotherapy in patientswith MUCl-positive advanced cancer[J]. J Gene Med2003,5(8):690-699．
[41] Wei YQ. Immutherapy of tumor with vaccines based on xenogeneious homologousmolecules[J]. Anticancer Drugs2002,13(3):229-235．
[42] Donald W. Kufe. Mucins in cancer: function, prognosis and therapy[J]. Nat Rev Cancer,2009,9(12):874-885.
[43] Brossart P, Schneider A, Dill P, et al. The epithelial tumor antigen MUC1is expressedin hematological malignancies and is recognized by MUC1-specific cytotoxicT-lymphocytes[J]. Cancer Res,2001,61(18):6846-6850.
[44]何易.实时定量观察MUCl基因在急性白血病表达的临床意义[D].中山大学,2008.
[45] Harrison SJ, Cook G, Nibbs RJ, et a1. Immunotherapy ofmultiple myeloma: the start ofa long and tortuous journey[J]. Expert Rev Anticancer Ther,2006,6(12),1769-1785．
[46]魏华萍,张轴,杨玲,等. MUC1粘蛋白在多发性骨髓瘤中的表达及其意义[J].白血病.淋巴瘤，2005,14(1):30-32.
[47]王欣,张轴,杨红,等.多发性骨髓瘤血清MUC1表达的临床意义[J].白血病.淋巴瘤,2008,17(5):348-350.
[48] Treon SP, Maimonis P, Bna D, et a1．Elevated soluble MUCl levels and decreasedanti-MUCl antibody levels in patients with multiple myeloma[J]. Blood,2000,96(9):3147-3153.
[49] Ross AA, Cooper BW, Lazarus HM. Incidence of tumor cell contamination inperipheral blood stem cell (PBSC) collections from breast cancer patients [J]. J Pro AmSoc Clin Oncol.1993,12:69
[50] Zhong YX, Kaul S, Eichner A, et al. Evaluation of carcinoembryonic antigen reversetranscriptase-polymerase chain reaction for the detection of breast cancer disseminationin bone marrow and peripheral blood [J]. J Cancer Res Clin Oncol.1999,125(12):669-674.
[51] Dockhorn-Dworniczak B, Sch fer K-L, Dantcheva R, et al. Diagnostic value of themolecular genetic detection of the t(11;22) translocation in Ewing’s tumors [J].Virchows Arch,1994,425(2):107-12.
[52] Ferreira CS, Papamichael K, Guilbault G, et al. DNA aptamers against the MUC1tumour marker: design of aptamer-antibody sandwich ELISA for the early diagnosis ofepithelial tumours [J]. Anal Bioanal Chem,2008,390(4):1039-1050.
[53] Klee G, Schreiber W. E. MUC1gene-derived glycoprotein assays for monitoring breastcancer (CA15-3, CA27.29, BR): are they measuring the same antigen[J]? Arch. Pathol.Lab. Med.2004,128:1131-1135.
[54]张之南,沈悌(主编)血液病诊断及疗效标准.第3版[M].北京:科学出版社,2007:1-268.
[55]丘立文,汤汉文,王亚娣,等.酶联免疫吸附试验间接抗体夹心检测SARS-CoV抗原方法的建立和应用[J].中华流行病血杂志,2005,26(4):277-281.
[56]田满福,韩波.检测CA199、CA125、CA153及CEA在肿瘤诊断中的意义[J].临床和实验医学杂志,2010,9(7):483-485.
[57]刘卫星,邱慧敏,许德凤.血清CA153等4种肿瘤标志物联合检测对肺癌的诊断价值[J].同济大学学报(医学版),2001,22(5):60.
[58] Shibayama T, Ueoka H, Nishii K, et al. Complenmentary roles of pro-gastrin releasingpeptide (proGRP) and neuron specific enolase (NSE) in diagnoses and prognosis ofsmall-cell lung cancer (SCLC)[J]. Lung Cancer,2001,32(1):61-69.
[59]曾波航,蔡燕燕,吕嘉春,等.小细胞肺癌肿瘤标志物:血清NSE与CYFRA21-1和CEA[J].现代临床医学生物工程杂志,2003,9(3):216-218.