血清半乳糖凝集素3 对胰腺癌的诊断价值

肖明兵 谢玲 倪温慨 陈不尤 陆翠华 李小彦 江枫 倪润洲

·论著·

血清半乳糖凝集素3 对胰腺癌的诊断价值

肖明兵 谢玲 倪温慨 陈不尤 陆翠华 李小彦 江枫 倪润洲

目的运用时间分解免疫荧光(TRFIA)法检测血清半乳糖凝集素3(Galectin-3，Gal-3)水平，并探讨Gal-3对胰腺癌的诊断价值。方法采用固相双抗体夹心法建立检测血清Gal-3的TRFIA，探讨最佳实验条件。在最适条件下检测胰腺癌、胰腺良性占位、胰腺炎患者及健康对照者血清Gal-3水平，并联合检测血清CEA及CA19-9水平。结果TRFIA法检测血清Gal-3的线性为0～100 μg/L，批内变异系数(CV)≤6.45%，批间CV≤8.68%，平均回收率为106.6%。胰腺癌患者血清Gal-3水平为4.93(0.85～23.80)μg/L，明显高于胰腺良性占位者的2.83(2.17～4.06)μg/L、胰腺炎患者的2.62(0.55～9.76)μg/L和健康者的1.88(0.59～3.94)μg/L(P值均<0.05)。以3.77 μg/L为界，其诊断胰腺癌的敏感性为75.5%，特异性达90.9%。Gal-3与CEA及CA19-9水平均无相关性(r=0.1321，P=0.3761；r=0.0920，P=0.5384)，Gal-3联合CEA或CA19-9检测，对胰腺癌的诊断敏感性可提高到92%。结论TRFIA法检测血清Gal-3具有较好的敏感性和稳定性；Gal-3有望成为新的胰腺癌标记物。

胰腺肿瘤； 半乳糖凝集素3； 时间分解免疫荧光测定； 血清

目前临床上常用的胰腺癌肿瘤标志物CA19-9和CEA的敏感性和特异性均不太高。如何提高胰腺癌早期定性诊断水平是临床重要课题。半乳糖凝集素-3(Galectin-3，Gal-3)是半乳凝素家族的一员，能识别糖蛋白和糖脂的特异性低聚糖结构，在某些肿瘤组织中高表达[1-2]，参与肿瘤细胞与血管内皮的黏附、血管生成和肿瘤免疫逃避，抑制肿瘤细胞凋亡。我们曾运用MALDI-TOF-MS技术，发现胰腺癌组织中Gal-3表达水平明显上调[3]。但迄今国内外尚未见胰腺癌血清Gal-3的研究报道。为此，本研究检测胰腺癌患者血清Gal-3水平，探讨其对胰腺癌的诊断价值。

材料与方法

一、研究对象

收集本院2008年10月至2010年10月住院的胰腺癌患者49例，男性29例，女性20例，中位年龄67岁；胰腺良性占位患者16例，男性9例，女性7例，中位年龄45岁；急性胰腺炎患者36例，男性22例，女性14例，中位年龄53岁。所有病例均经临床和(或)病理学证实。以体检健康者36例作为对照，男性18例，女性18例，中位年龄20岁。均在治疗前采集清晨空腹静脉血，分离血清后立即置-80℃冰箱保存待测。

二、血清Gal-3水平检测

采用自行建立的固相双抗体夹心TRFIA法检测。用包被稀释液(0.05 mol/L碳酸钠-碳酸氢钠缓冲液，pH9.6，)将鼠抗人Gal-3单抗(Santa Cruz公司)稀释至4 000 μg/L，聚苯乙烯板每孔加入100 μl 4℃包被过夜。弃包被抗体，PBS洗涤后每孔加入含10 g/L BSA的PBS封闭液200 μl/L， 37℃孵育1 h。弃封闭液，洗涤后每孔分别加入系列稀释的Gal-3标准蛋白(ABR公司)、待测血清样本50 μl，以单加封闭液为空白对照，37℃孵育1 h。洗涤后加入生物素化羊抗人Gal-3多抗100 μl(80 μg/L，Santa Cruz公司)37℃孵育1 h。洗涤后每孔加入用含10 g/L BSA的PBS稀释的稀土离子(Eu3+)-标链亲和素100 μl(600 μg/L，PE公司)37℃避光孵育1 h。洗涤后每孔加入荧光增强液200 μl(PE公司)，室温避光缓慢水平摇动5 min。在VICTORTM X5型自动时间分辨荧光检测仪上设定。激发光波长为337 nm，发射光波长为615 nm，延时200 μs，测定615 nm处的荧光值(A615值)。以Gal-3标准蛋白的荧光值绘制标准曲线，根据样品的荧光值计算Gal-3的浓度。

三、血清CEA和CA19-9水平检测

采用化学发光免疫检测法，检测试剂盒购自美国雅培公司，按说明书操作。

四、统计学处理

采用Stata8.0及SPSS15.0统计软件进行分析。血清Gal-3、CEA及CA19-9值用中位数表示，各组间的差异采用秩和检验；率的比较采用χ2检验，当理论频数小于5时，采用Fisher′s确切概率法；血清Gal-3、CEA及CA19-9三者间的相关分析采用Spearman等级相关方法。界值的确定用受试者工作特征曲线(ROC)。P<0.05为差异具有统计学意义。

结 果

一、Gal-3检测最适条件的确立

将抗Gal-3单抗、生物素化的羊抗人Gal-3多抗及Eu3+-标链亲和素分别系列稀释，以12.5 μg/L的Gal-3标准蛋白为阳性对照，封闭液为阴性对照，设3个复孔，读取各孔荧光值。当抗Gal-3单抗、多抗及Eu3+-标链亲和素浓度分别为4000、80及600 μg/L时，阳性对照与阴性对照平均荧光值之比(P/N值)最大(8.05)，为最适工作浓度。

二、检测方法学评价

将Gal-3标准蛋白系列稀释，以上述最适条件测定，在0.78～100 μg/L范围内成线性关系(图1)。

图1 GAL-3 检测剂量-反应曲线图

将Gal-3标准蛋白稀释至0 μg/L附近，作为检测零点样品，重复测量20次，计算其荧光均值及标准差。以荧光均值加上2倍的标准差所得的荧光值代入标准曲线方程计算得出的浓度为其最低检测量。本法灵敏度为0.181 μg/L。

将Gal-3标准蛋白稀释成3.13、6.25、12.5 μg/L作为质控样品，分三批次进行测定，每批次各设8个复孔，批内变异系数(CV)≤6.45%，批间CV≤8.68%(表1)。

取3份胰腺癌患者血清混合，配制两份Gal-3浓度为12.5 μg/L及3.1 μg/L的标准品，按下法配制试验样品：基础样品为血清0.9 ml加蒸馏水0.1 ml；分析样品为血清0.9 ml加标准品(12.5 μg/L或3.1 μg/L)0.1 ml。按上述最适条件测定，每份样品设8个复孔，求均值。两分析样品回收浓度分别为1.29、0.34 μg/L，回收率分别为103.5%及109.7%，平均回收率为106.6%。

三、血清Gal-3、CEA、CA19-9水平及三者间相关分析

因Gal-3、CEA及CA19-9水平均呈偏态分布，故以中位数表示。胰腺癌患者血清Gal-3、CEA及CA19-9水平均明显高于其他各组(表2，P值均<0.05)。

表1 不同浓度Gal-3各批次的荧光值

表2 各组血清Gal-3、CEA及CA19-9水平(中位数，范围)

注：经秩和检验，a与其他各组比，Z值分别为3.778、4.874及6.379，P值分别为0.0002、0.0000及0.0000；b与其他各组比，Z值分别为4.723、4.839及5.898，P值均为0.0000；c与其他各组比，Z值分别为3.007、3.508及3.899，P值分别为0.0026、0.0005及0.0001

根据ROC曲线(图2)，Gal-3、CEA及CA19-9对良、恶性胰腺疾病的诊断界值分别为3.77 μg/L、3.82 μg/L及41.61 kU/L，诊断胰腺癌的敏感性在70%左右，特异性在90%以上(表3)。Gal-3与CEA、CA19-9之间无相关性(r=0.1321，P=0.3761；r=0.0920，P=0.5384)，而CEA与CA19-9之间呈正相关(r=0.3982，P=0.0056)。Gal-3联合CA19-9或Gal-3联合CEA检测可明显提高对胰腺癌的诊断敏感性，以Gal-3+CA19-9略优于Gal-3+CEA，但两者无明显差异(表3)。

图2 Gal-3、CEA及CA19-9诊断胰腺癌的ROC曲线

Gal-3是一种糖结合蛋白，分子质量约30 000，对β-半乳糖苷具有亲和性。上皮细胞、血管内皮细胞[4]、活化的巨噬细胞[5]及树突状细胞等[6]均表达Gal-3，它在细胞质内合成，但可以运到胞核或分泌到细胞外，参与不同的生物过程。Gal-3在多种肿瘤中高表达，如结直肠癌[7]、胃癌[8]、肝癌[9]、甲状腺癌[10]、垂体腺瘤[10]、肾透明细胞癌[11]、浸润性乳腺癌[12]、舌鳞状细胞癌[13]、恶性嗜铬细胞瘤[2]、上皮性卵巢癌[14]、膀胱癌[15]等，其表达的增加与肿瘤的转移和疾病进展呈正相关。

TRFIA技术利用3价稀土离子(Eu3+)代替荧光物质、放射性核素或酶为示踪物，标记抗体、抗原、激素、多肽、蛋白质、核酸探针及生物细胞，抗原抗体反应后用检测仪测定反应产物中的荧光强度，判断分析物浓度。检测系统可实现全部自动化，同时利用波长和时间两种分辨有效地排除了非特异荧光，大大提高了分析灵敏度。具有操作简便、灵敏度高、不受样品自然荧光干扰、示踪物稳定、标准曲线范围宽、无放射性污染、标记物存储时间长等优点[16]。本研究建立的血清Gal-3的TRFIA检测法，具有分析线性范围宽、灵敏度高、重复性及回收率好等特点，符合临床体外诊断试剂检测的要求。

表3 Gal-3联合CEA及CA19-9检测诊断胰腺癌的价值

本研究结果显示，胰腺癌患者血清Gal-3明显高于健康人及良性胰腺疾病患者，其诊断胰腺癌的敏感性与CA19-9及CEA相仿，且与CEA及CA19-9均无相关性。Gal-3联合CEA或CA19-9检测。可提高诊断敏感性，表明Gal-3对胰腺癌的诊断具有较好的临床价值。

[1] Htwe TT, Karim N, Wong J, et al. Differential expression of galectin-3 in advancing thyroid cancer cells: a clue toward understanding tumour progression and metastasis. Singapore Med J,2010,51:856-859.

[2] Saffar H, Sanii S, Heshmat R, et al. Expression of galectin-3, nm-23, and cyclooxygenase-2 could potentially discriminate between benign and malignant pheochromocytoma. Am J Clin Pathol, 2011,135:454-460.

[3] Chen JH, Ni RZ, Xiao MB, et al. Comparative proteomic analysis of differentially expressed proteins in human pancreatic cancer tissue. Hepatobiliary Pancreat Dis Int, 2009,8:193-200.

[4] Khaldoyanidi SK, Glinsky VV, Sikora L, et al. MDA-MB-435 human breast carcinoma cell homo-and heterotypic adhesion under flow conditions is mediated in part by Thomsen-Friedenreich antigengalectin-3 interactions.J Biol Chem, 2003, 278:4127-4134.

[5] Kim K, Mayer EP, Nachtigal M. Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins.Biochim Biophys Acta, 2003, 1641:13-23.

[6] Vray B, Camby I, Vercruysse V, et al. Up-regulation of galectin-3 and its ligands by Trypanosoma cruzi infection with modulation of adhesion and migration of murine dendritic cells. Glycobiology, 2004, 14:647-657.

[7] Zaia Povegliano L, Oshima CT, de Oliveira Lima F, et al. Immunoexpression of galectin-3 in colorectal cancer and its relationship with survival. J Gastrointest Cancer, 2011,42:217-221.

[8] Okada K, Shimura T, Suehiro T, et al. Reduced galectin-3 expression is an indicator of unfavorable prognosis in gastric cancer. Anticancer Res, 2006, 26:1369-1376.

[9] Matsuda Y, Yamagiwa Y, Fukushima K, et al. Expression of galectin-3 involved in prognosis of patients with hepatocellular carcinoma. Hepatol Res, 2008, 38:1098-1111.

[10] Righi A, Jin L, Zhang S, et al. Identification and consequences of galectin-3 expression in pituitary tumors. Mol Cell Endocrinol, 2010, 326:8-14.

[11] Sakaki M, Fukumori T, Fukawa T, et al. Clinical significance of Galectin-3 in clear cell renal cell carcinoma. J Med Invest, 2010, 57:152-157.

[12] Koo JS, Jung W. Clinicopathlogic and immunohistochemical characteristics of triple negative invasive lobular carcinoma. Yonsei Med J, 2011, 52:89-97.

[13] Alves PM, Godoy GP, Gomes DQ, et al. Significance of galectins-1, -3, -4 and -7 in the progression of squamous cell carcinoma of the tongue. Pathol Res Pract, 2011, 207:236-240.

[14] Kim MK, Sung CO, Do IG, et al. Overexpression of Galectin-3 and its clinical significance in ovarian carcinoma. Int J Clin Oncol, 2011, 16:352-358.

[15] Canesin G,Gonzalez-Peramato P,Palou J,et al.Galectin-3 expression is associated with bladder cancer progression and clinical outcome.Tumour Biol,2010,31:277-285.

[16] Andoh T, Nagasawa H. Development of a time-resolved fluoroimmunoassay for insulins and its application to monitoring of insulin secretion induced by feeding in the barfin flounder, Verasper moseri. Gen Comp Endocrinol, 2002, 125:365-374.

Dignosisvalueofserumglypican-3forpancreascancer

XIAOMing-bing,XIELin,NIWen-kai,CHENBu-you,LUCui-hua,LIXiao-yan,JIANGFeng,NIRun-zhou.
DepartmentofGastroenterology,AffiliatedHospitalofNantongUniversity,Nantong226001,China

NIRun-zhou,Email:nirz@163.com

ObjectiveTo establish the time-resolved fluoroimmunoassay (TRFIA) method for the detection of serum galectin-3 and investigate the clinical value of serum galectin-3 for the diagnosis of pancreas cancer.MethodsMonoclonal anti-human galectin-3 antibody and biotinylated polyclonal antibody were used to establish the sandwich TRFIA for detection of serum galectin-3. The optimal experimental condition was studied. Serum levels of galectin-3, CEA and CA19-9 in the patients with pancreatic cancer, benign pancreatic mass, pancreatitis, and healthy controls were measured. The diagnostic value of serum galectin-3, CEA and CA19-9 for pancreas cancer was studied.ResultsThe linearity of the TRFIA for detection of serum galectin-3 ranged between 0 to 100 μg/L. The within-run CV and between-run CV were ≤6.45% and ≤8.68%, respectively, and the average recovery was 106.6%. The level of serum galectin-3 was 4.93(0.85～23.80)μg/L in pancreatic cancer group, which were significantly higher than those in benign pancreatic mass [2.83(2.17～4.06)μg/L], pancreatitis [2.62(0.55～9.76)μg/L], and healthy controls group [1.88(0.59～3.94)μg/L](P<0.05). By using 3.77 μg/L as the cut-off point, the sensitivity, specificity for the diagnosis of pancreatic cancer was 75.5% and 90.9%. The levels of Gal 3 and CEA, CA19-9 was not correlated (r=0.1321,P=0.3761;r=0.0920,P=0.5384). Combined determination of galactin-3 and CEA, CA19-9 levels could increase the diagnostic sensitivity to 92%.ConclusionsTRFIA method for the detection of galactin-3 is sensitive and stable. Galectin-3 could be a potentially novel serum tumor marker of pancreatic cancer.

Pancreatic neoplasms; Galectin-3; Time-resolved fluoroimmunoassay; Serum

10.3760/cma.j.issn.1674-1935.2012.02.001

江苏省“六大人才高峰”资助项目(2006073)；江苏省卫生厅资助项目(H200923)；南通市科技计划资助项目(S2010012)

226001 南通，南通大学附属医院消化内科

倪润洲，Email: nirz@163.com

2011-06-12)

(本文编辑：屠振兴)