肖明兵 謝玲 倪溫慨 陳不尤 陸翠華 李小彥 江楓 倪潤(rùn)洲

·論著·

血清半乳糖凝集素3 對(duì)胰腺癌的診斷價(jià)值

肖明兵 謝玲 倪溫慨 陳不尤 陸翠華 李小彥 江楓 倪潤(rùn)洲

目的運(yùn)用時(shí)間分解免疫熒光(TRFIA)法檢測(cè)血清半乳糖凝集素3(Galectin-3，Gal-3)水平，并探討Gal-3對(duì)胰腺癌的診斷價(jià)值。方法采用固相雙抗體夾心法建立檢測(cè)血清Gal-3的TRFIA，探討最佳實(shí)驗(yàn)條件。在最適條件下檢測(cè)胰腺癌、胰腺良性占位、胰腺炎患者及健康對(duì)照者血清Gal-3水平，并聯(lián)合檢測(cè)血清CEA及CA19-9水平。結(jié)果TRFIA法檢測(cè)血清Gal-3的線性為0～100 μg/L，批內(nèi)變異系數(shù)(CV)≤6.45%，批間CV≤8.68%，平均回收率為106.6%。胰腺癌患者血清Gal-3水平為4.93(0.85～23.80)μg/L，明顯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為界，其診斷胰腺癌的敏感性為75.5%，特異性達(dá)90.9%。Gal-3與CEA及CA19-9水平均無(wú)相關(guān)性(r=0.1321，P=0.3761；r=0.0920，P=0.5384)，Gal-3聯(lián)合CEA或CA19-9檢測(cè)，對(duì)胰腺癌的診斷敏感性可提高到92%。結(jié)論TRFIA法檢測(cè)血清Gal-3具有較好的敏感性和穩(wěn)定性；Gal-3有望成為新的胰腺癌標(biāo)記物。

胰腺腫瘤； 半乳糖凝集素3； 時(shí)間分解免疫熒光測(cè)定； 血清

目前臨床上常用的胰腺癌腫瘤標(biāo)志物CA19-9和CEA的敏感性和特異性均不太高。如何提高胰腺癌早期定性診斷水平是臨床重要課題。半乳糖凝集素-3(Galectin-3，Gal-3)是半乳凝素家族的一員，能識(shí)別糖蛋白和糖脂的特異性低聚糖結(jié)構(gòu)，在某些腫瘤組織中高表達(dá)[1-2]，參與腫瘤細(xì)胞與血管內(nèi)皮的黏附、血管生成和腫瘤免疫逃避，抑制腫瘤細(xì)胞凋亡。我們?cè)\(yùn)用MALDI-TOF-MS技術(shù)，發(fā)現(xiàn)胰腺癌組織中Gal-3表達(dá)水平明顯上調(diào)[3]。但迄今國(guó)內(nèi)外尚未見(jiàn)胰腺癌血清Gal-3的研究報(bào)道。為此，本研究檢測(cè)胰腺癌患者血清Gal-3水平，探討其對(duì)胰腺癌的診斷價(jià)值。

材料與方法

一、研究對(duì)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，中位年齡67歲；胰腺良性占位患者16例，男性9例，女性7例，中位年齡45歲；急性胰腺炎患者36例，男性22例，女性14例，中位年齡53歲。所有病例均經(jīng)臨床和(或)病理學(xué)證實(shí)。以體檢健康者36例作為對(duì)照，男性18例，女性18例，中位年齡20歲。均在治療前采集清晨空腹靜脈血，分離血清后立即置-80℃冰箱保存待測(cè)。

二、血清Gal-3水平檢測(cè)

采用自行建立的固相雙抗體夾心TRFIA法檢測(cè)。用包被稀釋液(0.05 mol/L碳酸鈉-碳酸氫鈉緩沖液，pH9.6，)將鼠抗人Gal-3單抗(Santa Cruz公司)稀釋至4 000 μg/L，聚苯乙烯板每孔加入100 μl 4℃包被過(guò)夜。棄包被抗體，PBS洗滌后每孔加入含10 g/L BSA的PBS封閉液200 μl/L， 37℃孵育1 h。棄封閉液，洗滌后每孔分別加入系列稀釋的Gal-3標(biāo)準(zhǔn)蛋白(ABR公司)、待測(cè)血清樣本50 μl，以單加封閉液為空白對(duì)照，37℃孵育1 h。洗滌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。洗滌后每孔加入用含10 g/L BSA的PBS稀釋的稀土離子(Eu3+)-標(biāo)鏈親和素100 μl(600 μg/L，PE公司)37℃避光孵育1 h。洗滌后每孔加入熒光增強(qiáng)液200 μl(PE公司)，室溫避光緩慢水平搖動(dòng)5 min。在VICTORTM X5型自動(dòng)時(shí)間分辨熒光檢測(cè)儀上設(shè)定。激發(fā)光波長(zhǎng)為337 nm，發(fā)射光波長(zhǎng)為615 nm，延時(shí)200 μs，測(cè)定615 nm處的熒光值(A615值)。以Gal-3標(biāo)準(zhǔn)蛋白的熒光值繪制標(biāo)準(zhǔn)曲線，根據(jù)樣品的熒光值計(jì)算Gal-3的濃度。

三、血清CEA和CA19-9水平檢測(cè)

采用化學(xué)發(fā)光免疫檢測(cè)法，檢測(cè)試劑盒購(gòu)自美國(guó)雅培公司，按說(shuō)明書(shū)操作。

四、統(tǒng)計(jì)學(xué)處理

采用Stata8.0及SPSS15.0統(tǒng)計(jì)軟件進(jìn)行分析。血清Gal-3、CEA及CA19-9值用中位數(shù)表示，各組間的差異采用秩和檢驗(yàn)；率的比較采用χ2檢驗(yàn)，當(dāng)理論頻數(shù)小于5時(shí)，采用Fisher′s確切概率法；血清Gal-3、CEA及CA19-9三者間的相關(guān)分析采用Spearman等級(jí)相關(guān)方法。界值的確定用受試者工作特征曲線(ROC)。P<0.05為差異具有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

一、Gal-3檢測(cè)最適條件的確立

將抗Gal-3單抗、生物素化的羊抗人Gal-3多抗及Eu3+-標(biāo)鏈親和素分別系列稀釋?zhuān)?2.5 μg/L的Gal-3標(biāo)準(zhǔn)蛋白為陽(yáng)性對(duì)照，封閉液為陰性對(duì)照，設(shè)3個(gè)復(fù)孔，讀取各孔熒光值。當(dāng)抗Gal-3單抗、多抗及Eu3+-標(biāo)鏈親和素濃度分別為4000、80及600 μg/L時(shí)，陽(yáng)性對(duì)照與陰性對(duì)照平均熒光值之比(P/N值)最大(8.05)，為最適工作濃度。

二、檢測(cè)方法學(xué)評(píng)價(jià)

將Gal-3標(biāo)準(zhǔn)蛋白系列稀釋?zhuān)陨鲜鲎钸m條件測(cè)定，在0.78～100 μg/L范圍內(nèi)成線性關(guān)系(圖1)。

圖1 GAL-3 檢測(cè)劑量-反應(yīng)曲線圖

將Gal-3標(biāo)準(zhǔn)蛋白稀釋至0 μg/L附近，作為檢測(cè)零點(diǎn)樣品，重復(fù)測(cè)量20次，計(jì)算其熒光均值及標(biāo)準(zhǔn)差。以熒光均值加上2倍的標(biāo)準(zhǔn)差所得的熒光值代入標(biāo)準(zhǔn)曲線方程計(jì)算得出的濃度為其最低檢測(cè)量。本法靈敏度為0.181 μg/L。

將Gal-3標(biāo)準(zhǔn)蛋白稀釋成3.13、6.25、12.5 μg/L作為質(zhì)控樣品，分三批次進(jìn)行測(cè)定，每批次各設(shè)8個(gè)復(fù)孔，批內(nèi)變異系數(shù)(CV)≤6.45%，批間CV≤8.68%(表1)。

取3份胰腺癌患者血清混合，配制兩份Gal-3濃度為12.5 μg/L及3.1 μg/L的標(biāo)準(zhǔn)品，按下法配制試驗(yàn)樣品：基礎(chǔ)樣品為血清0.9 ml加蒸餾水0.1 ml；分析樣品為血清0.9 ml加標(biāo)準(zhǔn)品(12.5 μg/L或3.1 μg/L)0.1 ml。按上述最適條件測(cè)定，每份樣品設(shè)8個(gè)復(fù)孔，求均值。兩分析樣品回收濃度分別為1.29、0.34 μg/L，回收率分別為103.5%及109.7%，平均回收率為106.6%。

三、血清Gal-3、CEA、CA19-9水平及三者間相關(guān)分析

因Gal-3、CEA及CA19-9水平均呈偏態(tài)分布，故以中位數(shù)表示。胰腺癌患者血清Gal-3、CEA及CA19-9水平均明顯高于其他各組(表2，P值均<0.05)。

表1 不同濃度Gal-3各批次的熒光值

表2 各組血清Gal-3、CEA及CA19-9水平(中位數(shù)，范圍)

注：經(jīng)秩和檢驗(yàn)，a與其他各組比，Z值分別為3.778、4.874及6.379，P值分別為0.0002、0.0000及0.0000；b與其他各組比，Z值分別為4.723、4.839及5.898，P值均為0.0000；c與其他各組比，Z值分別為3.007、3.508及3.899，P值分別為0.0026、0.0005及0.0001

根據(jù)ROC曲線(圖2)，Gal-3、CEA及CA19-9對(duì)良、惡性胰腺疾病的診斷界值分別為3.77 μg/L、3.82 μg/L及41.61 kU/L，診斷胰腺癌的敏感性在70%左右，特異性在90%以上(表3)。Gal-3與CEA、CA19-9之間無(wú)相關(guān)性(r=0.1321，P=0.3761；r=0.0920，P=0.5384)，而CEA與CA19-9之間呈正相關(guān)(r=0.3982，P=0.0056)。Gal-3聯(lián)合CA19-9或Gal-3聯(lián)合CEA檢測(cè)可明顯提高對(duì)胰腺癌的診斷敏感性，以Gal-3+CA19-9略?xún)?yōu)于Gal-3+CEA，但兩者無(wú)明顯差異(表3)。

圖2 Gal-3、CEA及CA19-9診斷胰腺癌的ROC曲線

Gal-3是一種糖結(jié)合蛋白，分子質(zhì)量約30 000，對(duì)β-半乳糖苷具有親和性。上皮細(xì)胞、血管內(nèi)皮細(xì)胞[4]、活化的巨噬細(xì)胞[5]及樹(shù)突狀細(xì)胞等[6]均表達(dá)Gal-3，它在細(xì)胞質(zhì)內(nèi)合成，但可以運(yùn)到胞核或分泌到細(xì)胞外，參與不同的生物過(guò)程。Gal-3在多種腫瘤中高表達(dá)，如結(jié)直腸癌[7]、胃癌[8]、肝癌[9]、甲狀腺癌[10]、垂體腺瘤[10]、腎透明細(xì)胞癌[11]、浸潤(rùn)性乳腺癌[12]、舌鱗狀細(xì)胞癌[13]、惡性嗜鉻細(xì)胞瘤[2]、上皮性卵巢癌[14]、膀胱癌[15]等，其表達(dá)的增加與腫瘤的轉(zhuǎn)移和疾病進(jìn)展呈正相關(guān)。

TRFIA技術(shù)利用3價(jià)稀土離子(Eu3+)代替熒光物質(zhì)、放射性核素或酶為示蹤物，標(biāo)記抗體、抗原、激素、多肽、蛋白質(zhì)、核酸探針及生物細(xì)胞，抗原抗體反應(yīng)后用檢測(cè)儀測(cè)定反應(yīng)產(chǎn)物中的熒光強(qiáng)度，判斷分析物濃度。檢測(cè)系統(tǒng)可實(shí)現(xiàn)全部自動(dòng)化，同時(shí)利用波長(zhǎng)和時(shí)間兩種分辨有效地排除了非特異熒光，大大提高了分析靈敏度。具有操作簡(jiǎn)便、靈敏度高、不受樣品自然熒光干擾、示蹤物穩(wěn)定、標(biāo)準(zhǔn)曲線范圍寬、無(wú)放射性污染、標(biāo)記物存儲(chǔ)時(shí)間長(zhǎng)等優(yōu)點(diǎn)[16]。本研究建立的血清Gal-3的TRFIA檢測(cè)法，具有分析線性范圍寬、靈敏度高、重復(fù)性及回收率好等特點(diǎn)，符合臨床體外診斷試劑檢測(cè)的要求。

表3 Gal-3聯(lián)合CEA及CA19-9檢測(cè)診斷胰腺癌的價(jià)值

本研究結(jié)果顯示，胰腺癌患者血清Gal-3明顯高于健康人及良性胰腺疾病患者，其診斷胰腺癌的敏感性與CA19-9及CEA相仿，且與CEA及CA19-9均無(wú)相關(guān)性。Gal-3聯(lián)合CEA或CA19-9檢測(cè)?？商岣咴\斷敏感性，表明Gal-3對(duì)胰腺癌的診斷具有較好的臨床價(jià)值。

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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

江蘇省“六大人才高峰”資助項(xiàng)目(2006073)；江蘇省衛(wèi)生廳資助項(xiàng)目(H200923)；南通市科技計(jì)劃資助項(xiàng)目(S2010012)

226001 南通，南通大學(xué)附屬醫(yī)院消化內(nèi)科

倪潤(rùn)洲，Email: nirz@163.com

2011-06-12)

(本文編輯：屠振興)