抗survivin抗體與survivin mRNA在結(jié)直腸癌患者外周血中的表達(dá)及其臨床意義

施朕善,康馬飛,李文見(jiàn)

(桂林醫(yī)學(xué)院附屬醫(yī)院腫瘤內(nèi)科,廣西桂林 541001)

抗survivin抗體與survivin mRNA在結(jié)直腸癌患者外周血中的表達(dá)及其臨床意義

施朕善,康馬飛△,李文見(jiàn)

(桂林醫(yī)學(xué)院附屬醫(yī)院腫瘤內(nèi)科,廣西桂林 541001)

目的探討結(jié)直腸癌(CRC)患者外周血中抗survivin抗體?survivin mRNA的表達(dá)情況及其臨床意義?方法 選擇結(jié)直腸癌患者116例,依據(jù)所接受治療手段,分為根治手術(shù)組?姑息手術(shù)組?姑息化療組?對(duì)照組為30例健康體檢者?分別于患者治療前后抽取外周血,應(yīng)用ELISA和半定量RT-PCR檢測(cè)抗survivin抗體和survivin mRNA的表達(dá)水平?結(jié)果結(jié)直腸癌患者與健康者外周血中抗survivin抗體陽(yáng)性率分別為40.5%和0,survivin mRNA陽(yáng)性率分別為76.7%和0,差異均有統(tǒng)計(jì)學(xué)意義(P=0.000)?抗survivin抗體和survivin mRNA表達(dá)水平隨腫瘤分期的增高而增高,腫瘤直徑大于5cm者較直徑小于或等于5cm者高(分別P=0.045?P=0.003),有淋巴結(jié)轉(zhuǎn)移者較無(wú)淋巴結(jié)轉(zhuǎn)移者顯著增高(分別P=0.040?P=0.000);根治手術(shù)后較術(shù)前明顯降低(分別P=0.005?P=0.004),根治術(shù)后表達(dá)水平高者轉(zhuǎn)移或復(fù)發(fā)率顯著高于表達(dá)水平低者(分別P=0.001?P=0.004);表達(dá)水平在姑息手術(shù)前后差異無(wú)統(tǒng)計(jì)學(xué)意義(分別P=0.709?P=0.669)?行姑息性化療的患者中,有效組表達(dá)水平較化療前明顯降低(分別P=0.043?P=0.032);化療無(wú)效組抗survivin抗體水平變化無(wú)統(tǒng)計(jì)學(xué)意義(P=0.135),而survivin mRNA表達(dá)較前顯著升高(P=0.039)?結(jié)論外周血抗survivin抗體和survivin mRNA檢測(cè)可能對(duì)CRC的診斷?預(yù)后估計(jì)?化療療效預(yù)測(cè)及隨訪(fǎng)有一定的臨床意義?

結(jié)直腸腫瘤;診斷;survivin

結(jié)直腸癌(colorectal cancer,CRC)是常見(jiàn)消化道惡性腫瘤之一,CRC的早期診斷?轉(zhuǎn)移或復(fù)發(fā)監(jiān)測(cè)是臨床中的重要問(wèn)題,尋找方便有效的標(biāo)志物顯得尤為重要?survivin作為凋亡抑制蛋白家族成員之一,已被證實(shí)在包括CRC在內(nèi)的多種惡性腫瘤中過(guò)表達(dá)[1]?目前的研究大多數(shù)僅為觀察腫瘤組織中的survivin表達(dá)情況,不利于動(dòng)態(tài)觀察其表達(dá)變化情況?研究表明,腫瘤患者外周血中有抗survivin抗體[2]?為探討外周血抗survivin抗體以及survivin mRNA在CRC發(fā)生過(guò)程中的臨床意義,本組對(duì)116例CRC患者進(jìn)行了上述指標(biāo)的檢測(cè),現(xiàn)報(bào)道如下?

1 資料與方法

1.1 一般資料 收集2009年10月至2011年6月期間在本院住院治療的CRC患者116例,其中,結(jié)腸癌42例,直腸癌74例;年齡27～84歲,中位58歲?所有患者均經(jīng)病理學(xué)診斷,臨床資料完整?依據(jù)所接受治療手段,分為根治手術(shù)組?姑息手術(shù)組和姑息化療組?對(duì)照組納入從本院體檢中心收集的30例健康人,年齡31～80歲,中位56歲?

1.2 標(biāo)本的采集與處理 手術(shù)組于手術(shù)前1天和術(shù)后第2周末清晨空腹采集外周血5mL,其中,根治手術(shù)組每3月隨訪(fǎng)一次,抽外周血5mL;姑息化療組于化療前及化療2周期后采集外周血5mL;對(duì)照組空腹采集外周血5mL?所采集血液標(biāo)本中,其中2mL使用無(wú)抗凝劑的真空管采集,以3 000r/min離心10min,取上清液,-20℃凍存,用于檢測(cè)抗survivin抗體;剩余3mL使用含有乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)抗凝劑真空管采集,室溫靜置30min,用于提取總RNA?

1.3 抗survivin抗體的測(cè)定 應(yīng)用雙位點(diǎn)夾心酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay,ELISA)測(cè)定血清中抗survivin抗體的水平,采用R&D公司提供的人survivin抗體定量檢測(cè)試劑盒?

1.4 survivin mRNA的檢測(cè) 應(yīng)用康為世紀(jì)公司血液RNA提取試劑盒(Cat.NO:CW0538),按照說(shuō)明書(shū)提取總RNA?紫外分光光度計(jì)進(jìn)行定量并檢測(cè)其純度,1.5%瓊脂糖電泳鑒定RNA完整性?應(yīng)用TaKaRa公司的PrimeScript RT reagent Kit(TaKaRa Code:DDR037A)按說(shuō)明書(shū)進(jìn)行反轉(zhuǎn)錄?反轉(zhuǎn)錄條件為37℃15min,85℃5s?以β-actin作內(nèi)參基因,應(yīng)用Primer Premier 5.0軟件設(shè)計(jì)引物?Survivin mRNA上?下游引物分別為5＇-GTCCCTGGCTCCTCTACTG-3＇,5＇-GACGCTTCCTATCACTCTATTC-3＇,產(chǎn)物為174bp;β-actin上?下游引物 分 別 為 5＇-AGAGCCTCGCCTTTGCCGAT-3＇,5＇-TGCCAGATTTTCTCCATGTCGT-3＇,產(chǎn)物為 313bp?所有引物由Invitrigen公司合成?使用TaKaRa公司的Premix Ex Taq試劑盒進(jìn)行PCR,經(jīng)過(guò)預(yù)變性95℃30s,34個(gè)循環(huán)(變性95℃5s,退火60℃30s,延伸72℃50s),終延伸72℃2min,其中survivin循環(huán)數(shù)為34,退火溫度60℃,β-actin循環(huán)數(shù)為28,退火溫度62℃?所得產(chǎn)物進(jìn)行2.5%瓊脂糖電泳鑒定(圖1),并應(yīng)用上海培清JS-6800凝膠成像分析系統(tǒng),分別測(cè)定β-actin和survivin條帶的光密度值,并計(jì)算survivin的表達(dá)系數(shù),即survivin的光密度值與β-actin的光密度值之比?

1.5 隨訪(fǎng) 起點(diǎn)為首次檢測(cè)survivin時(shí)間,終點(diǎn)為發(fā)現(xiàn)復(fù)發(fā)或轉(zhuǎn)移時(shí)間?每3個(gè)月隨訪(fǎng)1次?2009年10月至2011年6月期間共隨訪(fǎng)行CRC根治術(shù)患者75例,失訪(fǎng)2例?

1.6 統(tǒng)計(jì)學(xué)處理 應(yīng)用SPSS18.0軟件包,計(jì)量資料采用t檢驗(yàn)或方差分析,計(jì)數(shù)資料采用χ2檢驗(yàn),P0.05為差異有統(tǒng)計(jì)學(xué)意義?

2 結(jié) 果

2.1 抗survivin抗體及survivin mRNA在CRC患者及正常人外周血中的表達(dá) 116例CRC患者血清中抗survivin抗體的濃度為10.66～96.02ng/mL,對(duì)照組為10.36～21.77ng/mL,以±2s為截?cái)嘀?則陽(yáng)性率分別為40.5%?0,二者差異具有統(tǒng)計(jì)學(xué)意義(P=0.000)?兩組survivin mRNA陽(yáng)性率分別為76.7%?0,差異有統(tǒng)計(jì)學(xué)意義(P=0.000),見(jiàn)表1?圖1?

表1 抗survivin抗體及Survivin mRNA在CRC組及對(duì)照組外周血中的表達(dá)

續(xù)表2 抗survivin抗體及survivin mRNA表達(dá)與結(jié)直腸癌臨床病理特征

表3 手術(shù)前后外周血抗survivin抗體及survivin mRNA水平變化

表4 化療前后外周血抗survivin抗體及survivin mRNA水平變化

2.2 抗survivin抗體及survivin mRNA表達(dá)與CRC臨床病理特征的關(guān)系 CRC患者外周血中抗survivin抗體及survivin mRNA表達(dá)與年齡?性別?腫瘤類(lèi)型?組織學(xué)分型無(wú)關(guān),而與分期?腫瘤大小及淋巴結(jié)是否轉(zhuǎn)移顯著相關(guān)?抗survivin抗體表達(dá)水平及survivin mRNA陽(yáng)性率隨著腫瘤分期的增高而增高;腫瘤直徑大于5cm者,二者表達(dá)水平較直徑小于或等于5cm者高,差異有統(tǒng)計(jì)學(xué)意義(P0.05);有淋巴結(jié)轉(zhuǎn)移者抗survivin抗體水平及survivin mRNA陽(yáng)性率較無(wú)淋巴結(jié)轉(zhuǎn)移者顯著增高,見(jiàn)表2?

2.3 手術(shù)對(duì)結(jié)直腸癌患者外周血中抗survivin抗體及sur-vivin mRNA水平的影響 74例CRC患者行根治手術(shù)后血清中抗survivin抗體水平較術(shù)前明顯降低(P=0.005),其中48例survivin mRNA陽(yáng)性者根治術(shù)后survivin mRNA表達(dá)水平較術(shù)前亦明顯降低(P=0.004);其中13例患者行根治術(shù)后復(fù)發(fā)或轉(zhuǎn)移,復(fù)發(fā)或轉(zhuǎn)移患者外周血中二者表達(dá)水平均較前顯著升高(分別P=0.024?P=0.048)?14例患者行姑息手術(shù)前后,抗survivin抗體?survivin mRNA表達(dá)水平比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)?根治術(shù)后,抗survivin抗體和survivin mRNA表達(dá)水平與是否復(fù)發(fā)或轉(zhuǎn)移相關(guān),復(fù)發(fā)或轉(zhuǎn)移者根治術(shù)后其表達(dá)水平顯著高于無(wú)復(fù)發(fā)或轉(zhuǎn)移者(分別P=0.001,P=0.004),見(jiàn)表3?

2.4 抗survivin抗體及survivin mRNA水平與化療療效的關(guān)系 化療有效組患者(完全緩解+部分緩解)化療2周期后血清抗survivin抗體及survivin mRNA水平較化療前明顯降低(P=0.043,P=0.032),而化療無(wú)效組患者(疾病穩(wěn)定+疾病進(jìn)展)抗survivin抗體有上升趨勢(shì),但其水平變化差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.135),而survivin mRNA表達(dá)較前顯著升高(P=0.039),見(jiàn)表4?

圖1 PCR產(chǎn)物電泳圖

2.5 抗survivin抗體與survivin mRNA的相關(guān)性分析 對(duì)CRC患者外周血中抗survivin抗體和survivin mRNA進(jìn)行Pearson相關(guān)分析,結(jié)果顯示二者表達(dá)水平呈正相關(guān)(r=0.776,P=0.000)?

3 討 論

3.1 抗survivin抗體和survivin mRNA與CRC的診斷 survivin為凋亡抑制蛋白家族成員之一,由于survivin啟動(dòng)子在正常細(xì)胞中基本是靜止的,而在腫瘤細(xì)胞中過(guò)度表達(dá)[3],使其有高度的組織表達(dá)特異性,在除胸腺?睪丸?分泌期子宮內(nèi)膜?胎盤(pán)外的分化成熟的正常成人組織中不表達(dá)或低表達(dá)外,包括結(jié)直腸癌在內(nèi)的絕大多數(shù)惡性腫瘤組織中survivin高表達(dá)[4]?研究認(rèn)為survivin可作為腫瘤標(biāo)志物[5-6]?

survivin蛋白可被自身固有T細(xì)胞所識(shí)別,具有抗原性,血清中其抗體稱(chēng)為抗survivin抗體,因此從腫瘤患者血清中檢測(cè)抗survivin抗體可用于腫瘤診斷[7]?本研究中30例健康人外周血中無(wú)抗survivin抗體和survivin mRNA表達(dá),而116例結(jié)直腸癌患者的陽(yáng)性表達(dá)率分別為40.5%?76.9%,提示抗survivin抗體和survivin mRNA表達(dá)可能為篩查CRC的指標(biāo)之一?

結(jié)直腸癌的發(fā)生過(guò)程涉及多個(gè)癌基因激活和抑癌基因失活的復(fù)雜過(guò)程?survivin主要表達(dá)于黏膜層腺體底部,而在浸潤(rùn)的淋巴細(xì)胞?纖維細(xì)胞?平滑肌細(xì)胞和神經(jīng)細(xì)胞中無(wú)表達(dá),survivin表達(dá)是結(jié)腸癌發(fā)生的早期事件,其表達(dá)情況與腫瘤分化程度明顯相關(guān)[8]?Kawasaki等[9]分別對(duì)不同病理時(shí)期的組織標(biāo)本進(jìn)行檢測(cè),發(fā)現(xiàn)survivin表達(dá)的陽(yáng)性率從增生性息肉?低度增生不良腺瘤?高度增生不良腺瘤到腺癌組織標(biāo)本中逐步升高?這表明,survivin在結(jié)腸癌的演變過(guò)程中逐步表達(dá)出來(lái),并且其表達(dá)過(guò)程貫穿于整個(gè)結(jié)腸癌的發(fā)生?發(fā)展過(guò)程?本研究結(jié)果顯示,抗survivin抗體及survivin mRNA隨著腫瘤分期的增高其表達(dá)水平增高;有淋巴結(jié)轉(zhuǎn)移者抗survivin抗體水平及survivin mRNA陽(yáng)性率較無(wú)淋巴結(jié)轉(zhuǎn)移者高,這與Pavlidou等[10]的研究結(jié)果一致?外周血中survivin表達(dá)與腫瘤負(fù)荷有關(guān),腫瘤越大,其表達(dá)水平越高?

尋找腫瘤標(biāo)志物篩查和早期發(fā)現(xiàn)轉(zhuǎn)移和復(fù)發(fā)對(duì)提高治愈率或延長(zhǎng)生存期有重要意義?Sarela等[11]發(fā)現(xiàn)survivin高表達(dá)是增加病理分期的獨(dú)立因素,結(jié)腸癌根治術(shù)后復(fù)發(fā)的死亡率可用腫瘤組織survivin表達(dá)水平和淋巴結(jié)轉(zhuǎn)移情況進(jìn)行預(yù)測(cè),survivin陰性表達(dá)患者的5年生存率為94.4%,而陽(yáng)性表達(dá)者僅為44.8%?本研究中根治術(shù)組患者的外周血中抗survivin抗體和survivin mRNA表達(dá)水平明顯降低,而姑息手術(shù)后其表達(dá)水平有下降,但差異無(wú)統(tǒng)計(jì)學(xué)意義?13例根治術(shù)后轉(zhuǎn)移或復(fù)發(fā)者其外周血中抗survivin抗體和survivin mRNA表達(dá)水平較根治術(shù)后明顯升高,這表明在隨訪(fǎng)過(guò)程中定期進(jìn)行抗survivin抗體和survivin mRNA檢測(cè)有意義?若抗survivin抗體或survivin mRNA水平升高,提示可能轉(zhuǎn)移或復(fù)發(fā)?

3.2 抗survivin抗體和survivin mRNA與CRC化療療效

化療藥物發(fā)揮作用的重要機(jī)制之一是誘導(dǎo)腫瘤細(xì)胞凋亡,而survivin的過(guò)表達(dá)抑制了腫瘤細(xì)胞凋亡,成為導(dǎo)致化療耐藥原因之一?survivin可以有效地維持微小血管網(wǎng)的結(jié)構(gòu),促進(jìn)血管內(nèi)皮生長(zhǎng)因子對(duì)內(nèi)皮細(xì)胞的保護(hù)功能,拮抗藥物對(duì)血管內(nèi)皮細(xì)胞所產(chǎn)生的凋亡作用[12]?survivin可能作為新輔助化療耐藥的指標(biāo)之一[13]?本研究顯示,在姑息化療中有效組化療后抗survivin抗體及survivin mRNA表達(dá)水平較化療前下降,具有統(tǒng)計(jì)學(xué)意義?化療無(wú)效組患者血清抗survivin抗體水平有上升趨勢(shì),但無(wú)統(tǒng)計(jì)學(xué)意義,而survivin mRNA表達(dá)水平顯著升高?姑息化療前后,survivin抗體和survivin mRNA表達(dá)水平變化可能作為化療療效評(píng)定指標(biāo)之一?

綜上所述,在CRC篩查?手術(shù)前后?姑息性化療前后及隨訪(fǎng)過(guò)程中進(jìn)行抗survivin抗體和survivin mRNA檢測(cè),對(duì)CRC的診斷?療效預(yù)測(cè)?轉(zhuǎn)移或復(fù)發(fā)監(jiān)測(cè)具有一定的臨床意義?

[1] Temme A,Rieger M,Reber F,et al.Localization,dynamics,an function of survivin revealed by expression of functional survivin DsRed fusion proteins in the living cell[J].Mol Biol Cell,2003,14(1):78-92.

[2] Yagihashi A,Asanuma K,Nakamura M,et al.Detection of anti-survivin antibody in gastrointestinal cancer patients[J].Clin Chem,2001,47(9):1729-1731.

[3] Giodini A,Kallio MJ,Wall NR,et al.Regulation of microtubule stability and mitotic progression by surviving[J].Cancer Res,2002,62(9):2462-2467.

[4] Park YN,Chae KJ,Kim YB.Apoptosis and proliferation in hepatoeareinogenesis related to cirrhosis.[J].Cancer,2001,92(11):2733-2738.

[5] Adamkov M,Halasova E,Kajo K,et al.Survivin:apro mising biomarker in breast carcinoma[J].Neoplasma,2010,57(6):572-577.

[6] Eto M,Kodama S,Uemura N,et al.Antibody responses to survivin and their clinical significance in patients with head and neck cancer[J].Head Neck,2007,29(12):1128-1135.

[7] Chen JS,Chen KT,Fan WC,et al.Combined analysis of survivin autoantibody and carcinoembryonic antigen biomarkers for improved detection of colorectal cancer[J].Clin Chem Lab Med,2010,48(5):719-725.

[8] Svec J,Ergang P,Mandys V,et al.Expression profiles of proliferative and antiapoptotic genes in sporadic and colitis-related mouse colon cancer models [J].Int J Exp Pathol,2010,91(1):44-53.

[9] Kawasaki H,Toyoda M,Shinohara H,et al.Expression of surviving correlates with apoptosis,proliferation,and angiogenesis during human colorectal tumorigenesis [J].Cancer,2001,91(11):2026-2032.

[10]Pavlidou A,Dalamaga M,Kroupis C,et al.Survivin iso-forms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR[J].World J Gastroenterol,2011,17(12):1614-1621.

[11]Sarela AI,Macadam RCA,Farmery SM,et al.Expression of the antiapoptosis gene,Survivin,predicts death from recurrent colorectal carcinoma[J].Gut,2000,46(5):645-650.

[12]Tran J,Master Z,Yu J,et al.A role of survivin in chemoresistancee of endothelial cells mediated by VEGF[J].Proc Natl Acad Sci,2002,99(7):4349-4354.

[13]Horisberger K,Erben P,Str?bel P,et al.Annexin and survivin in locally advanced rectal cancer:indicators of resistance to preoperative chemoradiotherapy[J].Onkologie,2010,33(8-9):439-444.

Expression of anti-survivin antibody and survivin mRNA in peripheral blood of colorectal cancer and its clinical significance

, ,
(,,, 541001,)

ObjectiveTo explore the expression of anti-survivin antibody and survivin mRNA in peripheral blood of patients with colorectal cancer and its clinical significance.Methods116patients with colorectal cancer were divided into radical surgery group,palliative surgery group and palliative chemotherapy group,and 30healthy people were taken as controls.Peripheral blood was collected before and after treatment,and the expression levels of anti-survivin antibody and survivin mRNAwere detected by ELISA and semi-quantitative RT-PCR respectively.ResultsThe positive rates of anti-survivin antibody were respectively 40.5%and 0,and survivin mRNAwere respectively 76.7%and 0,in peripheral blood of patients with colorectal cancer and healthy people,and differences were significant(P=0.000).The expression levels of anti-survivin antibody and survivin mRNA increased with the advance of tumor stage,tumors>5cm in diameter were significantly higher than those≤5cm(P=0.045,P=0.003),those with lymph node metastasis were significantly higher than those without lymph node metastasis(P=0.040,P=0.000),those after radical surgery were significantly lower than those preoperation(P=0.005,P=0.004),those with a high expression were significantly higher in metastatic rate than those with a low expression after radical resection(P=0.001,P=0.004).There was no significant difference of expression levels between before and after palliative surgery(P=0.709,P=0.669).In palliative chemotherapy groups,the expression levels after chemotherapy were significantly lower than before chemotherapy in the effective group(P=0.043,P=0.032),anti-survivin antibody was no significant change(P=0.135),but survivin mRNAexpression in those after chemotherapy was significantly higher than those before chemotherapy in the ineffective group(P=0.039).ConclusionThe detection of anti-survivin antibody and survivin mRNA in peripheral blood may have certain clinical significance to the diagnosis,prognostic estimation,efficacy prediction of chemotherapy,and follow-up of colorectal cancer.

colorectal neoplasms;diagnosis;survivin

10.3969/j.issn.1671-8348.2012.16.006

A

1671-8348(2012)16-1575-04

△通訊作者,Tel:13517837025;E-mail:kmfgl@163.com?

2011-10-04

2011-11-22)

?臨床研究?