抗survivin抗體與survivin mRNA在結直腸癌患者外周血中的表達及其臨床意義

施朕善,康馬飛,李文見

(桂林醫學院附屬醫院腫瘤內科,廣西桂林 541001)

抗survivin抗體與survivin mRNA在結直腸癌患者外周血中的表達及其臨床意義

施朕善,康馬飛△,李文見

(桂林醫學院附屬醫院腫瘤內科,廣西桂林 541001)

目的探討結直腸癌(CRC)患者外周血中抗survivin抗體?survivin mRNA的表達情況及其臨床意義?方法 選擇結直腸癌患者116例,依據所接受治療手段,分為根治手術組?姑息手術組?姑息化療組?對照組為30例健康體檢者?分別于患者治療前后抽取外周血,應用ELISA和半定量RT-PCR檢測抗survivin抗體和survivin mRNA的表達水平?結果結直腸癌患者與健康者外周血中抗survivin抗體陽性率分別為40.5%和0,survivin mRNA陽性率分別為76.7%和0,差異均有統計學意義(P=0.000)?抗survivin抗體和survivin mRNA表達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),有淋巴結轉移者較無淋巴結轉移者顯著增高(分別P=0.040?P=0.000);根治手術后較術前明顯降低(分別P=0.005?P=0.004),根治術后表達水平高者轉移或復發率顯著高于表達水平低者(分別P=0.001?P=0.004);表達水平在姑息手術前后差異無統計學意義(分別P=0.709?P=0.669)?行姑息性化療的患者中,有效組表達水平較化療前明顯降低(分別P=0.043?P=0.032);化療無效組抗survivin抗體水平變化無統計學意義(P=0.135),而survivin mRNA表達較前顯著升高(P=0.039)?結論外周血抗survivin抗體和survivin mRNA檢測可能對CRC的診斷?預后估計?化療療效預測及隨訪有一定的臨床意義?

結直腸腫瘤;診斷;survivin

結直腸癌(colorectal cancer,CRC)是常見消化道惡性腫瘤之一,CRC的早期診斷?轉移或復發監測是臨床中的重要問題,尋找方便有效的標志物顯得尤為重要?survivin作為凋亡抑制蛋白家族成員之一,已被證實在包括CRC在內的多種惡性腫瘤中過表達[1]?目前的研究大多數僅為觀察腫瘤組織中的survivin表達情況,不利于動態觀察其表達變化情況?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發生過程中的臨床意義,本組對116例CRC患者進行了上述指標的檢測,現報道如下?

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,結腸癌42例,直腸癌74例;年齡27～84歲,中位58歲?所有患者均經病理學診斷,臨床資料完整?依據所接受治療手段,分為根治手術組?姑息手術組和姑息化療組?對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 標本的采集與處理 手術組于手術前1天和術后第2周末清晨空腹采集外周血5mL,其中,根治手術組每3月隨訪一次,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;對照組空腹采集外周血5mL?所采集血液標本中,其中2mL使用無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抗體的測定 應用雙位點夾心酶聯免疫吸附法(enzyme-linked immunosorbent assay,ELISA)測定血清中抗survivin抗體的水平,采用R&D公司提供的人survivin抗體定量檢測試劑盒?

1.4 survivin mRNA的檢測 應用康為世紀公司血液RNA提取試劑盒(Cat.NO:CW0538),按照說明書提取總RNA?紫外分光光度計進行定量并檢測其純度,1.5%瓊脂糖電泳鑒定RNA完整性?應用TaKaRa公司的PrimeScript RT reagent Kit(TaKaRa Code:DDR037A)按說明書進行反轉錄?反轉錄條件為37℃15min,85℃5s?以β-actin作內參基因,應用Primer Premier 5.0軟件設計引物?Survivin mRNA上?下游引物分別為5＇-GTCCCTGGCTCCTCTACTG-3＇,5＇-GACGCTTCCTATCACTCTATTC-3＇,產物為174bp;β-actin上?下游引物 分 別 為 5＇-AGAGCCTCGCCTTTGCCGAT-3＇,5＇-TGCCAGATTTTCTCCATGTCGT-3＇,產物為 313bp?所有引物由Invitrigen公司合成?使用TaKaRa公司的Premix Ex Taq試劑盒進行PCR,經過預變性95℃30s,34個循環(變性95℃5s,退火60℃30s,延伸72℃50s),終延伸72℃2min,其中survivin循環數為34,退火溫度60℃,β-actin循環數為28,退火溫度62℃?所得產物進行2.5%瓊脂糖電泳鑒定(圖1),并應用上海培清JS-6800凝膠成像分析系統,分別測定β-actin和survivin條帶的光密度值,并計算survivin的表達系數,即survivin的光密度值與β-actin的光密度值之比?

1.5 隨訪 起點為首次檢測survivin時間,終點為發現復發或轉移時間?每3個月隨訪1次?2009年10月至2011年6月期間共隨訪行CRC根治術患者75例,失訪2例?

1.6 統計學處理 應用SPSS18.0軟件包,計量資料采用t檢驗或方差分析,計數資料采用χ2檢驗,P0.05為差異有統計學意義?

2 結 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 116例CRC患者血清中抗survivin抗體的濃度為10.66～96.02ng/mL,對照組為10.36～21.77ng/mL,以±2s為截斷值,則陽性率分別為40.5%?0,二者差異具有統計學意義(P=0.000)?兩組survivin mRNA陽性率分別為76.7%?0,差異有統計學意義(P=0.000),見表1?圖1?

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

續表2 抗survivin抗體及survivin mRNA表達與結直腸癌臨床病理特征

表3 手術前后外周血抗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表達與CRC臨床病理特征的關系 CRC患者外周血中抗survivin抗體及survivin mRNA表達與年齡?性別?腫瘤類型?組織學分型無關,而與分期?腫瘤大小及淋巴結是否轉移顯著相關?抗survivin抗體表達水平及survivin mRNA陽性率隨著腫瘤分期的增高而增高;腫瘤直徑大于5cm者,二者表達水平較直徑小于或等于5cm者高,差異有統計學意義(P0.05);有淋巴結轉移者抗survivin抗體水平及survivin mRNA陽性率較無淋巴結轉移者顯著增高,見表2?

2.3 手術對結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患者行根治手術后血清中抗survivin抗體水平較術前明顯降低(P=0.005),其中48例survivin mRNA陽性者根治術后survivin mRNA表達水平較術前亦明顯降低(P=0.004);其中13例患者行根治術后復發或轉移,復發或轉移患者外周血中二者表達水平均較前顯著升高(分別P=0.024?P=0.048)?14例患者行姑息手術前后,抗survivin抗體?survivin mRNA表達水平比較,差異無統計學意義(P0.05)?根治術后,抗survivin抗體和survivin mRNA表達水平與是否復發或轉移相關,復發或轉移者根治術后其表達水平顯著高于無復發或轉移者(分別P=0.001,P=0.004),見表3?

2.4 抗survivin抗體及survivin mRNA水平與化療療效的關系 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),而化療無效組患者(疾病穩定+疾病進展)抗survivin抗體有上升趨勢,但其水平變化差異無統計學意義(P=0.135),而survivin mRNA表達較前顯著升高(P=0.039),見表4?

圖1 PCR產物電泳圖

2.5 抗survivin抗體與survivin mRNA的相關性分析 對CRC患者外周血中抗survivin抗體和survivin mRNA進行Pearson相關分析,結果顯示二者表達水平呈正相關(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啟動子在正常細胞中基本是靜止的,而在腫瘤細胞中過度表達[3],使其有高度的組織表達特異性,在除胸腺?睪丸?分泌期子宮內膜?胎盤外的分化成熟的正常成人組織中不表達或低表達外,包括結直腸癌在內的絕大多數惡性腫瘤組織中survivin高表達[4]?研究認為survivin可作為腫瘤標志物[5-6]?

survivin蛋白可被自身固有T細胞所識別,具有抗原性,血清中其抗體稱為抗survivin抗體,因此從腫瘤患者血清中檢測抗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抗survivin抗體和survivin mRNA表達,而116例結直腸癌患者的陽性表達率分別為40.5%?76.9%,提示抗survivin抗體和survivin mRNA表達可能為篩查CRC的指標之一?

結直腸癌的發生過程涉及多個癌基因激活和抑癌基因失活的復雜過程?survivin主要表達于黏膜層腺體底部,而在浸潤的淋巴細胞?纖維細胞?平滑肌細胞和神經細胞中無表達,survivin表達是結腸癌發生的早期事件,其表達情況與腫瘤分化程度明顯相關[8]?Kawasaki等[9]分別對不同病理時期的組織標本進行檢測,發現survivin表達的陽性率從增生性息肉?低度增生不良腺瘤?高度增生不良腺瘤到腺癌組織標本中逐步升高?這表明,survivin在結腸癌的演變過程中逐步表達出來,并且其表達過程貫穿于整個結腸癌的發生?發展過程?本研究結果顯示,抗survivin抗體及survivin mRNA隨著腫瘤分期的增高其表達水平增高;有淋巴結轉移者抗survivin抗體水平及survivin mRNA陽性率較無淋巴結轉移者高,這與Pavlidou等[10]的研究結果一致?外周血中survivin表達與腫瘤負荷有關,腫瘤越大,其表達水平越高?

尋找腫瘤標志物篩查和早期發現轉移和復發對提高治愈率或延長生存期有重要意義?Sarela等[11]發現survivin高表達是增加病理分期的獨立因素,結腸癌根治術后復發的死亡率可用腫瘤組織survivin表達水平和淋巴結轉移情況進行預測,survivin陰性表達患者的5年生存率為94.4%,而陽性表達者僅為44.8%?本研究中根治術組患者的外周血中抗survivin抗體和survivin mRNA表達水平明顯降低,而姑息手術后其表達水平有下降,但差異無統計學意義?13例根治術后轉移或復發者其外周血中抗survivin抗體和survivin mRNA表達水平較根治術后明顯升高,這表明在隨訪過程中定期進行抗survivin抗體和survivin mRNA檢測有意義?若抗survivin抗體或survivin mRNA水平升高,提示可能轉移或復發?

3.2 抗survivin抗體和survivin mRNA與CRC化療療效

化療藥物發揮作用的重要機制之一是誘導腫瘤細胞凋亡,而survivin的過表達抑制了腫瘤細胞凋亡,成為導致化療耐藥原因之一?survivin可以有效地維持微小血管網的結構,促進血管內皮生長因子對內皮細胞的保護功能,拮抗藥物對血管內皮細胞所產生的凋亡作用[12]?survivin可能作為新輔助化療耐藥的指標之一[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表達水平較化療前下降,具有統計學意義?化療無效組患者血清抗survivin抗體水平有上升趨勢,但無統計學意義,而survivin mRNA表達水平顯著升高?姑息化療前后,survivin抗體和survivin mRNA表達水平變化可能作為化療療效評定指標之一?

綜上所述,在CRC篩查?手術前后?姑息性化療前后及隨訪過程中進行抗survivin抗體和survivin mRNA檢測,對CRC的診斷?療效預測?轉移或復發監測具有一定的臨床意義?

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

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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?