抗survivin抗体与survivin mRNA在结直肠癌患者外周血中的表达及其临床意义

施朕善,康马飞,李文见

(桂林医学院附属医院肿瘤内科,广西桂林 541001)

抗survivin抗体与survivin mRNA在结直肠癌患者外周血中的表达及其临床意义

施朕善,康马飞△,李文见

(桂林医学院附属医院肿瘤内科,广西桂林 541001)

目的探讨结直肠癌(CRC)患者外周血中抗survivin抗体､survivin mRNA的表达情况及其临床意义｡方法 选择结直肠癌患者116例,依据所接受治疗手段,分为根治手术组､姑息手术组､姑息化疗组｡对照组为30例健康体检者｡分别于患者治疗前后抽取外周血,应用ELISA和半定量RT-PCR检测抗survivin抗体和survivin mRNA的表达水平｡结果结直肠癌患者与健康者外周血中抗survivin抗体阳性率分别为40.5%和0,survivin mRNA阳性率分别为76.7%和0,差异均有统计学意义(P=0.000)｡抗survivin抗体和survivin mRNA表达水平随肿瘤分期的增高而增高,肿瘤直径大于5cm者较直径小于或等于5cm者高(分别P=0.045､P=0.003),有淋巴结转移者较无淋巴结转移者显著增高(分别P=0.040､P=0.000);根治手术后较术前明显降低(分别P=0.005､P=0.004),根治术后表达水平高者转移或复发率显著高于表达水平低者(分别P=0.001､P=0.004);表达水平在姑息手术前后差异无统计学意义(分别P=0.709､P=0.669)｡行姑息性化疗的患者中,有效组表达水平较化疗前明显降低(分别P=0.043､P=0.032);化疗无效组抗survivin抗体水平变化无统计学意义(P=0.135),而survivin mRNA表达较前显著升高(P=0.039)｡结论外周血抗survivin抗体和survivin mRNA检测可能对CRC的诊断､预后估计､化疗疗效预测及随访有一定的临床意义｡

结直肠肿瘤;诊断;survivin

结直肠癌(colorectal cancer,CRC)是常见消化道恶性肿瘤之一,CRC的早期诊断､转移或复发监测是临床中的重要问题,寻找方便有效的标志物显得尤为重要｡survivin作为凋亡抑制蛋白家族成员之一,已被证实在包括CRC在内的多种恶性肿瘤中过表达[1]｡目前的研究大多数仅为观察肿瘤组织中的survivin表达情况,不利于动态观察其表达变化情况｡研究表明,肿瘤患者外周血中有抗survivin抗体[2]｡为探讨外周血抗survivin抗体以及survivin mRNA在CRC发生过程中的临床意义,本组对116例CRC患者进行了上述指标的检测,现报道如下｡

1 资料与方法

1.1 一般资料 收集2009年10月至2011年6月期间在本院住院治疗的CRC患者116例,其中,结肠癌42例,直肠癌74例;年龄27～84岁,中位58岁｡所有患者均经病理学诊断,临床资料完整｡依据所接受治疗手段,分为根治手术组､姑息手术组和姑息化疗组｡对照组纳入从本院体检中心收集的30例健康人,年龄31～80岁,中位56岁｡

1.2 标本的采集与处理 手术组于手术前1天和术后第2周末清晨空腹采集外周血5mL,其中,根治手术组每3月随访一次,抽外周血5mL;姑息化疗组于化疗前及化疗2周期后采集外周血5mL;对照组空腹采集外周血5mL｡所采集血液标本中,其中2mL使用无抗凝剂的真空管采集,以3 000r/min离心10min,取上清液,-20℃冻存,用于检测抗survivin抗体;剩余3mL使用含有乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)抗凝剂真空管采集,室温静置30min,用于提取总RNA｡

1.3 抗survivin抗体的测定 应用双位点夹心酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)测定血清中抗survivin抗体的水平,采用R&D公司提供的人survivin抗体定量检测试剂盒｡

1.4 survivin mRNA的检测 应用康为世纪公司血液RNA提取试剂盒(Cat.NO:CW0538),按照说明书提取总RNA｡紫外分光光度计进行定量并检测其纯度,1.5%琼脂糖电泳鉴定RNA完整性｡应用TaKaRa公司的PrimeScript RT reagent Kit(TaKaRa Code:DDR037A)按说明书进行反转录｡反转录条件为37℃15min,85℃5s｡以β-actin作内参基因,应用Primer Premier 5.0软件设计引物｡Survivin mRNA上､下游引物分别为5＇-GTCCCTGGCTCCTCTACTG-3＇,5＇-GACGCTTCCTATCACTCTATTC-3＇,产物为174bp;β-actin上､下游引物 分 别 为 5＇-AGAGCCTCGCCTTTGCCGAT-3＇,5＇-TGCCAGATTTTCTCCATGTCGT-3＇,产物为 313bp｡所有引物由Invitrigen公司合成｡使用TaKaRa公司的Premix Ex Taq试剂盒进行PCR,经过预变性95℃30s,34个循环(变性95℃5s,退火60℃30s,延伸72℃50s),终延伸72℃2min,其中survivin循环数为34,退火温度60℃,β-actin循环数为28,退火温度62℃｡所得产物进行2.5%琼脂糖电泳鉴定(图1),并应用上海培清JS-6800凝胶成像分析系统,分别测定β-actin和survivin条带的光密度值,并计算survivin的表达系数,即survivin的光密度值与β-actin的光密度值之比｡

1.5 随访 起点为首次检测survivin时间,终点为发现复发或转移时间｡每3个月随访1次｡2009年10月至2011年6月期间共随访行CRC根治术患者75例,失访2例｡

1.6 统计学处理 应用SPSS18.0软件包,计量资料采用t检验或方差分析,计数资料采用χ2检验,P0.05为差异有统计学意义｡

2 结 果

2.1 抗survivin抗体及survivin mRNA在CRC患者及正常人外周血中的表达 116例CRC患者血清中抗survivin抗体的浓度为10.66～96.02ng/mL,对照组为10.36～21.77ng/mL,以±2s为截断值,则阳性率分别为40.5%､0,二者差异具有统计学意义(P=0.000)｡两组survivin mRNA阳性率分别为76.7%､0,差异有统计学意义(P=0.000),见表1､图1｡

表1 抗survivin抗体及Survivin mRNA在CRC组及对照组外周血中的表达

续表2 抗survivin抗体及survivin mRNA表达与结直肠癌临床病理特征

表3 手术前后外周血抗survivin抗体及survivin mRNA水平变化

表4 化疗前后外周血抗survivin抗体及survivin mRNA水平变化

2.2 抗survivin抗体及survivin mRNA表达与CRC临床病理特征的关系 CRC患者外周血中抗survivin抗体及survivin mRNA表达与年龄､性别､肿瘤类型､组织学分型无关,而与分期､肿瘤大小及淋巴结是否转移显著相关｡抗survivin抗体表达水平及survivin mRNA阳性率随着肿瘤分期的增高而增高;肿瘤直径大于5cm者,二者表达水平较直径小于或等于5cm者高,差异有统计学意义(P0.05);有淋巴结转移者抗survivin抗体水平及survivin mRNA阳性率较无淋巴结转移者显著增高,见表2｡

2.3 手术对结直肠癌患者外周血中抗survivin抗体及sur-vivin mRNA水平的影响 74例CRC患者行根治手术后血清中抗survivin抗体水平较术前明显降低(P=0.005),其中48例survivin mRNA阳性者根治术后survivin mRNA表达水平较术前亦明显降低(P=0.004);其中13例患者行根治术后复发或转移,复发或转移患者外周血中二者表达水平均较前显著升高(分别P=0.024､P=0.048)｡14例患者行姑息手术前后,抗survivin抗体､survivin mRNA表达水平比较,差异无统计学意义(P0.05)｡根治术后,抗survivin抗体和survivin mRNA表达水平与是否复发或转移相关,复发或转移者根治术后其表达水平显著高于无复发或转移者(分别P=0.001,P=0.004),见表3｡

2.4 抗survivin抗体及survivin mRNA水平与化疗疗效的关系 化疗有效组患者(完全缓解+部分缓解)化疗2周期后血清抗survivin抗体及survivin mRNA水平较化疗前明显降低(P=0.043,P=0.032),而化疗无效组患者(疾病稳定+疾病进展)抗survivin抗体有上升趋势,但其水平变化差异无统计学意义(P=0.135),而survivin mRNA表达较前显著升高(P=0.039),见表4｡

图1 PCR产物电泳图

2.5 抗survivin抗体与survivin mRNA的相关性分析 对CRC患者外周血中抗survivin抗体和survivin mRNA进行Pearson相关分析,结果显示二者表达水平呈正相关(r=0.776,P=0.000)｡

3 讨 论

3.1 抗survivin抗体和survivin mRNA与CRC的诊断 survivin为凋亡抑制蛋白家族成员之一,由于survivin启动子在正常细胞中基本是静止的,而在肿瘤细胞中过度表达[3],使其有高度的组织表达特异性,在除胸腺､睾丸､分泌期子宫内膜､胎盘外的分化成熟的正常成人组织中不表达或低表达外,包括结直肠癌在内的绝大多数恶性肿瘤组织中survivin高表达[4]｡研究认为survivin可作为肿瘤标志物[5-6]｡

survivin蛋白可被自身固有T细胞所识别,具有抗原性,血清中其抗体称为抗survivin抗体,因此从肿瘤患者血清中检测抗survivin抗体可用于肿瘤诊断[7]｡本研究中30例健康人外周血中无抗survivin抗体和survivin mRNA表达,而116例结直肠癌患者的阳性表达率分别为40.5%､76.9%,提示抗survivin抗体和survivin mRNA表达可能为筛查CRC的指标之一｡

结直肠癌的发生过程涉及多个癌基因激活和抑癌基因失活的复杂过程｡survivin主要表达于黏膜层腺体底部,而在浸润的淋巴细胞､纤维细胞､平滑肌细胞和神经细胞中无表达,survivin表达是结肠癌发生的早期事件,其表达情况与肿瘤分化程度明显相关[8]｡Kawasaki等[9]分别对不同病理时期的组织标本进行检测,发现survivin表达的阳性率从增生性息肉､低度增生不良腺瘤､高度增生不良腺瘤到腺癌组织标本中逐步升高｡这表明,survivin在结肠癌的演变过程中逐步表达出来,并且其表达过程贯穿于整个结肠癌的发生､发展过程｡本研究结果显示,抗survivin抗体及survivin mRNA随着肿瘤分期的增高其表达水平增高;有淋巴结转移者抗survivin抗体水平及survivin mRNA阳性率较无淋巴结转移者高,这与Pavlidou等[10]的研究结果一致｡外周血中survivin表达与肿瘤负荷有关,肿瘤越大,其表达水平越高｡

寻找肿瘤标志物筛查和早期发现转移和复发对提高治愈率或延长生存期有重要意义｡Sarela等[11]发现survivin高表达是增加病理分期的独立因素,结肠癌根治术后复发的死亡率可用肿瘤组织survivin表达水平和淋巴结转移情况进行预测,survivin阴性表达患者的5年生存率为94.4%,而阳性表达者仅为44.8%｡本研究中根治术组患者的外周血中抗survivin抗体和survivin mRNA表达水平明显降低,而姑息手术后其表达水平有下降,但差异无统计学意义｡13例根治术后转移或复发者其外周血中抗survivin抗体和survivin mRNA表达水平较根治术后明显升高,这表明在随访过程中定期进行抗survivin抗体和survivin mRNA检测有意义｡若抗survivin抗体或survivin mRNA水平升高,提示可能转移或复发｡

3.2 抗survivin抗体和survivin mRNA与CRC化疗疗效

化疗药物发挥作用的重要机制之一是诱导肿瘤细胞凋亡,而survivin的过表达抑制了肿瘤细胞凋亡,成为导致化疗耐药原因之一｡survivin可以有效地维持微小血管网的结构,促进血管内皮生长因子对内皮细胞的保护功能,拮抗药物对血管内皮细胞所产生的凋亡作用[12]｡survivin可能作为新辅助化疗耐药的指标之一[13]｡本研究显示,在姑息化疗中有效组化疗后抗survivin抗体及survivin mRNA表达水平较化疗前下降,具有统计学意义｡化疗无效组患者血清抗survivin抗体水平有上升趋势,但无统计学意义,而survivin mRNA表达水平显著升高｡姑息化疗前后,survivin抗体和survivin mRNA表达水平变化可能作为化疗疗效评定指标之一｡

综上所述,在CRC筛查､手术前后､姑息性化疗前后及随访过程中进行抗survivin抗体和survivin mRNA检测,对CRC的诊断､疗效预测､转移或复发监测具有一定的临床意义｡

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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

△通讯作者,Tel:13517837025;E-mail:kmfgl@163.com｡

2011-10-04

2011-11-22)

•临床研究•