CN102206710B - 预测肝癌术后转移与复发的实时pcr微阵列芯片试剂盒 - Google Patents

预测肝癌术后转移与复发的实时pcr微阵列芯片试剂盒 Download PDF

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CN102206710B
CN102206710B CN 201110091076 CN201110091076A CN102206710B CN 102206710 B CN102206710 B CN 102206710B CN 201110091076 CN201110091076 CN 201110091076 CN 201110091076 A CN201110091076 A CN 201110091076A CN 102206710 B CN102206710 B CN 102206710B
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CN102206710A (zh
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周闯
钦伦秀
叶青海
董琼珠
贾户亮
周海君
王冠
张晓飞
付丽云
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Luo Jie
SHANGHAI SHINES PHARMACEUTICALS CO Ltd
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Zhongshan Hospital Fudan University
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Abstract

本发明提供了一种预测肝癌术后转移与复发的实时PCR微阵列芯片试剂盒，其特征在于，包括CXCL10基因扩增引物及荧光标记探针、FKBP10基因扩增引物及荧光标记探针、ASPH基因扩增引物及荧光标记探针、SPP2基因扩增引物及荧光标记探针和ENO2基因扩增引物及荧光标记探针。本发明发现了CXCL10基因、FKBP10基因、ASPH基因、SPP2基因和ENO2基因可以作为预测肝癌术后转移与复发的标志物，并提供了相应的试剂盒，通过实时定量PCR微阵列芯片技术对肝癌患者术中切除的癌组织进行mRNA水平的检测，具有高通量、高敏感性和高均一性等优点。相对于芯片技术或者分子杂交，该方法简便、快速且经济实用。

Description

预测肝癌术后转移与复发的实时PCR微阵列芯片试剂盒
技术领域
本发明涉及一种预测肝癌术后转移与复发的实时PCR微阵列芯片试剂盒，属于诊断试剂技术领域。
背景技术
原发性肝细胞癌（简称肝癌）是世界上最常见、恶性程度最高的肿瘤之一，位居全球恶性肿瘤发病率第5位、死因第3位；每年新发病564,000例，死亡549,000例，我国占其中一半以上，是我国恶性肿瘤中第2位的杀手。手术是目前肝癌主要的治疗手段，然而术后高转移与复发率（5年复发率达70％，小肝癌也达50％）已成为进一步提高远期疗效的瓶颈，也是攻克肝癌的重要关键。近年来，肝癌转移与复发研究备受关注，虽有所进展，但仍存在许多问题。
如何及早、准确地预测肝癌的转移潜能是困扰临床的一大难题。根据复旦大学肝癌研究所研究发现伴转移肝癌与不伴转移肝癌之间存在153个显著差异基因，基于此在国际上首次建立了能正确预测病人有无肝内转移的多分子预测模型，可将20例(100%)建立模型标本进行准确分类，经小样本验证，可以准确预测另外20例待测标本中的至少18例，预测准确率达90% (Nat Med 2003)。本发明对上述研究进行了大样本独立验证得到优化的预测模型，可对肝癌转移潜能进行准确预测，从而能对根治术后肝癌患者转移、复发风险进行准确的预测和评估，对高风险患者进行重点监测和有效干预，减少复发、转移，进一步改善患者预后。
发明内容
本发明的目的是提供一种预测肝癌术后转移与复发的实时PCR微阵列芯片试剂盒，用于对肝癌术后患者转移与复发风险进行预测和风险评估，对高风险患者进行重点监测并指导术后治疗，进一步降低术后转移与复发，改善患者预后。
为了达到上述目的，本发明提供了一种预测肝癌术后转移与复发的实时PCR微阵列芯片试剂盒，其特征在于，包括CXCL10基因扩增引物及荧光标记探针、FKBP10基因扩增引物及荧光标记探针、ASPH基因扩增引物及荧光标记探针、SPP2基因扩增引物及荧光标记探针和ENO2基因扩增引物及荧光标记探针。
所述的CXCL10基因的扩增引物序列正义链为TGATGCAGGTACAGCGTACAGT，扩增引物序列反义链为TCCAGTCTCAGCACCATGAATC，荧光标记探针序列为CTGCCATTCTGATTTGCTGCCTTAT。
所述的FKBP10基因的扩增引物序列正义链为CGACTTTGTCCGCTACCACTAC，扩增引物序列反义链为ACCAGAGCCGACGTAGGTGT，荧光标记探针序列为CACCCTGCTGGACGGCACCTC。
所述的ASPH基因的扩增引物序列正义链为CTGTCTGAGCGACGCTTCAA，扩增引物序列反义链为GGAGCCATCGAGACCTACCA，荧光标记探针序列为AGGTGGCCAGCCTACCTGATGTCC。
所述的SPP2基因的扩增引物序列正义链为CCGGCACAGAGCAAGAATAA，扩增引物序列反义链为AAGCAGCAGCTCCTTGAACA，荧光标记探针序列为TTGAGTAACGGCCTTGAGGTGTCCC。
所述的ENO2基因的扩增引物序列正义链为CCAACTCCAGCATCAGGTTGT，扩增引物序列反义链为TGAAGGCAGTGGACCACATC，荧光标记探针序列为AACTCCACCATCGCGCCAGCC。
优选地，所述的试剂盒中还包括反转录系统、扩增系统和384微孔板组成。
所述的反转录系统由5×反转录体系缓冲液、反转录酶、脱氧核糖核酸混合物以及随机的6核苷酸引物组成。
所述的扩增系统由含Taq酶的信使核糖核酸表达定量检测混合液组成。
本发明的优点为：
（1）本发明发现了CXCL10基因、FKBP10基因、ASPH基因、SPP2基因和ENO2基因可以作为预测肝癌术后转移与复发的标志物，并给出了预测模型，该预测模型可对肝癌患者术后转移与复发进行准确的预测和风险评估，将有助于早期识别或预测高危患者，对其进行重点检测和有效干预，为临床个体化干预治疗提供有效的依据。
（2）本发明提供了相应的试剂盒，通过实时定量PCR微阵列芯片技术对肝癌患者术中切除的癌组织进行mRNA水平的检测，具有高通量、高敏感性和高均一性等特征。相对于芯片技术或者分子杂交，该方法简便、快速且经济实用。
（3）本发明的预测准确率明显优于目前用于预测肝癌患者术后转移与复发的临床病理指标，如甲胎蛋白、肿瘤大小，包膜有无等。而且检测的是患者手术切除下来的癌组织，对患者没有额外损伤。
附图说明
图1为肝癌转移预测模型对380例行根治性切除术的肝癌患者生存预测分析结果图。
具体实施方式
实施例1
一种预测肝癌术后转移与复发的实时PCR微阵列芯片试剂盒，由以下试剂组成：
（1） 反转录系统：由5×反转录体系缓冲液、反转录酶、脱氧核糖核酸混合物以及随机的6核苷酸引物组成。所用试剂组分来源于PrimeScriptTM RT reagent kit perfect Real time反转录试剂盒(Takara公司生产，型号：DDR037A) ；
（2） 扩增系统：由含Taq酶的信使核糖核酸(mRNA)表达定量检测混合液(Applied Biosystems公司生产，型号：TaqMan® Gene expression Master mix 4369016)组成；
（3） 384微孔板；
l CXCL10基因、FKBP10基因、ASPH基因、SPP2基因和ENO2基因的扩增引物和Taqman荧光标记探针混合液，溶剂为DEPC(焦碳酸二乙酯)处理过的水，引物和探针的具体序列如下：
Figure DEST_PATH_IMAGE002A
2、使用上述试剂盒来预测肝癌术后生存率的具体步骤如下：
2.1、380例肝癌患者手术切除肝癌组织总RNA的抽提和纯化：
将380例患者切除的肝癌组织与TRIzol试剂按比例75毫克：1mL混合，用匀浆器匀浆；将匀浆液在室温下孵育5分钟，按1：0.2的体积比加入氯仿，盖严，用手摇晃15秒钟，室温下孵育2.5分钟；于12000×g、4℃条件下离心15分钟，离心后混合液分成三层，取上层水相，按照每1mL TRIzol试剂加入0.5mL的比例加入异丙醇，混匀，20℃下静置10分钟，于12000×g、4℃条件下离心10分钟，RNA沉淀形成胶状物沉在管底管壁；倒掉上清液，按照1mL TRIzol试剂加入1mL的比例加入75%乙醇，振荡混匀；于7500×g、4℃条件下离心5分钟，弃上清液，用移液器吸干试管内残留酒精，室温下自然干燥RNA沉淀10分钟；用DEPC处理过的水重新溶解RNA；用Nanodrop分光光度计检测RNA浓度及A260/A280的比值，当A260/A280的比值为1.9-2.1时，进入下一步；
2.2、采用甲醛变性琼脂糖凝胶电泳对组织标本总RNA样品进行质量检测：
将10×MOPS（3-(N-吗啡啉)丙磺酸RNA变性缓冲液）缓冲液l0mL、0.1% DEPC水70mL、37%甲醛20mL与RNA琼脂糖1.0g混合制备甲醛变性琼脂糖凝胶；用DEPC处理过的水将10×MOPS缓冲液稀释成1×MOPS缓冲液配制电泳缓冲液；将步骤2.1得到的RNA样品5.5µL、10×MOPS缓冲液1.0µL、37%甲醛3.5µL以及去离子甲酰胺10.0µL混合，配成电泳样品，65℃孵育5分钟，冰上冷却；将电泳槽内注入电泳缓冲液，置入甲醛变性琼脂糖凝胶；电泳样品内加入2µL 10×RNA加样缓冲液Gel Loading BufferⅡ(Ambion公司)以及0.1µL EB（溴化乙锭），混合均匀后加入上样孔内，电压100V条件下电泳30分钟，紫外线凝胶分析仪下观测，拍照；当检验证实RNA没有降解时，进入下一步；
2.3、cDNA的合成：
将本发明试剂盒中的2µL 5×反转录体系缓冲液、0.5µL 反转录酶、0.5µL 脱氧核糖核酸混合物 (50µM)和2µL随机的6核苷酸引物(100 µM)，以及步骤2.2中得到的组织样本模板RNA 5.0ul(总量为1µg)充分混合均匀，冰上孵育5分钟。
反转录反应程序如下：37℃，15分钟；85℃，5秒钟。
2.4、实时定量PCR的检测
将步骤2.3中所得产物用DEPC水稀释20倍，将本发明试剂盒中的0.5µL 扩增引物(18µM)和Taqman荧光标记探针（5µM）混合液与5µL含Taq酶的信使核糖核酸(mRNA)表达定量检测混合液混合，加入4.5µL步骤2.3中所得产物稀释液。
实时定量PCR反应程序如下：第一步：95℃，10分钟。第二步：95℃，15秒；60℃，60秒(40个循环)。实验选取TBP（TATA box binding protein）为内参照基因，其荧光标记探针和引物（购于Applied Biosystems公司 型号：4331182），试验条件同上。
2.5、预测原发性肝癌病人术后复发情况分析：
通过传统的△Ct法（即目的基因的Ct值减去内参照基因的Ct值），计算目标基因在癌旁组织中的表达量。
5个预测基因列表及其贡献度如下所示：
基因名称 贡献度 P值
CXCL10 0.1603053435 0.132
FKBP10 0.2595419847 0.061
ASPH 0.1221374046 0.223
SPP2 0.320610687 0.012
ENO2 0.1374045802 0.157
（1）利用libsvm算法对5个预测基因做预测模型，并选用径向基核函数（radical basis function）：
其中，表示径向基核函数，表示某个病人的基因表达值，表示每个基因的表达值，核参数=16。
（2）预测计算公式如下：
P=∑Yi+C
其中，表示径向基核函数，Yi为基因的贡献度，C为惩罚因子（为LIBSVM中对错误分类的惩罚常数）。根据libsvm的预测结果和基因的贡献度加权投票后的总分P值，如果P值大于等于0.879为高风险，小于0.879为低风险。
运用该预测模型对380例行根治性切除术的肝癌患者进行术后生存预测分析并分组，发现高风险组生存预后比低风险组显著差（P=9.3e-08，如图1所示，为肝癌转移预测模型对380例行根治性切除术的肝癌患者生存预测分析结果图），可以对患者术后生存进行准确预测。该模型总的预测准确性：70.21 %，特异性：68.12%，敏感性：72.83 %。
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Claims (4)

1.一种预测肝癌术后转移与复发的实时PCR微阵列芯片试剂盒，其特征在于，包括CXCL10基因扩增引物及荧光标记探针、FKBP10基因扩增引物及荧光标记探针、ASPH基因扩增引物及荧光标记探针、SPP2基因扩增引物及荧光标记探针和ENO2基因扩增引物及荧光标记探针；所述的CXCL10基因的扩增引物序列正义链为TGATGCAGGTACAGCGTACAGT，扩增引物序列反义链为TCCAGTCTCAGCACCATGAATC，荧光标记探针序列为CTGCCATTCTGATTTGCTGCCTTAT；所述的FKBP10基因的扩增引物序列正义链为CGACTTTGTCCGCTACCACTAC，扩增引物序列反义链为ACCAGAGCCGACGTAGGTGT，荧光标记探针序列为CACCCTGCTGGACGGCACCTC；所述的ASPH基因的扩增引物序列正义链为CTGTCTGAGCGACGCTTCAA，扩增引物序列反义链为GGAGCCATCGAGACCTACCA，荧光标记探针序列为AGGTGGCCAGCCTACCTGATGTCC；所述的SPP2基因的扩增引物序列正义链为CCGGCACAGAGCAAGAATAA，扩增引物序列反义链为AAGCAGCAGCTCCTTGAACA，荧光标记探针序列为TTGAGTAACGGCCTTGAGGTGTCCC；所述的ENO2基因的扩增引物序列正义链为CCAACTCCAGCATCAGGTTGT，扩增引物序列反义链为TGAAGGCAGTGGACCACATC，荧光标记探针序列为AACTCCACCATCGCGCCAGCC。
2.如权利要求1所述的预测肝癌术后转移与复发的实时PCR微阵列芯片试剂盒，其特征在于，所述的试剂盒中还包括反转录系统、扩增系统和384微孔板。
3.如权利要求2所述的预测肝癌术后转移与复发的实时PCR微阵列芯片试剂盒，其特征在于，所述的反转录系统由5×反转录体系缓冲液、反转录酶、脱氧核糖核酸混合物以及随机的6核苷酸引物组成。
4.如权利要求2所述的预测肝癌术后转移与复发的实时PCR微阵列芯片试剂盒，其特征在于，所述的扩增系统由含Taq酶的信使核糖核酸表达定量检测混合液组成。
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