CN103911451A - 筛选抗血管生成靶向药物治疗肺癌目标人群的方法 - Google Patents

筛选抗血管生成靶向药物治疗肺癌目标人群的方法 Download PDF

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CN103911451A
CN103911451A CN201410143242.6A CN201410143242A CN103911451A CN 103911451 A CN103911451 A CN 103911451A CN 201410143242 A CN201410143242 A CN 201410143242A CN 103911451 A CN103911451 A CN 103911451A
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蒋晓东
丁曼华
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Abstract

本发明公开了一种筛选抗血管生成靶向药物治疗肺癌目标人群的方法，通过采用荧光原位杂交技术(FISH)检测VEGFR2基因(KDR)及NRP-1基因拷贝获益(copy number gains，CNG)情况，最终所获结果结合患者的临床病理资料进行分析，指导病人治疗及评价预后情况。

Description

筛选抗血管生成靶向药物治疗肺癌目标人群的方法
技术领域
本发明属于医药领域，具体而言，提供了一筛选抗血管生成靶向药物治疗肺癌目标人群的方法。
背景技术
肺癌所致死亡率已位居各肿瘤之首，寻求更有效的治疗方法迫在眉睫。目前抗血管生成靶向治疗的出现给肺癌患者带来了曙光，然而其治疗效果并不令人满意，同样是非小细胞肺癌，采用同样的治疗方案和剂量，会产生截然不同的治疗结果和毒副反应。究其原因在于个体间差异，因而在抗血管生成靶向药物治疗肺癌的过程中，如何选择合适的病人进行治疗是目前研究的关键。血管内皮细胞生长因子受体2(VEGFR2)、神经纤毛蛋白-1(NRP-1)可以作为抗血管生成治疗的主要靶点指导临床治疗，为此我们要筛选出该优势人群。
发明内容
本发明采用荧光原位杂交技术(FISH)检测VEGFR2基因(KDR)及NRP-1基因拷贝获益(copy number gains，CNG)情况，最终所获结果结合患者的临床病理资料进行分析，指导病人治疗及评价预后情况)。为了实现本发明的目的，拟采用如下技术方案：
本发明涉及一种筛选抗血管生成靶向药物治疗肺癌目标人群的方法，采用荧光原位杂交技术(FISH)检测VEGFR2基因(KDR)及NRP-1基因拷贝获益(copy number gains，CNG)情况，最终所获结果结合患者的临床病理资料进行分析，其特征在于包括如下步骤：
将待测标本蜡块重新制备成4μm厚连续切片，放置70℃烘烤10min，切片放入二甲苯脱蜡3×15min，依次放入100％，100％，90％和70％乙醇各脱苯复水5min；去离子水洗2×2min；切片置于预处理液PT1盒中98℃孵育15min；去离子水洗2×2min，室温晾干；切片滴加胃蛋白酶ES1于37℃反应13min；Wash Buffer SSC洗5min，去离子水洗1min。切片依次放入70％，90％和100％乙醇各脱水3min，3min，5min，室温晾干；每张切片滴加5uL FISH杂交液，杂交液中探针体系中各成分比例为目的基因(KDR基因/NRP-1基因)：对照基因：杂交液为＝1∶1∶3；
将原位杂交专用盖玻片的保护膜揭开后，盖在切片上并用胶暂时性封片，77℃反应10min，转入37℃杂交过夜约16h；揭掉胶，在37℃的1×Wash BufferA中放置1-3min并小心移去盖玻片，37℃1×Wash BufferA洗2×5min；切片依次放入70％，90％和100％乙醇分别脱水3min、3min、5min，室温避光并晾干；切片滴加10uL DAPI，室温避光反应15min；透明指甲油封片，荧光显微镜下观察，拍照，观察结果。
附图说明
图1KDR CNG(FISH法，1000倍油镜)
A鳞癌CNG(+)，B腺癌CNG(+)，C鳞癌CNG(-)，D腺癌CNG(-)。红色信号为KDR基因探针，绿色信号为对照。
图2NRP-1CNG(FISH法，1000倍油镜)
A鳞癌CNG(+)，B腺癌CNG(+)，C鳞癌CNG(-)，D腺癌CNG(-)。红色信号为NRP-1基因探针，绿色信号为对照。
图3NSCLC患者总体生存率(OS)和无进展生存期(PFS)的Kaplan-Meier生存曲线图.
A：Kaplan-Meier生存曲线图显示VEGFR2CNG(+)的NSCLC患者有较短的OS(P＝0.0008)B：NRP-1CNG(+)的NSCLC患者有较短的OS(P＝0.0231)C：Kaplan-Meier生存曲线图显示VEGFR2CNG(+)的NSCLC患者有较差的PFS(P＝0.0480)D：NRP-1CNG(+)的NSCLC患者有较差的PFS(P＝0.0315)
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步的详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定发明。
实施例1：
2009年01月至2012年11月间，选择在我院胸外科手术切除并经病理学确诊的NSCLC石蜡包埋标本40例，有完整的临床资料。选择同期10例肺部炎性假瘤组织石蜡包埋标本为对照。
标本蜡块重新制备成4μm厚连续切片。将原位杂交切片放置70℃烘烤10min，切片放入二甲苯脱蜡3×15min，依次放入100％，100％，90％和70％乙醇各脱苯复水5min。去离子水洗2×2min。切片置于预处理液PT1盒(购于美国Empire Genomics公司)中(提前放入98℃预热)98℃孵育15min。去离子水洗2×2min，并确保室温晾干。切片滴加胃蛋白酶ES1于37℃反应13min。Wash Buffer SSC洗5min，去离子水洗1min。切片依次放入70％，90％和100％乙醇各脱水3min，3min，5min，并确保室温晾干。每张切片滴加5uL FISH杂交液，杂交液中探针体系中各成分比例为目的基因(KDR基因/NRP-1基因)：对照基因：杂交液为＝1∶1∶3。
将原位杂交专用盖玻片的保护膜揭开后，盖在切片上并用胶暂时性封片，77℃反应10min，转入37℃杂交过夜约16h。揭掉胶，在37℃的1×Wash BufferA中放置1-3min并小心移去盖玻片，37℃1×Wash BufferA洗2×5min。切片依次放入70％，90％和100％乙醇分别脱水3min、3min、5min，室温避光并确保晾干。切片滴加10uL DAPI，室温避光反应15min。透明指甲油封片，荧光显微镜下观察，拍照，观察结果见附图。KDR、NRP-1CNG(+)与对照组比较差异有统计学意义(分别为χ2＝4.39，P＝0.036；χ2＝3.95，P＝0.046)。生存分析结果显示，VEGFR2及NRP-1CNG(+)与对照组间的总生存期(OS)及无进展生存期(PFS)差异均有统计学意义(P＜0.05)。(注：以上均应在避光条件下进行)
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (1)

1.一种筛选抗血管生成靶向药物治疗肺癌目标人群的方法，采用荧光原位杂交技术(FISH)检测VEGFR2基因(KDR)及NRP-1基因拷贝获益(copy number gains，CNG)情况，最终所获结果结合患者的临床病理资料进行分析，其特征在于包括如下步骤：
将待测标本蜡块重新制备成4μm厚连续切片，放置70℃烘烤10min，切片放入二甲苯脱蜡3×15min，依次放入100％，100％，90％和70％乙醇各脱苯复水5min；去离子水洗2×2min；切片置于预处理液PT1盒中98℃孵育15min；去离子水洗2×2min，室温晾干；切片滴加胃蛋白酶ES1于37℃反应13min；Wash Buffer SSC洗5min，去离子水洗1min；切片依次放入70％，90％和100％乙醇各脱水3min，3min，5min，室温晾干；每张切片滴加5uL FISH杂交液，杂交液中探针体系中各成分比例为目的基因(KDR基因/NRP-1基因)：对照基因：杂交液为＝1∶1∶3；
将原位杂交专用盖玻片的保护膜揭开后，盖在切片上并用胶暂时性封片，77℃反应10min，转入37℃杂交过夜约16h；揭掉胶，在37℃的1×Wash BufferA中放置1-3min并小心移去盖玻片，37℃1×Wash BufferA洗2×5min；切片依次放入70％，90％和100％乙醇分别脱水3min、3min、5min，室温避光并晾干；切片滴加10uL DAPI，室温避光反应15min；透明指甲油封片，荧光显微镜下观察，拍照，观察结果。
CN201410143242.6A 2014-04-11 2014-04-11 筛选抗血管生成靶向药物治疗肺癌目标人群的方法 Pending CN103911451A (zh)

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Application publication date: 20140709