CN113528665A - 一种筛选小细胞肺癌药物新靶点的方法及其应用 - Google Patents
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Abstract
本发明公开了小细胞肺癌药物靶点领域的一种筛选小细胞肺癌药物新靶点的方法及其应用，通过设计不同的引物，然后通过qPCR实验发现了生长抑素受体2(SSTR2)在小细胞肺癌细胞表面高表达，发现在非小细胞肺癌细胞系NCI‑H69和NCI‑H446上高表达SSTR2蛋白，表明SSTR2可以作为一个潜在的药物靶点，有助于广大药物开发者针对小细胞肺癌进行靶向药物的研发。
Description
技术领域
本发明涉及小细胞肺癌药物靶点领域，具体是一种筛选小细胞肺癌药物新靶点的方法 及其应用。
背景技术
小细胞肺癌约占总肺癌种类的15％-20％，相较于其他类型的肺癌，小细胞肺癌通常有 更好的化疗及放疗疗效，然而，由于小细胞肺癌确诊时多数已有肿瘤的广泛扩散，因此， 小细胞肺癌往往预后较差；人小细胞肺癌细胞NCI-H69细胞，又称H69细胞，源自患有小细胞肺癌的白人男性，有多种癌相关基因表达，如c-myb,v-fes,v-fms,c-raf 1, Ha-ras,Ki-ras and N-ras等；NCI-H446细胞是从一位小细胞肺癌患者的胸水中建立的， NCI-H446细胞是小细胞肺癌的生化和形态学上的变种，表达神经元特有的烯醇酶和脑部 肌酸激酶同功酶，其C-myc DNA序列扩增约20倍，c-myc RNA比正常细胞增加15倍，G 蛋白偶联受体家族(GPCRs)是目前已知的、最大的受体家族，也是现代药物开发中研究 得最多的领域，生长抑素受体(SSTR)是GPCRs家族的成员之一，分SSTR1—5五个亚 型。
目前全球范围内还没有针对小细胞肺癌的靶向药物上市，主要原因是目前没有发现小 细胞肺癌的有效靶点。
因此，本发明提供了一种筛选小细胞肺癌药物新靶点的方法及其应用，以解决上述背 景技术中提出的问题。
发明内容
本发明的目的在于提供一种筛选小细胞肺癌药物新靶点的方法及其应用，以解决上述 背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：
一种筛选小细胞肺癌药物新靶点的方法及其应用，通过设计不同的引物，然后通过qPCR实验发现了生长抑素受体2(SSTR2)在小细胞肺癌细胞表面高表达。
一种筛选小细胞肺癌药物新靶点的方法，其实验步骤为：
S1：通过RNA提取试剂盒提取小细胞肺癌细胞系NCI-H69和NCI-H446细胞总RNA，测定其含量；
S2：按照下表准备逆转录反应液，补水至终体积为20μL。轻柔混匀准备好的逆转录反应液，短暂离心：
| 混合液组分 | 体积 | 质量或20μl浓度 |
| SureScript RTase Mix(20×) | 1μL | 1× |
| SureScript RT Reaction Buffer(5×) | 4μL | 1× |
| Total RNA或者poly A RNA | 1μg或者10ng | |
| ddH<sub>2</sub>O(RNase/DNase free) | 补水至20μL |
S3：反转录反应程序设置：
| 温度 | 时间 |
| 25℃ | 5min |
| 42℃ | 30min |
| 85℃ | 5min |
| 4℃ | hold |
S4：按照下表内容，在4℃(冰上)或者室温准备qPCR反应液：
S5：轻柔混匀qPCR预混液并短暂离心，按照反应体系说明将预混液加入PCR反应管中，短暂离心确保预混反应液填充满PCR反应管底部；
S6：根据下表设置二步法qPCR程序进行qPCR反应：
作为本发明再进一步的方案：引物序列如下：
有益效果
与现有技术相比，本发明的有益效果是：
通过荧光定量聚合酶链式反应(qPCR)技术，发现在非小细胞肺癌细胞系NCI-H69和NCI-H446上高表达SSTR2蛋白，表明SSTR2可以作为一个潜在的药物靶点，有助于 广大药物开发者针对小细胞肺癌进行靶向药物的研发。
附图说明
图1为小细胞肺癌NCI-H69中SSTR1—5表达量的检测；
图2为小细胞肺癌NCI-H446中SSTR1—5表达量的检测。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地 描述。
请参阅图1～2，本发明实施例中，一种筛选小细胞肺癌药物新靶点的方法及其应用， 通过设计不同的引物，然后通过qPCR实验发现了生长抑素受体2(SSTR2)在小细胞肺癌细胞表面高表达。
一种筛选小细胞肺癌药物新靶点的方法，其实验步骤为：
S1：通过RNA提取试剂盒提取小细胞肺癌细胞系NCI-H69和NCI-H446细胞总RNA，测定其含量；
S2：按照下表准备逆转录反应液，补水至终体积为20μL。轻柔混匀准备好的逆转录反应液，短暂离心：
| 混合液组分 | 体积 | 质量或20μl浓度 |
| SureScript RTase Mix(20×) | 1μL | 1× |
| SureScript RT Reaction Buffer(5×) | 4μL | 1× |
| Total RNA或者poly A RNA | 1μg或者10ng | |
| ddH<sub>2</sub>O(RNase/DNase free) | 补水至20μL |
S3：反转录反应程序设置：
S4：按照下表内容，在4℃(冰上)或者室温准备qPCR反应液：
S5：轻柔混匀qPCR预混液并短暂离心，按照反应体系说明将预混液加入PCR反应管中，短暂离心确保预混反应液填充满PCR反应管底部；
S6：根据下表设置二步法qPCR程序进行qPCR反应：
本实施例中，引物序列如下：
结果分析：我们选取了两个小细胞肺癌细胞系NCI-H69和NCI-H446，设计特定的引物， 通过qPCR技术，鉴定生长抑素受体五种不同亚型SSTR1—5的表达情况，结果表明，两个细胞系中均只有SSTR2高表达，与其他亚型相比有极显著性差异(P<0.001)，因此SSTR2 是小细胞肺癌潜在的一个药物靶点，可以针对该靶点开发相应的抗肿瘤靶向药物，如单克 隆抗体(Antibody)，抗体偶联药物(Antibody Drug-Conjugates,ADC)，多肽偶联药 物(Peptide Drug-Conjugates,PDC)等。
Claims (3)
1.一种筛选小细胞肺癌药物新靶点的方法及其应用，通过设计不同的引物，然后通过qPCR实验发现了生长抑素受体2(SSTR2)在小细胞肺癌细胞表面高表达。
2.根据权利要求1所述的一种筛选小细胞肺癌药物新靶点的方法，其实验步骤为：
S1：通过RNA提取试剂盒提取小细胞肺癌细胞系NCI-H69和NCI-H446细胞总RNA，测定其含量；
S2：按照下表准备逆转录反应液，补水至终体积为20μL。轻柔混匀准备好的逆转录反应液，短暂离心：
S3：反转录反应程序设置：
S4：按照下表内容，在4℃(冰上)或者室温准备qPCR反应液：
S5：轻柔混匀qPCR预混液并短暂离心，按照反应体系说明将预混液加入PCR反应管中，短暂离心确保预混反应液填充满PCR反应管底部；
S6：根据下表设置二步法qPCR程序进行qPCR反应。
3.根据权利要求1所述的一种筛选小细胞肺癌药物新靶点的方法及其应用，其特征在于：引物序列如下：
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101439182A (zh) * | 2008-12-18 | 2009-05-27 | 北京大学 | 一种生长抑素受体介导的肿瘤靶向药物组合物 |
| US20130287681A1 (en) * | 2010-09-08 | 2013-10-31 | Board Of Regents, The University Of Texas System | Somatostatin receptor-based cancer therapy |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101439182A (zh) * | 2008-12-18 | 2009-05-27 | 北京大学 | 一种生长抑素受体介导的肿瘤靶向药物组合物 |
| US20130287681A1 (en) * | 2010-09-08 | 2013-10-31 | Board Of Regents, The University Of Texas System | Somatostatin receptor-based cancer therapy |
Non-Patent Citations (2)
| Title |
|---|
| KERRY A. WHALEN 等: "Targeting the Somatostatin Receptor 2 with the Miniaturized Drug Conjugate, PEN-221: A Potent and Novel Therapeutic for the Treatment of Small Cell Lung Cancer", 《MOL CANCER THER》, vol. 18, no. 11, pages 1927 * |
| 王健 等: "奥曲肽紫杉醇偶联物靶向治疗小细胞肺癌的实验研究", 《山东大学学报(医学版)》, vol. 49, no. 3, pages 28 * |
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