CN112553287A - 检测vegf位点靶向药对肠癌类器官影响的方法 - Google Patents

检测vegf位点靶向药对肠癌类器官影响的方法 Download PDF

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CN112553287A
CN112553287A CN202011413257.1A CN202011413257A CN112553287A CN 112553287 A CN112553287 A CN 112553287A CN 202011413257 A CN202011413257 A CN 202011413257A CN 112553287 A CN112553287 A CN 112553287A
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陈益莹
饶栓
蒋鑫
蒋沁悦
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Abstract

本发明公开了一种检测VEGF位点靶向药对肠癌类器官影响的方法，该方法包括如下步骤：1)以传代消化液消化15‑25min；2)以微器官为单位均匀分配入微孔板中；3)类器官铺板培养22‑26h后换为含药培养基；4)采用CellTiter‑Glo试剂盒检测用药后类器官活力。本发明的检测VEGF位点靶向药对肠癌类器官影响的方法具有：温和的消化液减少此步骤对类器官的伤害；以微器官为单位进行药筛，更好的模拟临床用药后体内癌组织的反应；铺板后正常培养22‑26h，使类器官生长状态稳定后接受药物作用，更贴近体内用药状态；采用CellTiter‑Glo试剂盒检测细胞活力，结果更高效和准确。

Description

检测VEGF位点靶向药对肠癌类器官影响的方法
技术领域
本发明涉及生物医药领域，尤其涉及一种检测VEGF位点靶向药对肠癌类器官影响的方法。
背景技术
目前，检测VEGF位点靶向药对肠癌类器官影响的方法主要有以下几种：
1)癌症细胞系用药实验
细胞系体外生长环境与体内环境有显著不同，且多为商业化的通用细胞系，个性化程度低，无法准确模拟实际肿瘤对药物的反应，也无法做到个性化。
2)临床人体用药疗效统计分析
临床用药时间周期通常以年为单位，试验周期长，且成本高昂。
3)肠癌类器官用药实验
目前已使用肠癌类器官为模型检测多种常用的肠癌化疗药物(不包括本专利所检测药物)的效果，证实与临床效果接近，参考文献如下：
Schütte M,Risch T,Abdavi-Azar N,Boehnke K,Schumacher D,Keil M,Yildiriman R,Jandrasits C,Borodina T,Amstislavskiy V,Worth CL,Schweiger C,Liebs S,Lange M,Warnatz HJ,Butcher LM,Barrett JE,Sultan M,Wierling C,Golob-Schwarzl N,Lax S,Uranitsch S,Becker M,Welte Y,Regan JL,Silvestrov M,Kehler I,Fusi A,Kessler T,Herwig R,Landegren U,Wienke D,Nilsson M,Velasco JA,Garin-Chesa P,Reinhard C,Beck S,Sch fer R,Regenbrecht CR,Henderson D,Lange B,Haybaeck J,Keilholz U,Hoffmann J,Lehrach H,Yaspo ML.Molecular dissection ofcolorectal cancer in pre-clinical models identifies biomarkers predictingsensitivity to EGFR inhibitors.Nat Commun.2017Feb 10；8:14262.doi:10.1038/ncomms14262.PMID:28186126；PMCID:PMC5309787。
发明内容
针对现有技术中存在的上述不足，本发明提供了一种检测VEGF位点靶向药对肠癌类器官影响的方法，该方法更快速和直观的反映靶向VEGF位点的靶向药对肠癌类器官的作用，该方法使用成本更低，且该方法能得到更接近临床且具有个性化的药效结果。
为了解决上述技术问题，本发明采用了如下技术方案：
检测VEGF位点靶向药对肠癌类器官影响的方法，该方法包括如下步骤：
1)以传代消化液消化15-25min；
2)以微器官为单位均匀分配入微孔板中；
3)类器官铺板培养22-26h后换为含药培养基；
4)采用CellTiter-Glo试剂盒检测用药后类器官活力。
作为本发明的一种优选方案，步骤1)中的消化具体步骤为：选择状态稳定的类器官，去除培养板中培养基，并使用HBSS清洗1-2次，加入1.5mL传代消化液至培养板中，吹打基质胶，混匀后放入37℃，5％CO2培养箱15-25min，终止消化后以1200rpm，4℃的条件离心4min，弃上清。
作为本发明的一种优选方案，步骤3)中的铺板具体步骤为：将复融的基质胶与类器官沉淀按13-25个/μl的比例混匀，以8μl/孔加入384孔细胞培养板中，放入37℃，5％CO2培养箱，凝固10-15min。
作为本发明的一种优选方案，步骤3)中的加培养基具体步骤为：待胶凝固后每孔加入20uL预热的自制培养基，放入37℃，5％CO2培养箱。
作为本发明的一种优选方案，步骤3)中的加药换液具体步骤为：培养24h后换为含药培养基，并每隔3天换液1次；其中，所用药物及其浓度如下：
药物：贝伐单抗或雷莫芦单抗；
浓度：100μM、25μM、6.25μM、1.56μM或0.39μM。
作为本发明的一种优选方案，步骤4)中的检测具体步骤为：加药6天后，放于22-25℃室温平衡30min，加入与培养试剂等量的CellTiter-Glo3D检测试剂，在微孔震荡仪上震荡5min，充分混匀，室温放置25min后，使用酶标仪检测发光值。
与现有技术相比，本发明具有如下优点：
1、温和的消化液减少此步骤对类器官的伤害。
2、以微器官为单位进行药筛，更好的模拟临床用药后体内癌组织的反应。
3、铺板后正常培养22-26h，使类器官生长状态稳定后接受药物作用，更贴近体内用药状态。
4、采用CellTiter-Glo试剂盒检测细胞活力，结果更高效和准确。
附图说明
图1是样本一在不同浓度贝伐单抗下的相对存活率图；
图2是样本二在不同浓度贝伐单抗下的相对存活率图。
具体实施方式
检测VEGF位点靶向药对肠癌类器官影响的方法，该方法包括如下步骤：
1.消化：选择状态稳定的类器官，去除培养板中培养基，并使用HBSS清洗1-2次，加入1.5mL传代消化液至培养板中，吹打基质胶，混匀后放入37℃，5％CO2培养箱15-25min，终止消化后以1200rpm，4℃的条件离心4min，弃上清。
2.铺板：将复融的基质胶与类器官沉淀按13-25个/μl的比例混匀，以8μl/孔加入384孔细胞培养板中，放入37℃，5％CO2培养箱，凝固10-15min。
3.加培养基：待胶凝固后每孔加入20uL预热的自制培养基，放入37℃，5％CO2培养箱。
4.加药换液：培养24h后换为含药培养基，并每隔3天换液1次。
其中，本实例所用药物及其浓度如下：
药物：贝伐单抗或雷莫芦单抗；
浓度：100μM、25μM、6.25μM、1.56μM或0.39μM。
5.检测：加药6天后，放于22-25℃的室温平衡30min，加入与培养试剂等量的CellTiter-Glo3D检测试剂，在微孔震荡仪上震荡5min，充分混匀，室温放置25min后，使用酶标仪检测发光值。
结果分析：贝伐单抗作用于不同患者的肠癌类器官均显示无效。由于其靶点VEGF的受体位于肿瘤血管内皮细胞表面，而肠癌类器官模型中无血管内皮细胞，所以其理论结果与实验结果一致，贝伐单抗对单纯的肠癌类器官无效，如图1和图2所示。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (6)

1.检测VEGF位点靶向药对肠癌类器官影响的方法，其特征在于，该方法包括如下步骤：
1)以传代消化液消化15-25min；
2)以微器官为单位均匀分配入微孔板中；
3)类器官铺板培养22-26h后换为含药培养基；
4)采用CellTiter-Glo试剂盒检测用药后类器官活力。
2.如权利要求1所述的检测VEGF位点靶向药对肠癌类器官影响的方法，其特征在于，步骤1)中的消化具体步骤为：选择状态稳定的类器官，去除培养板中培养基，并使用HBSS清洗1-2次，加入1.5mL传代消化液至培养板中，吹打基质胶，混匀后放入37℃，5％CO2培养箱15-25min，终止消化后以1200rpm，4℃的条件离心4min，弃上清。
3.如权利要求1所述的检测VEGF位点靶向药对肠癌类器官影响的方法，其特征在于，步骤3)中的铺板具体步骤为：将复融的基质胶与类器官沉淀按13-25个/μl的比例混匀，以8μl/孔加入384孔细胞培养板中，放入37℃，5％CO2培养箱，凝固10-15min。
4.如权利要求1所述的检测VEGF位点靶向药对肠癌类器官影响的方法，其特征在于，步骤3)中的加培养基具体步骤为：待胶凝固后每孔加入20uL预热的自制培养基，放入37℃，5％CO2培养箱。
5.如权利要求3所述的检测VEGF位点靶向药对肠癌类器官影响的方法，其特征在于，步骤3)中的加药换液具体步骤为：培养24h后换为含药培养基，并每隔3天换液1次；其中，所用药物及其浓度如下：
药物：贝伐单抗或雷莫芦单抗；
浓度：100μM、25μM、6.25μM、1.56μM或0.39μM。
6.如权利要求1所述的检测VEGF位点靶向药对肠癌类器官影响的方法，其特征在于，步骤4)中的检测具体步骤为：加药6天后，放于22-25℃室温平衡30min，加入与培养试剂等量的CellTiter-Glo3D检测试剂，在微孔震荡仪上震荡5min，充分混匀，室温放置25min后，使用酶标仪检测发光值。
CN202011413257.1A 2020-12-02 2020-12-02 检测vegf位点靶向药对肠癌类器官影响的方法 Pending CN112553287A (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103739710A (zh) * 2014-01-26 2014-04-23 中国人民解放军军事医学科学院基础医学研究所 一种抗vegf抗体及其应用
CN109069632A (zh) * 2016-04-15 2018-12-21 伊莱利利公司 用于治疗结直肠癌的雷莫芦单抗和merestinib的组合
CN109655606A (zh) * 2019-01-11 2019-04-19 华东师范大学 一种利用3d类器官评价药物肠毒性的检测方法
CN109952315A (zh) * 2016-11-16 2019-06-28 伊莱利利公司 用于转移性结直肠癌的治疗
CN111534564A (zh) * 2020-04-17 2020-08-14 上海市第十人民医院 一种基于肠道类器官筛选药物的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103739710A (zh) * 2014-01-26 2014-04-23 中国人民解放军军事医学科学院基础医学研究所 一种抗vegf抗体及其应用
CN109069632A (zh) * 2016-04-15 2018-12-21 伊莱利利公司 用于治疗结直肠癌的雷莫芦单抗和merestinib的组合
CN109952315A (zh) * 2016-11-16 2019-06-28 伊莱利利公司 用于转移性结直肠癌的治疗
CN109655606A (zh) * 2019-01-11 2019-04-19 华东师范大学 一种利用3d类器官评价药物肠毒性的检测方法
CN111534564A (zh) * 2020-04-17 2020-08-14 上海市第十人民医院 一种基于肠道类器官筛选药物的方法

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