CN107976534A - 一种抗肿瘤药物快速药效筛选方法及其专用装置 - Google Patents
一种抗肿瘤药物快速药效筛选方法及其专用装置 Download PDFInfo
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Abstract
本发明提供了一种抗肿瘤药物快速药效测试方法包括以下步骤:1.取病人来源的新鲜肿瘤组织或小鼠PDX模型的肿瘤组织;2.将步骤1获得的肿瘤组织进行组织消化处理,并分选,以获得肿瘤单细胞;3.将步骤2所获得的肿瘤单细胞置于管状装置中;4.将步骤3所获得的管子植入动物体内后,并按临床给药途径给药;5.检测药效敏感性。
Description
技术领域
本发明涉及药物药效筛选领域,尤其涉及一种抗肿瘤药物快速药效筛选方法及其专用装置。
背景技术
目前,个体化精准医疗被越来越多的应用在临床上,尤其是肿瘤治疗领域。化疗是目前对晚期转移性癌症患者的一种主要治疗方式,但由于化疗药物均缺乏肿瘤细胞特异性,在杀伤肿瘤细胞的同时,也杀伤大量骨髓及其他增殖旺盛正常细胞,有较严重的不良反应。同时传统的化疗方法在进行多次化疗后容易产生耐药性,这会影响化疗效果。由此针对患者个体化精准医疗需求越来越迫切。个体化精准医疗一方面提高治疗的精确性,另一方面也可以有效地降低患者用药的随机性和盲目性,从而减轻患者用药的伤害。近年来,与临床病人用药疗效相关性高的动物模型为病人来源肿瘤移植物模型(patient-derivedxenograft model,PDX模型)广泛应用于精准医疗中。北美已有多家大型临床医疗中心通过建立PDX模型来进行临床前药物研发和药物筛选以及指导癌症患者的个性化精准用药。
目前应用于抗肿瘤药物筛选最好的技术是在病人来源肿瘤移植物模型(patient-derived xenograft model,PDX模型)上进行药效实验。PDX模型较好地保持了患者原发肿瘤的生物学特性。该模型是将病人新鲜的瘤组织直接移植到免疫缺陷小鼠(常用裸鼠或SCID小鼠)体内而建立的肿瘤模型,此类模型保持了与病人相似的遗传特性和肿瘤异质性。目前,运用PDX模型进行个体化精准医疗的主要难点在于模型建成周期与药敏测试花费时间长以及PDX模型的移植成瘤率较低。例如原代移植就需要2~3个月,而随后药物敏感测试时间也需要3~4个月。另外,PDX模型的操作技术要求很高,标本从手术室获得后需要外科医生、组织学家和研究者密切配合。因此,对PDX模型抗肿瘤药物药敏测试方法的进行改进也是精准医疗领域中一个急需解决的问题。
发明内容
为了克服以上传统PDX模型药敏测试存在的技术问题,发明人开发了一种抗肿瘤药物快速筛选或测试药效的筛选方法:
本发明提供的抗肿瘤药物快速药效测试方法包括以下步骤:
1.取病人来源的新鲜肿瘤组织或小鼠PDX模型的肿瘤组织;
2.将步骤1获得的肿瘤组织进行组织消化处理,并分选,以获得肿瘤单细胞;
3.将步骤2所获得的肿瘤单细胞置于管状装置中;
4.将步骤3所获得的管子植入动物体内后,并按临床给药途径给药;
5.检测药效敏感性。
优选地,步骤5是结合体外药敏检测的方法确定药物的药效敏感性。
优选地,动物选用小鼠,还优选是免疫缺陷小鼠,还优选是裸鼠或SCID小鼠。
优选地,步骤4所述的临床给药途径为口服灌胃或尾静脉注射。
本发明另一方面还提供了一种管状装置,其为直径为2mm,所述管状装置能够允许小于500KD大小的分子通透。该管状装置的优点是:其放置在动物体内以后,所述装置内的液体可与小鼠体液中的营养物质进行交换,因此,肿瘤细胞可以利用小鼠体内的这些营养维持生长。
本发明另一方面还提供了一种试剂盒,其包含直径为2mm的管状装置,所述管状装置能够允许小于500KD大小的分子通透。该管状装置可以分离移植到动物体内。
有益效果:由于肿瘤细胞在体外不容易生长,此种方法借助小鼠作为营养提供者,使肿瘤细胞在体内生长,并利用体外检测细胞的生长状态,两周即可做出药效结果。经过验证,该方法与传统PDX所得到的药效结果有极高的相关性。本发明可以结合体内和体外实验技术进行快速抗肿瘤药物药效评价,可用于快速高效的检测抗肿瘤药物的药效。
与本发明还极大地缩短了传统PDX模型的药效实验所需的时间,及临床实验的成本。总之,本发明具有快速,操作便捷,费用低,可重复性强,易于推广的优点。
附图说明
图1为本发明的测试方法的流程示意图;
图2为抗肿瘤药物S-1使用GAPF155模型在不同胃癌临床病人肿瘤细胞通过传统PDX方法的肿瘤生长曲线图;
图3为抗肿瘤药物S-1使用GAPF155模型在不同胃癌临床病人肿瘤细胞通过本发明Mini-PDX方法药效数据图;
图4为抗肿瘤药物S-1使用GAPF157模型在不同胃癌临床病人肿瘤细胞通过传统PDX方法的肿瘤生长曲线图;
图5为抗肿瘤药物S-1使用GAPF157模型在不同胃癌临床病人肿瘤细胞通过本发明Mini-PDX方法药效数据图;
图6为抗肿瘤药物S-1通过GAPF161模型在不同胃癌临床病人肿瘤细胞通过传统PDX方法肿瘤生长曲线图;
图7为抗肿瘤药物S-1通过GAPF161模型在不同胃癌临床病人肿瘤细胞通过本发明Mini-PDX方法药效数据图。
具体实施方式
为了更好地说明本发明,下面参照附图予以说明。
本发明的测试方法也可称为Mini-PDX方法。
实验动物
从供应商订购4-5周龄的雌性Nu/Nu小鼠,饲养于SPF级别动物房,实验开始前动物至少适应性饲养三天。
样本的采集
取新鲜收集的临床肿瘤手术样本或小鼠PDX样本于运输保存管中,冰上运输,在尽可能短的时间内运至实验室。
单个肿瘤细胞分离
1.在生物安全柜中去除非肿瘤组织和坏死组织;
2.将组织切成小块,PBS冲洗并收集组织块,离心去上清;PBS清洗,离心3去上清,使用胶原酶37℃消化肿瘤块1-2小时;
3.用血清培养基1:1稀释终止消化,收集细胞悬液;
4.离心去除上清,使用PBS重悬细胞,清洗一次;
5.PBS重悬细胞,计数,调整细胞密度至1×108/ml;加入肿瘤细胞分选磁珠,室温孵育30min;
6.加入PBS清洗一次细胞,并用2ml PBS重悬。将细胞加入磁柱中,进行分选,收集分选出的肿瘤细胞;
7.离心去除上清,细胞培养液重悬,计数,调整细胞密度至1×106/ml。
细胞罐装,接种及活力检测
在生物安全柜中取出管子,检查管子有无破损或变形,每根管子约2cm长度大小,用PBS反复冲洗3-5遍,最后将管中液体吹掉;
用200uL移液枪将肿瘤细胞悬液加入管中,管子加入密封;
用肿瘤穿刺针将纤维管接种至实验小鼠背部皮下,并设置给药组和对照组,分别进行小鼠的给药处理;
实验进行5-7天,实验结束后安乐死小鼠;
小鼠体内取出的管子,用移液枪吹打出里面的细胞,并进行活力检测。
数据分析
根据对照组细胞的活力值,计算出给药组细胞活力增殖百分比,进而评价相应药物的药效。
图1显示了为抗肿瘤药物快速药效测试方法的流程图。首先,从临床或PDX模型上收取肿瘤组织,然后用组织消化液消化,获得单个肿瘤细胞,并将肿瘤细胞罐装至管中,接种到小鼠皮下,给药处理,5-7天后取出管子,进行细胞活力测试。
图2-7显示了为传统PDX药效测试与本发明Mini-PDX药效测试的对比结果。
实施例一
图2和3使用了GAPF155模型。图2-3的结果显示了在临床上病人对S-1敏感;
实施例二
图4和5使用了GAPF157模型。图4-5的结果显示了在临床上病人对S-1敏感;
实施例三
图6和7是使用了GAPF161模型,图6和7显示在临床上病人对S-1不敏感。
传统PDX和本发明Mini-PDX药效数据显示,S-1在GAPF155模型(图2和3)和GAPF157模型(图4和5)均有非常好的抗肿瘤活性,而S-1在GAPF161模型(图6和7)无抗肿瘤活性,与临床结果一致。
上述实施例仅仅是通过举例的方式描述。在不偏离本发明所附权利要求限定的保护范围的情况下,可有各种变体。
Claims (6)
1.一种抗肿瘤药物快速药效测试方法包括以下步骤:
1.取病人来源的新鲜肿瘤组织或小鼠PDX模型的肿瘤组织;
2.将步骤1获得的肿瘤组织进行组织消化处理,并分选,以获得肿瘤单细胞;
3.将步骤2所获得的肿瘤单细胞置于管状装置中;
4.将步骤3所获得的管子植入动物体内后,并按临床给药途径给药;
5.检测药效敏感性。
2.如权利要求1所述的方法,其特征在于,步骤4中所述动物为小鼠。
3.如权利要求1所述的方法,其特征在于,所述步骤5是用体外药敏检测方法来确定药物的药效敏感性。
4.如权利要求1所述的方法,其特征在于,所述临床给药途径为口服灌胃或尾静脉注射。
5.一种抗肿瘤药物快速药效测试方法专用管状装置,其特征在于,其为直径为2mm,所述管状装置能够允许小于500KD大小的分子通透。
6.一种用于抗肿瘤药物快速药效测试方法的试剂盒,其包含直径为2mm的管状装置,所述管状装置能够允许小于500KD大小的分子通透。
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CN108351348A (zh) * | 2016-10-21 | 2018-07-31 | 上海立迪生物技术股份有限公司 | 一种药物筛选方法及其装置 |
CN109479814A (zh) * | 2018-11-14 | 2019-03-19 | 苏州致诺优生物医学有限公司 | 抗肺癌肿瘤药物的筛选模型 |
CN112980690A (zh) * | 2019-12-17 | 2021-06-18 | 华东数字医学工程研究院 | Pdx模型孵育装置和抗肿瘤药物筛选方法 |
CN113142135A (zh) * | 2021-04-09 | 2021-07-23 | 山东第一医科大学附属省立医院(山东省立医院) | 一种消化道肿瘤pdx模型及标准化模型库的构建方法 |
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