CN115418353A - 一种结直肠腹膜转移癌类器官移瘤模型构建及其应用 - Google Patents
一种结直肠腹膜转移癌类器官移瘤模型构建及其应用 Download PDFInfo
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Abstract
本发明公开了一种结直肠癌腹膜转移癌类器官培养方法、利用所述方法获得的类器官。还公开了利用结直肠癌腹膜转移癌类器官构建结直肠癌腹膜转移癌异种移植模型的方法。进一步公开了上述结直肠癌腹膜转移癌类器官、异种移植模型在结直肠癌腹膜转移癌药物筛选、结直肠癌腹膜转移癌发病机制研究中的应用。
Description
技术领域
本发明涉及医药领域,具体涉及结直肠腹膜转移癌类器官移瘤模型构建及其应用。
发明背景
现有结直肠癌(colorectal cancer,CRC)腹膜转移癌(peritoneal metastaticcolorectal cancer,pmCRC)PDX模型构建主要依赖于人源肿瘤细胞系或者腹膜转移癌患者新鲜组织消化成单细胞后进行裸鼠皮下移植或者腹腔内注射进行构建。
人源肿瘤细胞系不能反映临床病人的异质性,不能体现患者肿瘤的组织病理和基因组特性。此种结肠癌小鼠模型要么制备周期长、成瘤均一度较差;要么未能完全模拟自然转移过程、并发症多;要么制备操作复杂、成功率较低、模型不稳定。
pmCRC患者新鲜组织PDX模型构建。如果采用的是腹腔内注射,可能由于肠外腹腔多处种植,即使成功致瘤,也易于出现因接种部位肿瘤组织呈团块状生长过大,造成肠腔梗阻,导致小鼠死亡。并且只能反映体内的肿瘤病理变化进展,敏感药物筛选实验缺乏体外实验的验证,药敏结果可能缺乏一定的稳定性。
发明内容
为解决上述问题,本发明提供了一种构建pmCRC类器官模型(patient-derivedmetastatic organoids,PDMO)的构建方法及其应用。
具体地,本发明第一方面提供了一种结直肠癌腹膜转移癌类器官培养方法,包括如下步骤:1)获取结直肠癌腹膜转移癌组织样本,组织破碎和消化;2)过滤获得单细胞和细胞团悬液;3)基质胶重悬细胞或细胞团;4)待基质胶凝固,加入CRC类器官完全培养基,培养获得直肠癌腹膜转移癌类器官。
在某些实施方式中,所述步骤1)中在含有FBS、PS、IX型胶原酶和II型组织蛋白消化酶的消化液中进行消化。优选地,消化液中含1-5%FBS、0.1-5%PS、25-100U/ml IX型胶原酶和50-200g/mL II型组织蛋白消化酶;更优选地,消化液中含2.5%FBS、1%PS、75U/mlIX型胶原酶和125g/mL II型组织蛋白消化酶。
在某些实施方式中,所述步骤1)中消化后采用基础培养基中止消化。
在某些实施方式中,所述基础培养基包括Advanced DMEM/F12、PS、HEPES和GlutaMAX;优选地,所述基础培养基包括Advanced DMEM/F12、0.1-5%PS、1-20mM HEPES和0.5-10mM GlutaMAX;更优选地,所述基础培养基包括Advanced DMEM/F12、1%PS、10mMHEPES和2mM GlutaMAX。
在某些实施方式中,所述CRC类器官完全培养基包括基础培养基,含R-spondin-1重组蛋白的培养基,Noggin,1×B27,n-Acetyl Cysteine,Nicotinamide,EGF,Gastrin,A83-01,SB202190,Prostaglandine E2和Primocin;优选地,所述CRC类器官完全培养基包括基础培养基,10-30%含R-spondin-1重组蛋白的培养基,50-200ng/ml Noggin,1×B27,0.25-2mM n-Acetyl Cysteine,1-50mM Nicotinamide,10-100ng/ml EGF,1-50nMGastrin,100-1000nM A83-01,1-10μM SB202190,1-50nM Prostaglandine E2和10-200μg/ml Primocin;更优选地,所述CRC类器官完全培养基包括基础培养基,20%含R-spondin-1重组蛋白的培养基,100ng/ml Noggin,1×B27,1.25mM n-Acetyl Cysteine,10mMNicotinamide,50ng/ml EGF,10nM Gastrin,500nM A83-01,5μM SB202190,10nMProstaglandine E2和100μg/ml Primocin。
在某些实施方式中,所述基础培养基包括Advanced DMEM/F12、PS、HEPES和GlutaMAX;优选地,所述基础培养基包括Advanced DMEM/F12、0.1-5%PS、1-20mM HEPES和0.5-10mM GlutaMAX;更优选地,所述基础培养基包括Advanced DMEM/F12、1%PS、10mMHEPES和2mM GlutaMAX。
本发明第二方面提供了根据本发明第一方面所述的方法培养获得的结直肠癌腹膜转移癌类器官。
本发明第三方面提供了一种结直肠癌腹膜转移癌异种移植模型的构建方法,包括如下步骤:a.将结直肠癌腹膜转移癌类器官消化成单细胞,然后用基质胶和肿瘤类器官培养基的混合物重悬;b.用类器官悬液皮下注射小鼠,待类器官种植的皮下瘤形成;c.待瘤子长至适宜大小后,把瘤块切成小块接种至裸鼠盲肠上,缝合后待肿瘤生长,从而构建腹膜转移类器官异种移植模型。
在某些实施方式中,所述基质胶和肿瘤类器官培养基的混合物包括50%BME和50%。
本发明第四方面提供了根据本发明第三方面所述的方法培养获得的结直肠癌腹膜转移癌异种移植模型。
本发明地五方面提供了根据本发明第二方面所述的结直肠癌腹膜转移癌类器官或本发明第四方面所述的结直肠癌腹膜转移癌异种移植模型在结直肠癌腹膜转移癌药物筛选、结直肠癌腹膜转移癌发病机制研究中的应用。
本发明和现有技术相比,具有以下有益效果:
1、本发明成功构建了结直肠癌腹膜转移灶类器官生物库,该库包含了丰富的组织病理类型和分子基因类型的CRC来源类器官,为以后进一步的CRC基础及转化医学研究提供了充足的生物样本库。同时,类器官组织病理学及形态学表明类器官与来源的CRC保持高度的一致性和同源性,为后续预测化疗敏感性提供可能的理论基础。
2、本发明成功地从腹膜转移癌来源的类器官产生的移植瘤中建立了异种移植瘤的类器官,为抗肿瘤药物筛选和肿瘤进展基于肿瘤类器官模型探索结直肠癌腹膜转移机制的研究提供了体外和体内的两种研究模型
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式描述中所需要使用的附图作简单地介绍。
图1构建结直肠癌腹膜转移灶组织来源的类器官实验流程。
图2腹膜转移灶类器官原位移植瘤模型构建。A.将腹膜类器官移植瘤重悬经盲肠注射类器官。B.腹膜类器官移植瘤构建的宏观图。
图3腹膜转移类器官原位移植瘤组织H&E跟肿瘤上皮相关性蛋白的免疫组化染色。
具体实施方式
本发明提供了一种优化的构建pmCRC类器官模型(patient-derived metastaticorganoids,PDMO)的方法,解决人源肿瘤细胞系不具备临床病人异质性以及无法反映患者肿瘤组织病理以及基因组特性的问题。所述方法包括如下步骤:(1)将一部分肿瘤组织切成小块,在含有2.5%FBS、1%PS、75U/ml IX型胶原酶和125g/mL II型组织蛋白消化酶的消化缓冲液中消化30分钟,之后加入基础培养基(包括Advanced DMEM/F12、1%PS、10mM HEPES和2mM GlutaMAX)终止消化,用100μm细胞过滤器移除较大的组织碎片。1,000rpm,离心3分钟,沉淀用基础培养基重悬,1,000rpm,离心3分钟。这个过程重复两次,以清除组织碎片和胶原酶。(2)肿瘤细胞用BME(Basement Membrane Extract)基质胶重悬种入24孔细胞培养板放入细胞培养箱,20分钟后BME凝固,加入500μl CRC类器官完全培养基。CRC类器官完全培养基的组成是:基础培养基,20%含R-spondin-1重组蛋白的培养基,100ng/ml Noggin,1×B27,1.25mM n-Acetyl Cysteine,10mM Nicotinamide,50ng/ml EGF,10nM Gastrin,500nM A83-01,5μM SB202190,10nM Prostaglandine E2和100μg/ml Primocin。其中WNT激活子R-spondin1在体内诱导隐窝的增生。R-spondin1是肠干细胞的标志物LGR5的配体,是激活肠隐窝处WNT信号的必要因子。EGF信号通路与肠道增殖有关。Noggin可以诱导隐窝数目的增加。Y27632可以防止分离的肠细胞在脱离正常组织环境后发生失巢凋亡。通过烟酰胺(Nicotinamide)、A83-01(Alk抑制剂)、前列腺素E2(Prostaglandin E2)、SB202190(p38抑制剂)和胃泌素(Gastrin)的添加,来提高原发性和转移性肿瘤类器官的体外长期培养的成功率。(3)观察类器官形态结构,进行H&E以及免疫组化染色,比较类器官与亲本组织的组织病理学结构。
本发明还提供了利用基于上述方案获得的PDMO建成异种移植模型(PDMOX)的方案方法,包括如下步骤:(1)对上述培养方案培养成功的成对类器官消化成单细胞,然后用50%BME/50%类器官培养基以107cells/ml的浓度重悬。之后用200μl(2×106cells)的类器官悬液皮下注射BALB/c nude小鼠(6-8周,雄性)。(2)注射完成后每4天测量一次肿瘤体积,采用标准公式:length×width2/2计算肿瘤体积,待类器官种植的皮下瘤形成。(2)对于pmCRC类器官长成的皮下瘤,待瘤子长成约1*1.5cm后,把瘤块切成~0.1cm3小块,取小块接种至裸鼠盲肠上,缝合后待肿瘤生长,从而构建腹膜转移类器官异种移植模型。(3)对小鼠腹膜肿瘤组织切片,进行H&E以及免疫组化染色,比较pmCRC类器官来源PDX与亲本组织的组织病理学结构。
本发明进一步提供了利用基于上述方案获得的PDMO进行药物敏感性筛选的方法和流程,包括如下步骤:(1)类器官传代培养4-5天后,收集类器官,并使用40μm的细胞过滤器进行过滤,以消除大体积的类器官。(2)类器官在含有2%BME的类器官培养基(15-20,000类器官/ml)中重悬,种入的96孔板(Corning)中。在铺板后24h,加入化合物,每种化合物稀释到指定浓度,三个重复。药物浓度按照不同药物的性质而定(最大DMSO浓度为1%)。(3)在化合物处理6天后,根据CellTiter-Glo试剂盒使用说明,利用3D CTG试剂检测细胞活力,并将结果进行标准化,最后使用GraphPad Prism7.0b软件进行数据分析。体内:(1)根据体外类器官药敏实验数据,挑选对应的PDMOX采取相应的体内用药方案,不同药物的注射途径不同,主要包括腹腔内注射(intraperitoneal)以及静脉注射(intravenous)。
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施案例所描述的内容仅用于说明和解释本发明,并不用于限制权利要求书中所详细描述的本发明。除非特别说明,本发明采用的试剂、方法和设备如无特别说明,均为常规方法,所使用的试验材料如无特别说明,均可从商业公司获取。
本发明使用的试剂来源如下:
试剂来源
实施例1结直肠癌转移灶类器官的构建:
1.1类器官培养
将一部分肿瘤组织切成小块,在含有2.5%FBS、1%PS、75U/ml IX型胶原酶和125g/mL II型组织蛋白消化酶的消化缓冲液中消化30分钟,之后加入基础培养基(包括Advanced DMEM/F12、1%PS、10mM HEPES和2mM GlutaMAX)终止消化,用100μm细胞过滤器移除较大的组织碎片。1,000rpm,离心3分钟,沉淀用基础培养基重悬,1,000rpm,离心3分钟。这个过程重复两次,以清除组织碎片和胶原酶。
肿瘤细胞用BME(Basement Membrane Extract)基质胶重悬种入24孔细胞培养板放入细胞培养箱,20分钟后BME凝固,加入500μl CRC类器官完全培养基。CRC类器官完全培养基的组成是:基础培养基,20%含R-spondin-1培养基,100ng/ml Noggin,1×B27,1.25mMn-Acetyl Cysteine,10mM Nicotinamide,50ng/ml EGF,10nM Gastrin,500nM A83-01,5μMSB202190,10nM Prostaglandine E2和100μg/ml Primocin。
构建结直肠癌腹膜转移灶组织来源的类器官实验流程如图1所示。
实施例2:结直肠癌转移灶类器官异种移植瘤模型的构建
2.1.腹膜转移类器官移植瘤模型(PDMOX)的构建
如附图2,对于腹膜转移来源的组织,待其皮下移植瘤大小长成约1*1.5cm后,再把瘤块切成~0.1cm3小块,取小块接种至裸鼠盲肠上(A),缝合后待肿瘤生长,从而构建腹膜转移类器官异种移植模型。由附图2可见,待盲肠种植3周后,小鼠腹部可见肿块凸起,解剖后可见腹膜上存在肿瘤肿块(B-C),表明腹膜转移类器官异种移植模型构建成功。
2.3.PDMOX组织免疫组化染色
对小鼠腹膜肿瘤组织切片,进行H&E以及免疫组化染色(CDX2和CK20分子),比较pmCRC类器官来源PDX与亲本组织的组织病理学结构。如附图3,展示的是PDMOX组织的H&E跟肿瘤上皮特异性蛋白的免疫组化染色。由附图3可知,PDMOX不同程度表达CDX2和CK20,保留了其来源肿瘤的特性。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。
Claims (10)
1.结直肠癌腹膜转移癌类器官培养方法,其特征在于,包括如下步骤:1)获取结直肠癌腹膜转移癌组织样本,组织破碎和消化;2)过滤获得单细胞和细胞团悬液;3)基质胶重悬细胞或细胞团;4)待基质胶凝固,加入CRC类器官完全培养基,培养获得直肠癌腹膜转移癌类器官。
2.根据权利要求1所述的培养方法,其特征在于,所述步骤1)中在含有FBS、PS、IX型胶原酶和II型组织蛋白消化酶的消化液中进行消化;优选地,消化液中含1-5%FBS、0.1-5%PS、25-100U/ml IX型胶原酶和50-200g/mLII型组织蛋白消化酶;更优选地,消化液中含2.5%FBS、1%PS、75U/ml IX型胶原酶和125g/mL II型组织蛋白消化酶。
3.根据权利要求2中所述的培养方法,其特征在于,所述步骤1)中消化后采用基础培养基中止消化。
4.根据权利要求4中所述的培养方法,其特征在于,所述基础培养基包括AdvancedDMEM/F12、PS、HEPES和GlutaMAX;优选地,所述基础培养基包括Advanced DMEM/F12、0.1-5%PS、1-20mM HEPES和0.5-10mM GlutaMAX;更优选地,所述基础培养基包括AdvancedDMEM/F12、1%PS、10mM HEPES和2mM GlutaMAX。
5.根据权利要求1所述的方法,其特征在于,所述CRC类器官完全培养基包括基础培养基,含R-spondin-1重组蛋白的培养基,Noggin,1×B27,n-Acetyl Cysteine,Nicotinamide,EGF,Gastrin,A83-01,SB202190,Prostaglandine E2和Primocin;优选地,所述CRC类器官完全培养基包括基础培养基,10-30%含R-spondin-1重组蛋白的培养基,50-200ng/mlNoggin,1×B27,0.25-2mMn-Acetyl Cysteine,1-50mM Nicotinamide,10-100ng/ml EGF,1-50nM Gastrin,100-1000nM A83-01,1-10μM SB202190,1-50nM Prostaglandine E2和10-200μg/ml Primocin;更优选地,所述CRC类器官完全培养基包括基础培养基,20%含R-spondin-1重组蛋白的培养基,100ng/ml Noggin,1×B27,1.25mMn-Acetyl Cysteine,10mMNicotinamide,50ng/ml EGF,10nM Gastrin,500nM A83-01,5μMSB202190,10nMProstaglandine E2和100μg/ml Primocin。
6.根据权利要求5所述的方法,其特征在于,所述基础培养基包括Advanced DMEM/F12、PS、HEPES和GlutaMAX;优选地,所述基础培养基包括Advanced DMEM/F12、0.1-5%PS、1-20mM HEPES和0.5-10mM GlutaMAX;更优选地,所述基础培养基包括Advanced DMEM/F12、1%PS、10mM HEPES和2mM GlutaMAX。
7.根据权利要求1-6任一项所述的方法培养获得的结直肠癌腹膜转移癌类器官。
8.一种结直肠癌腹膜转移癌异种移植模型的构建方法,其特征在于,包括如下步骤:a.将结直肠癌腹膜转移癌类器官消化成单细胞,然后用基质胶和肿瘤类器官培养基的混合物重悬;b.用类器官悬液皮下注射小鼠,待类器官种植的皮下瘤形成;c.待瘤子长至适宜大小后,把瘤块切成小块接种至裸鼠盲肠上,缝合后待肿瘤生长,从而构建腹膜转移类器官异种移植模型;优选地,基质胶和肿瘤类器官培养基的混合物包括50%BME和50%。
9.根据权利要求8所述的方法培养获得的结直肠癌腹膜转移癌异种移植模型。
10.根据权利要求7所述的结直肠癌腹膜转移癌类器官或权利要求9所述的结直肠癌腹膜转移癌异种移植模型在结直肠癌腹膜转移癌药物筛选、结直肠癌腹膜转移癌发病机制研究中的应用。
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