CN114774361B - 一种鼻腔鼻窦腺样囊性癌类器官培养基培养基及其应用 - Google Patents
一种鼻腔鼻窦腺样囊性癌类器官培养基培养基及其应用 Download PDFInfo
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- CN114774361B CN114774361B CN202210423291.XA CN202210423291A CN114774361B CN 114774361 B CN114774361 B CN 114774361B CN 202210423291 A CN202210423291 A CN 202210423291A CN 114774361 B CN114774361 B CN 114774361B
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Abstract
本公开涉及一种鼻腔鼻窦腺样囊性癌类器官培养基领域，具体涉及一种类器官培养基及其在鼻腔鼻窦腺样囊性癌类器官的培养中的应用。本公开的鼻腔鼻窦腺样囊性癌类器官培养基，由如下组分组成：Human R‑spond i n 1、HumanNogg i n、Human Wnt3a、N i cot i nami de、N‑acety l yste i ne、Gast i n I、Human EGF、Human FGF 10、Human FGF bas i c、Hydrocort i sone、Prostag l and i n E2、SB202190、Y‑27632D i hydroch l or i de、B27、A83‑01、I nsu l i n‑Transferr i n‑Se l en i um、L‑G l utmax、Tr i i odothyron i ne、HEPES。此外还提供了根据本公开鼻腔鼻窦腺样囊性癌类器官培养基培养腔鼻窦腺样囊性癌来源细胞的方法，所述方法包括：(1)获取腔鼻窦腺样囊性癌细胞；(2)将获取的细胞接种至所述培养基中培养。
Description
技术领域
本公开涉及一种鼻腔鼻窦腺样囊性癌类器官培养基领域，具体涉及一种鼻腔鼻窦腺样囊性癌类器官培养基及其在鼻腔鼻窦腺样囊性癌类器官的培养中的应用。
背景技术
鼻腔鼻窦腺癌分为唾液腺型腺癌和非唾液腺型腺癌。鼻窦腺癌可起源于呼吸道上皮或粘膜下层的黏液腺，大部分(尤其是唾液腺型)起源于黏液腺。恶性鼻腔鼻窦(包括鼻腔、鼻窦和鼻咽部，统称为鼻腔鼻窦)肿瘤相对少见，占所有上呼吸道恶性肿瘤的3％。鳞状细胞癌是最常见的鼻腔鼻窦恶性肿瘤，而腺样囊性癌(Adenoid cystic carcinoma，ACC)是第二常见的鼻腔鼻窦恶性肿瘤，也是鼻腔鼻窦中最常见的唾液腺型肿瘤。ACC占所有头颈部恶性肿瘤的1％-2％，占所有头颈部恶性唾液腺型肿瘤的10％-25％。据以往研究报道，ACC通常表现为生长缓慢，然而，该肿瘤也具有较高的神经周围浸润率、局部复发率和远处转移的迟发性的特点，进而导致临床病程延长。
目前，鼻腔鼻窦腺样囊性癌的主要治疗方法是手术。对于患者来说，完整的手术切除(切缘阴性)代表了其唯一最重要的预后因素。较宽的游离切缘的获得与多种因素有关，包括肿瘤部位、T分期、浸润方式和术前治疗。由于手术受到肿瘤组织周围重要结构(颅底、硬脑膜、颅神经或颈动脉等)的限制，难以确保阴性的手术切缘，已证实来自颅底附近部位的鼻腔鼻窦腺样囊性癌患者具有显着增加的局部复发风险。基于获得阴性切缘的困难以及肿瘤往黏膜下层和周围神经扩散的倾向性，往往需要对潜在传播途径及部位来设定照射野区域。有研究表明，单独或联合化疗的辅助放疗可用于清除手术减瘤后留下的阳性切缘或残留病灶。然而，接受手术及术后放疗的患者依旧显示出高复发率，在治疗5年后，复发率为45％-65％。因此，术后放疗可能会延缓而不是杜绝复发，且放疗后随之出现的严重不良反应导致患者生存质量极差，经济负担加剧。当前针对鼻腔鼻窦腺样囊性癌的全身性化学治疗研究较少，前期研究显示无论是单药还是多药联合化疗，其客观缓解率均不超过20％。近年来，大家纷纷将目光聚焦于姑息性系统治疗，尤其是以靶向治疗及免疫治疗为代表的生物治疗已成为继手术、放疗及化疗之后的第四种新型治疗方法。靶向治疗及免疫治疗现阶段仍处于基础实验和临床探索时期，由于缺乏体内外适合的鼻腔鼻窦腺样囊性癌疾病模型，极大地阻碍了其开发进程。因此，建立一种能够比较完美的重现亲本肿瘤异质性以及完成高通量药物筛选的体外研究模型势在必行。
类器官是指通过体外3D培养各类干细胞形成具有与类似于来源组织形态、结构及功能的培养物，能够在体外长期培养的条件下，保持与亲本组织高度一致的遗传信息和表型，也有着“瓶皿里的微型器官”的别称。与传统的2D细胞模型相比，类器官能够更真实的反映人体内细胞组成以及生理或病理性行为。肿瘤是一种高度异质性的疾病，肿瘤类器官作为肿瘤患者的体外“替身”，能够重现患者对化疗、放疗和靶向治疗的特异性和临床相关反应，准确预测疗效，将对鼻腔鼻窦腺样囊性癌患者制定个体化治疗方案起到关键作用，也为寻找新的治疗靶点和开发新型抗肿瘤药物提供了良好的技术平台。
现有技术中，虽然已有多种人源肿瘤组织(例如胃、肠、肝、肾、甲状腺等肿瘤组织)在不同培养条件下于体外成功培养成相应的肿瘤类器官的报道，但是暂无关于鼻腔鼻窦腺样囊性癌类器官培养方法的研究，尤其是鼻腔鼻窦腺样囊性癌类器官培养基配方以及具体的鼻腔鼻窦腺样囊性癌类器官培养操作流程尚无尝试和报道。此外，鼻腔鼻窦腺样囊性癌与其他癌种肿瘤微环境不同，其培养基及培养方法与其他肿瘤类器官相比更具创新性。
发明内容
为了解决相关技术中的问题，本公开实施例提供一种类器官培养基及其在鼻腔鼻窦腺样囊性癌类器官的培养中的应用。
第一方面，本公开实施例中提供了一种鼻腔鼻窦腺样囊性癌类器官培养基培养基，由如下组分组成：Human R-spondin 1、Human Noggin、Human Wnt3a、Nicotinamide、N-acetylysteine、Gastin I、Human EGF、Human FGF 10、Human FGF basic、Hydrocortisone、Prostaglandin E2、SB202190、Y-27632Dihydrochloride、B27、A83-01、Insulin-Transferrin-Selenium、L-Glutmax、Triiodothyronine、HEPES。
结合第一方面，各组分在培养基中的浓度为：Human R-spondin 110-20ng/ml、Human Noggin 5-15ng/ml、Human Wnt3a 15-40ng/ml、Nicotinamide 5-10mmol//L、N-acetylysteine 1-1.25mmol//L、Gastin I 2-10mmol//L、Human EGF 10-55ng/ml、HumanFGF 10 10-20ng/ml、Human FGF basic 5-10ng/ml、Hydrocortisone 100-175ng/ml、Prostaglandin E2 1-2μmol//L、SB202190 10-20ng/ml、Y-27632 Dihydrochloride 100-500nmol//L、B27 0.5-1X、A83-01 100-200nM、Insulin-Transferrin-Selenium 0.5-1X、L-Glutmax 0.5-1X、Triiodothyronine 100-500nmol//L、HEPES5-10mmol//L。需要说明的是，本发明的含量只是示意性的。
结合第一方面，各组分在培养基中的浓度如下：
在本公开中，Human R-spondin 1作为Wnt/β-catanin信号通路激活因子；HumanNoggin作为TGF-b信号通路抑制因子；Human Wnt3a作为Wnt/β-catanin信号通路激活因子；Nicotinamide作为辅酶前体；N-acetylysteine作为抗氧化剂；Gastin I促进肿瘤细胞增殖；Human EGF作为C-erbB、TGF-β信号通路抑制因子；Human FGF 10作为FGFR2bsignalling信号通路激活因子；Human FGF basic促进干细胞分化为中胚层谱系；Prostaglandin E2促进肿瘤干细胞生长；SB202190作为p38 MAPK signalling信号通路抑制因子；Y-27632 Dihydrochloride作为ROCK信号通路特异性抑制因子，减少失巢凋亡，提高类器官形成效率；B27insulin信号通路激活因子，适合肿瘤干细胞生长、扩增和维持；A-8301作为TGF-b信号通路抑制因子；Insulin-Transferrin-Selenium用于无血清细胞培养系统的营养补充剂；L-Glutmax作为营养补充剂；Triiodothyronine用于促进细胞生长发育；HEPES调节培养基酸碱度；Hydrocortisone用于促进细胞分化。
第二方面，本公开提供了前述鼻腔鼻窦腺样囊性癌培养基在培养腔鼻窦腺样囊性癌来源细胞中的应用。
第三方面，本公开提供了前述鼻腔鼻窦腺样囊性癌培养基培养腔鼻窦腺样囊性癌来源细胞的方法，所述方法包括：
(1)获取腔鼻窦腺样囊性癌细胞；
(2)将获取的细胞接种至所述培养基中培养。
结合第三方面，每隔14天更换一次所述培养基。
结合第三方面，所述获取腔鼻窦腺样囊性癌细胞的步骤如下：(1)获取鼻腔鼻窦腺样囊性癌组织；
(2)将鼻腔鼻窦腺样囊性癌组织进行破碎形成组织小块；
(3)对所述组织小块的组织粘液进行溶解获得干净的组织小块；
(4)对所述干净的组织小块进行组织消化，形成消化后的组织；
(5)对消化后的组织进行中止消化；
(6)对中止消化的组织进行离心，获取组织沉淀，对所述组织沉淀进行过滤后再离心，去除上清，加入裂解液裂解后再终止裂解，对细胞进行计数后，再离心得到腺样囊性癌细胞沉淀；
(7)根据总细胞量加入基质胶以及细胞计数结果加入Advanced DMEM/F12培养基，吹散混匀形成含有数个细胞的细胞团。
第四方面，本公开提供了根据前述方法提供的细胞。
第五方面，本公开提供了根据前述方法提供的细胞制备的鼻腔鼻窦腺样囊性癌类器官。
第六方面，本公开提供了根据前述鼻腔鼻窦腺样囊性癌类器官在药物筛选中的应用。
有益的技术效果：本发明的鼻腔鼻窦腺样囊性癌类器官的培养方法针对鼻腔鼻窦腺样囊性癌组织原代培养特性以及鼻腔鼻窦腺样囊性癌组织的病理生理学特征设计了最佳的实验操作流程。根据鼻腔鼻窦腺样囊性癌来源细胞的体外生长特点，选用了多种细胞因子成份按照相应的比例/终浓度进行调和，经过调和的培养基中细胞因子，细胞信号通路调控因子含量适宜，鼻腔鼻窦腺样囊性癌细胞能够有效的在3D培养体系中形成肿瘤类器官。通过使用本发明特有的鼻腔鼻窦腺样囊性癌类器官培养基，可以稳定的培养出高度拟似来源肿瘤组织形态结构、病理生理学特征、遗传表型的鼻腔鼻窦腺样囊性癌类器官。除了满足科学研究的需要，在临床用药指导方面，目前晚期转移性鼻腔鼻窦腺样囊性癌患者以及复发性鼻腔鼻窦腺样囊性癌患者没有合适的靶向药物，活检样本的体外3D培养为患者的药物用药指导提供了一个很好的选择。此外，本发明培养方法流程也可用于其余呼吸道癌种类器官的培养，形成一个标准规范化肿瘤类器官培养体系。同时，本发明的培养基可以完成鼻腔鼻窦腺样囊性癌类器官的传代培养，满足大规模复制鼻腔鼻窦腺样囊性癌类器官的需求，控制培养得到的类器官具有高度的一致性。
附图说明
结合附图，通过以下非限制性实施方式的详细描述，本公开的其它特征、目的和优点将变得更加明显。在附图中：
图1是不同原代培养方法得到的肿瘤组织培养物光镜图，其中左侧是本公开的类器官培养基培养的类器官，右侧是普通原代培养方法得到的培养物。
图2是根据本公开的培养基培养的鼻腔鼻窦腺样囊性癌类器官，从左到右依次是培养第1、3、5天的光镜图。
图3是由本培养基培养的类器官与缺少某种关键细胞因子的培养基培养的类器官数目与直径的对比
具体实施方式
下文中，将参考附图详细描述本公开的示例性实施例，以使本领域技术人员可容易地实现它们。此外，为了清楚起见，在附图中省略了与描述示例性实施例无关的部分。
在本公开中，应理解，诸如“包括”或“具有”等的术语旨在指示本说明书中所公开的特征、数字、步骤、行为、部件、部分或其组合的存在，并且不欲排除一个或多个其他特征、数字、步骤、行为、部件、部分或其组合存在或被添加的可能性。
另外还需要说明的是，在不冲突的情况下，本公开中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本公开。
第一实施方式：
一种鼻腔鼻窦腺样囊性癌细胞培养基，其各组分以及含量如下：Human R-spondin1 20ng/ml、Human Noggin 10ng/ml、Human Wnt3a 40ng/ml、Nicotinamide 5mmol/L、N-acetylysteine 1.25mmol/L、Gastin I 10mmol/L、Human EGF 55ng/ml、Human FGF 1020ng/ml、Human FGF basic 10ng/ml、Hydrocortisone 175ng/ml、Prostaglandin E2 1μmol/L、SB202190 20ng/ml、Y-27632 Dihydrochloride 500nmol/L、B27 50X稀释为0.5X、A83-01 200nmol/L、Insulin-Transferrin-Selenium 100X稀释为0.5X、L-Glutmax 100X稀释为1X、Triiodothyronine 500nmol/L、HEPES 10mmol/L。
第二实施方式：
利用第一实施方式的培养基培养腺样囊性癌细胞，具体培养步骤如下：
1、获取腔鼻窦腺样囊性癌细胞，具体方法如下:
(1)获取鼻腔鼻窦腺样囊性癌组织:将新鲜的鼻腔鼻窦腺样囊性癌组织转移至无菌50ml离心管，并加入5ml含2％双抗的0.9％NaCl溶液，振荡洗涤3-4次以清除组织表面杂质。
(2)将所述组织进行破碎形成组织小块:将洗涤后的肿瘤组织块转移至10cm培养皿中，向其中加入适量0.9％NaCl溶液，组织块表面湿润即可。用持针钳夹紧消毒后的刀片在冰上操作将组织反复切碎至大小＜0.8mm3组织碎末。
(3)对所述组织小块的组织粘液进行溶解获得干净的组织小块:向培养皿中加入2-3ml浓度为0.1mM的DTT溶液，使用装有已剪去末端的1ml枪头的移液枪将组织碎末混合液转移至50ml离心管中，反复吹打至组织粘液溶解后加入等量0.9％NaCl溶液中止(吹打时长一般不超过1min，具体情况视不同标本而定)；DTT溶液溶解完组织粘液后，以1300r转速离心3min。
去除上清后，加入1.5ml浓度为2.6mg/ml的I型胶原酶以及1.5ml浓度为300μg/ml的透明质酸酶。在37℃恒温摇床的条件下消化1.5h-2h左右，每隔20分钟将离心管取出，吹打组织混合液5次左右。吹打结束继续放置在37℃恒温摇床上。
(4)对消化后的组织进行中止消化：组织消化结束后，向离心管中加入1ml浓度为20mg/ml的Dispase II继续放置在37℃恒温摇床上消化10min，消化结束后加入4ml 0.9％NaCl液终止消化。
(5)对中止消化的组织进行离心，获取组织沉淀，对所述组织沉淀进行过滤后再离心，去除上清，加入裂解液裂解后再终止裂解，对细胞进行计数后，再离心得到腺样囊性癌细胞沉淀：
将离心管放入离心机内，以1300r转速离心3min；
离心结束去除上清，再加2-3ml 0.9％NaCl液吹打重悬消化过后的组织沉淀；
选用100μm滤网过滤组织混合液，得到含有腺样囊性癌细胞的滤液，将细胞滤液转移至新的15ml离心管中，以1500r转速离心3min，得到含有红细胞的腺样囊性癌细胞沉淀；
除去上清，向离心管中加入2ml红细胞裂解液，裂解5min后加入2ml0.9％NaCl液终止裂解，随后进行细胞计数，最后以1500r转速离心3min，得到腺样囊性癌细胞沉淀。
(6)根据总细胞量加入基质胶以及细胞计数结果加入Advanced DMEM/F12培养基，吹散混匀形成含有数个细胞的细胞团：
取出事先冰冻好的冰盒，为避免基质胶快速凝固，全程冰上操作。按照106总细胞量/10μl基质胶比例，以及Advanced DMEM/F12培养基/基质胶＝1：1.5的比例，根据细胞计数结果，加入计算量的预冷Advanced DMEM/F12培养基，吹散细胞沉淀并混匀。再加入计算量的基质胶，混匀至不分层，注意尽量不要产生气泡。
2.将获取的细胞接种至所述培养基中培养:
用200μl移液枪(事先已将200μl枪头置于4℃冰箱预冷)吸取含有细胞的基质胶混合液，将其接种至6cm培养皿中，每个胶滴大小约40μl。将培养皿静置2-3min，观察种植好的胶滴是否有流动迹象，若不流动，则放入37℃细胞孵箱，将培养皿倒置30min；
倒置结束后，向培养皿中加入事先已预热的前述细胞培养基，约4ml；
每隔2天在光镜下观察类器官生长情况并拍照记录；
每隔2-3天更换一次类器官培养基，培养出鼻腔鼻窦腺样囊性癌类器官；更换的培养基为前述培养基。
对照组1：
对比普通原代培养方法：采用传统的肿瘤组织原代培养方法以及普通的培养基(DMEM/F12+8％FBS)2D条件下培养的散在的鼻腔鼻窦腺样囊性癌细胞，置于37℃,5％CO2细胞培养箱中培养。每隔2-3天更换一次培养基。
表1：普通原代培养与类器官培养随着培养时间延长细胞活率的变化
从表1中可以看出，采用普通原代培养方法培养，细胞存活率随着培养时间的增加，存活率急速降低，而采用本公开中的方法培养，鼻腔鼻窦腺样囊性癌细胞存活率基本维持不变。
从图1中可以看出，在对照组1中，鼻腔鼻窦腺样囊性癌细胞活性随着培养时间的延长逐渐降低，细胞生长迟缓，成活率低，无法形成有立体结构的、具有异质细胞群的类器官。从图2中可以看出，本公开的鼻腔鼻窦腺样囊性癌类器官培养基培养出的类器官则呈现“实心”细胞团结构，细胞活性高，其直径随着培养时间的延长逐渐增大，类器官包含了肿瘤细胞、间质细胞等在内的多种细胞群体，是理想的体外疾病模型。
对照组2：
以第一实施方式的培养基为基础，去除该培养基中的Human R-spondin 1。
以该培养基培养细胞，制备类器官，培养方法与第二实施方式中的方法完全相同，仅仅所用的类器官培养基不同。
对照组3：
以第一实施方式的培养基为基础，去除该培养基中的Human FGF 10。
以该培养基培养细胞，制备类器官，培养方法与第二实施方式中的方法完全相同，仅仅所用的类器官培养基不同。
对照组4：
以第一实施方式的培养基为基础，去除该培养基中的Y-27632 Dihydrochloride。
以该培养基培养细胞，制备类器官，培养方法与第二实施方式中的方法完全相同，仅仅所用的类器官培养基不同。
对照组5：
以第一实施方式的培养基为基础，去除该培养基中的Insulin-Transferrin-Selenium。
以该培养基培养细胞，制备类器官，培养方法与第二实施方式中的方法完全相同，仅仅所用的类器官培养基不同。
从图3中可以看出，培养第5天，与正常类器官培养基相比，缺少Human R-spondin1、Human FGF 10、Y-27632 Dihydrochloride、Insulin-Transferrin-Selenium中的任何一种，均会均会导致类器官数目减少，直径变小。
以上描述仅为本公开的较佳实施例以及对所运用技术原理的说明。本领域技术人员应当理解，本公开中所涉及的发明范围，并不限于上述技术特征的特定组合而成的技术方案，同时也应涵盖在不脱离所述发明构思的情况下，由上述技术特征或其等同特征进行任意组合而形成的其它技术方案。例如上述特征与本公开中公开的(但不限于)具有类似功能的技术特征进行互相替换而形成的技术方案。
Claims (5)
1.一种鼻腔鼻窦腺样囊性癌类器官培养基培养基，由如下组分组成：Human R-spondin1、Human Noggin、Human Wnt3a、Nicotinamide、N-acetylysteine、Gastin I、Human EGF、Human FGF 10、Human FGF basic、Hydrocortisone、Prostaglandin E2、SB202190、Y-27632Dihydrochloride、B27、A83-01、Insulin-Transferrin-Selenium、L-Glutmax、Triiodothyronine、HEPES；
其中各组分在培养基中的浓度如下：
。
2.根据权利要求1所述的鼻腔鼻窦腺样囊性癌类器官培养基培养基在培养腔鼻窦腺样囊性癌来源细胞中的应用。
3.根据权利要求1所述的鼻腔鼻窦腺样囊性癌类器官培养基培养腔鼻窦腺样囊性癌来源细胞的方法，所述方法包括：
(1)获取腔鼻窦腺样囊性癌细胞；
(2)将获取的细胞接种至所述培养基中培养。
4.根据权利要求3中所述的方法，每隔14天更换一次所述培养基。
5.根据权利要求4或3所述的方法，其中所述获取腔鼻窦腺样囊性癌细胞的步骤如下：
(1)获取鼻腔鼻窦腺样囊性癌组织；
(2)将鼻腔鼻窦腺样囊性癌组织进行破碎形成组织小块；
(3)对所述组织小块的组织粘液进行溶解获得干净的组织小块；
(4)对所述干净的组织小块进行组织消化，形成消化后的组织；
(5)对消化后的组织进行中止消化；
(6)对中止消化的组织进行离心，获取组织沉淀，对所述组织沉淀进行过滤后再离心，去除上清，加入裂解液裂解后再终止裂解，对细胞进行计数后，再离心得到腺样囊性癌细胞沉淀；
(7)根据总细胞量加入基质胶以及细胞计数结果加入Advanced DMEM/F12培养基，吹散混匀形成含有数个细胞的细胞团。
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