CN113567404A - 一种分析肿瘤细胞耐药性的方法和试剂盒 - Google Patents

一种分析肿瘤细胞耐药性的方法和试剂盒 Download PDF

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CN113567404A
CN113567404A CN202110654859.4A CN202110654859A CN113567404A CN 113567404 A CN113567404 A CN 113567404A CN 202110654859 A CN202110654859 A CN 202110654859A CN 113567404 A CN113567404 A CN 113567404A
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窦红静
刘灵珊
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Abstract

本发明公开了一种分析肿瘤细胞耐药性的方法，该方法包括以下步骤：(a)提供一种二氧化硅纳米颗粒、聚苯乙烯‑co‑聚丙烯酸纳米颗粒或金属有机框架纳米颗粒；(b)将所述二氧化硅纳米颗粒、聚苯乙烯‑co‑聚丙烯酸纳米颗粒或金属有机框架纳米颗粒与肿瘤细胞共孵培养；(c)检测肿瘤细胞对所述二氧化硅纳米颗粒、聚苯乙烯‑co‑聚丙烯酸纳米颗粒或金属有机框架纳米颗粒的内吞性能。本发明的分析方法，能够清楚、直观、高效地分析出耐药性肿瘤细胞；提供的纳米粒子合成过程简便，耗费时间短，与肿瘤细胞共孵后，采用流式细胞仪进行检测，通过检测结果来判断肿瘤细胞的耐药性的强弱，以及耐药细胞所占的比例，方法简单高效。

Description

一种分析肿瘤细胞耐药性的方法和试剂盒
技术领域
本发明涉及肿瘤分析和治疗领域，尤其涉及一种分析肿瘤细胞耐药性的方法和试剂盒。
背景技术
癌症是全世界范围内威胁人类生命健康的问题，目前癌症的治疗方法主要有化疗、放射治疗、免疫治疗等。然而在癌症治疗过程中总是有不同程度的耐药产生，降低癌症治疗的效果。其中，化疗时发生的多药耐药是肿瘤化疗面临的主要挑战。化疗的多药耐药可由多种原因导致，最为常见的是p-糖蛋白的表达使得化疗药物被泵出细胞，化疗药物不能在细胞中累积达到杀死细胞的浓度，进而达不到治疗效果。所以，针对肿瘤化疗的多药耐药，分析其耐药细胞与非耐药细胞具有重要意义。
中国专利CN111440840A公开了一种利用多糖基纳米颗粒分析肿瘤细胞耐药性的方法。但是这种亲水性的纳米颗粒穿过脂溶性的细胞膜尚存在局限性，并且这种纳米颗粒合成过程较为复杂，实验条件要求高，耗时长。
有鉴于现有技术的缺陷，发明人引入了具有不同表面特性的纳米颗粒，分别是二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒和金属有机框架纳米颗粒。这些纳米颗粒合成过程简便，后处理耗费时间短，且表面特性更有利于穿过细胞膜并被细胞内吞。通过对纳米颗粒进行荧光标记或表面修饰，使纳米颗粒具有通过特异性的内吞性能来分析耐药性肿瘤细胞的能力，并以此为手段研究肿瘤耐药性的发生发展机制、以及肿瘤耐药细胞—非耐药细胞的相互作用机制，将对癌症的诊断和治疗均具有非常大的应用价值。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是如何更简单高效地分析出耐药性肿瘤细胞。
为实现上述目的，本发明提供了一种分析肿瘤细胞耐药性的方法，包括将二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒应用于肿瘤细胞的步骤。
进一步地，包括以下步骤：
(a)提供一种二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒；
(b)将所述二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒与肿瘤细胞共孵培养；
(c)检测肿瘤细胞对所述二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒的内吞性能。
进一步地，金属有机框架纳米颗粒包括ZIF-8纳米颗粒，ZIF-70纳米颗粒和ZIF-90纳米颗粒等。
进一步地，在步骤(b)之前还包括步骤(d)，步骤(d)为对二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒进行荧光分子修饰。
进一步地，步骤(d)中荧光分子选自荧光素、罗丹明、菁染料、香豆素和荧光无机纳米晶中的一种或多种。
进一步地，二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒与荧光分子按照质量比20-2000:1进行反应。
进一步地，步骤(b)中将二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒与肿瘤细胞共孵培养0.5-4h。
进一步地，耐药性肿瘤细胞包括人肺癌(A549)耐紫杉醇细胞、人肺癌(A549)耐顺铂细胞、人肺癌(A549)耐阿霉素细胞、人肺癌(A549)耐奥沙利铂细胞、人肺腺癌(PC-9)耐吉非替尼细胞、人白血病(K562)耐紫杉醇细胞、人白血病(K562)耐顺铂细胞、人白血病(K562)耐阿霉素细胞、人结肠癌(HCT-8)耐长春新碱细胞、人结肠癌(HCT-8)耐紫杉醇细胞、人结肠癌(HCT-8)耐氟尿嘧啶细胞、人结肠癌(HCT116)耐奥沙利铂细胞、人结肠癌(LoVo)耐阿霉素细胞、人结肠癌(HT-29)耐顺铂细胞、人乳腺癌(MCF-7)耐紫杉醇细胞、人乳腺癌(MCF-7)耐顺铂细胞、人乳腺癌(MCF-7)耐阿霉素细胞、人乳腺癌(MCF-7)耐多柔比星脂质体细胞、人乳腺癌(MDA-MB-231)耐阿霉素细胞、人卵巢癌(COC1)耐顺铂细胞、人卵巢癌(A2780)耐顺铂细胞、人卵巢癌(A2780)耐紫杉醇细胞、人卵巢癌(SKOV3)耐紫杉醇细胞、人卵巢癌(SKOV3)耐顺铂细胞、人肝癌(Bel)耐氟尿嘧啶细胞、人肝癌(SMMC7721)耐顺铂细胞、人胃癌(SGC7901)耐顺铂细胞、人肺鳞癌(SK-MES-1)耐顺铂细胞、人胰腺癌(PATU-8988)耐氟尿嘧啶细胞、人胰腺癌(Bxpc3)耐顺铂细胞、人膀胱癌(BIU-87)耐阿霉素细胞、人子宫颈癌(Hela)耐紫杉醇细胞、人子宫颈癌(Hela)耐顺铂细胞、人子宫颈癌(Hela)耐阿霉素细胞、人鼻咽癌(CNE2)耐顺铂细胞、人恶性黑色素瘤(A375)耐顺铂细胞、人膀胱移行细胞癌(T-24)耐顺铂细胞中的任意一种。
进一步地，非耐药肿瘤细胞包括人肺癌(A549)细胞、人白血病(K562)细胞、人结肠癌(HCT-8)细胞、人结肠癌(HCT116)细胞、人结肠癌(LoVo)细胞、人结肠癌(HT-29)细胞、人乳腺癌(MCF-7)细胞、人乳腺癌(MDA-MB-231)细胞、人卵巢癌(COC1)细胞、人卵巢癌(A2780)细胞、人卵巢癌(A2780)细胞、人卵巢癌(SKOV3)细胞、人肝癌(Bel)细胞、人肝癌(SMMC7721)细胞、人胃癌(SGC7901)细胞、人肺鳞癌(SK-MES-1)细胞、人胰腺癌(PATU-8988)细胞、人胰腺癌(Bxpc3)细胞、人膀胱癌(BIU-87)细胞、人子宫颈癌(Hela)细胞、人子宫颈癌(Hela)细胞、人子宫颈癌(Hela)细胞、人鼻咽癌(CNE2)细胞、人恶性黑色素瘤(A375)细胞、人膀胱移行细胞癌(T-24)细胞中的任意一种。
本发明还提供了一种分析肿瘤细胞耐药性的试剂盒，其包含二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒。
进一步地，金属有机框架纳米颗粒包括ZIF-8纳米颗粒，ZIF-70纳米颗粒和ZIF-90纳米颗粒等。
进一步地，二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒经由荧光分子修饰。
进一步地，荧光分子选自荧光素、罗丹明、菁染料、香豆素和荧光无机纳米晶中的一种或多种。
技术效果
本发明的分析方法，能够清楚、直观、高效地分析出耐药性肿瘤细胞；
本发明提供的纳米粒子合成过程简便，耗费时间短，与肿瘤细胞共孵后，采用流式细胞仪进行检测，通过检测结果来判断肿瘤细胞的耐药性的强弱，以及耐药细胞所占的比例，方法简单高效。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例的肿瘤耐药细胞和非耐药细胞的荧光强度结果图。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例，使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现，本发明的保护范围并非仅限于文中提到的实施例。
一、制备纳米颗粒
实施例1
将0.25g六水合硝酸锌(Zn(NO3)2·6H2O)溶于1mL水中，2.5g二甲基咪唑溶于10mL水中，分别得到六水合硝酸锌溶液和二甲基咪唑溶液。将菁染料Cy5.5琥珀酰亚胺溶于二甲基亚砜(DMSO)中，形成浓度为0.5mg/mL的菁染料Cy5.5琥珀酰亚胺二甲基亚砜溶液(Cy5.5-NHS DMSO溶液)。将1mL六水合硝酸锌溶液和总体积为7.7mL的Cy5.5-NHS DMSO溶液以及3.3mL去离子水加入反应容器，室温搅拌反应30min。按照纳米颗粒和荧光素的质量比为20-2000:1调节Cy5.5-NHS DMSO溶液的浓度。随后加入10mL二甲基咪唑溶液，室温继续搅拌反应1h。得到的产物经12000rpm，20min离心收集，随后用去离子水离心洗涤3次，得到菁染料Cy5.5标记的ZIF-8纳米颗粒。
实施例2
将4.9mg异硫氰酸荧光素(FITC)加入0.68mL 3-氨丙基三甲氧基硅烷(APTMS)中，再加入6.95mL乙醇，避光搅拌24h，得到产物FITC-APTMS。将FITC-APTMS，1mL硅酸四乙酯(TEOS)，5mL 28wt％氨水以及50mL乙醇加入反应瓶，室温避光搅拌反应10h。按照纳米颗粒和荧光素的质量比为20-2000:1调节FITC-APTMS的用量。反应结束后离心收集产物，并用乙醇离心洗涤3次，得到FITC标记的SiO2纳米颗粒。
实施例3
将180μL丙烯酸，45mL去离子水加入反应器，室温搅拌溶解，随后加入880μL苯乙烯，通氮气30min，随后加入0.03g过硫酸钾(溶解在5mL去离子水中)并升温至70℃，在氮气氛围下继续搅拌反应8h。反应结束后离心收集产物，并用去离子水洗涤3次，得到PS-co-PAA纳米颗粒。
将得到的PS-co-PAA纳米颗粒溶解在pH 5.5的MES缓冲液中，得到浓度为3mg/mL的PS-co-PAA溶液。在50mg的PS-co-PAA溶液加入6.4mg 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC·HCl)，室温搅拌10min，随后加入17.9mg N-羟基琥珀酰亚胺磺酸钠盐(sulfo-NHS)，继续反应3h，然后加入5-AF荧光素，继续避光反应至24h。按照纳米颗粒和荧光素的质量比为20-2000:1调节5-AF的用量。反应结束后离心收集产物，并用去离子水洗涤3次，得到5-AF标记的PS-co-PAA纳米颗粒。
二、利用纳米颗粒分析肿瘤细胞的耐药性
实施例4
人肺癌(A549)耐紫杉醇细胞与非耐药细胞混合接种在六孔板中，待细胞贴壁汇合度至70-80％时，吸出旧培养基，每孔加入2mL含有100μL菁染料Cy5.5标记的ZIF-8纳米颗粒的培养基，在培养箱中继续培养2h。消化收集细胞，用磷酸盐缓冲液以500g，5min的条件离心洗涤2次，最后用500μL磷酸盐缓冲液重悬细胞，在流式细胞仪上检测。比较人肺癌(A549)耐紫杉醇细胞与非耐药细胞的荧光强度，如图1所示，人肺癌(A549)耐紫杉醇细胞的荧光强度低于非耐药细胞的荧光强度，人肺癌(A549)耐紫杉醇细胞的比例为50.8％。
实施例5
人白血病(K562)耐紫杉醇细胞与非耐药细胞混合接种到六孔板中，共同培养12-24h后，每孔加入2mL含有100μL菁染料Cy5.5标记的ZIF-8纳米颗粒的培养基，在培养箱继续培养2h。收集细胞，用磷酸缓冲液以500g，5min的条件离心洗涤2次，最后用500μL磷酸盐缓冲液重悬细胞，在流式细胞仪上检测。比较人白血病(K562)耐紫杉醇细胞与非耐药细胞的荧光强度，人白血病(K562)耐紫杉醇细胞的荧光强度低于非耐药细胞的荧光强度。
实施例6
人白血病(K562)耐阿霉素细胞与非耐药细胞混合接种到六孔板中。培养12-24h后，每孔加入2mL含有100μL FITC标记的SiO2纳米颗粒的培养基，在培养箱继续培养2h。收集细胞，用磷酸缓冲液以500g，5min的条件离心洗涤2次，最后用500μL磷酸盐缓冲液重悬细胞，在流式细胞仪上检测。比较人白血病(K562)耐阿霉素细胞与非耐药细胞的荧光强度，人白血病(K562)耐阿霉素细胞的荧光强度低于非耐药细胞的荧光强度。
实施例7
人乳腺癌(MCF-7)耐紫杉醇细胞与非耐药细胞混合接种在六孔板中，培养过夜使其贴壁，待汇合度至70-80％时，每孔内的培养基更换为2mL含有100μL 5-AF标记的PS-co-PAA纳米颗粒的培养基，在培养箱中继续培养2h，消化收集细胞，用磷酸盐缓冲液以500g，5min的条件离心洗涤2次，最后用500μL磷酸盐缓冲液重悬细胞，在流式细胞仪上检测。比较人乳腺癌(MCF-7)耐紫杉醇细胞与非耐药细胞的荧光强度。人乳腺癌(MCF-7)耐紫杉醇细胞的荧光强度小于非耐药细胞的荧光强度。
实施例8
人结肠癌(HCT-8)耐长春新碱细胞与非耐药细胞混合接种在六孔板中，培养过夜使其贴壁，待汇合度至70-80％时，每孔内的培养基更换为2mL含有100μL5-AF标记的PS-co-PAA纳米颗粒的培养基，在培养箱中继续培养2h，消化收集细胞，用磷酸盐缓冲液以500g，5min的条件离心洗涤2次，最后用500μL磷酸盐缓冲液重悬细胞，在流式细胞仪上检测。比较人结肠癌(HCT-8)耐长春新碱细胞与非耐药细胞的荧光强度。人结肠癌(HCT-8)耐长春新碱细胞的荧光强度小于非耐药细胞的荧光强度。
实施例9
人卵巢癌(A2780)耐紫杉醇细胞与非耐药细胞混合接种在六孔板中，待细胞贴壁汇合度至70-80％时，吸出旧培养基，每孔加入2mL含有100μL FITC标记的SiO2纳米颗粒的培养基，在培养箱中继续培养2h。消化收集细胞，用磷酸盐缓冲液以500g，5min的条件离心洗涤2次，最后用500μL磷酸盐缓冲液重悬细胞，在流式细胞仪上检测。比较人卵巢癌(A2780)耐紫杉醇细胞与非耐药细胞的荧光强度。人卵巢癌(A2780)耐紫杉醇细胞的荧光强度小于非耐药细胞的荧光强度。
实施例10
人卵巢癌(SKOV3)耐顺铂细胞与非耐药细胞混合接种在六孔板中，待细胞贴壁汇合度至70-80％时，吸出旧培养基，每孔加入2mL含有100μL菁染料Cy5.5标记的ZIF-8纳米颗粒的培养基，在培养箱中继续培养2h。消化收集细胞，用磷酸盐缓冲液以500g，5min的条件离心洗涤2次，最后用500μL磷酸盐缓冲液重悬细胞，在流式细胞仪上检测。比较人卵巢癌(SKOV3)耐顺铂细胞与非耐药细胞的荧光强度。人卵巢癌(SKOV3)耐顺铂细胞的荧光强度小于非耐药细胞的荧光强度。
实施例11
人子宫颈癌(Hela)耐紫杉醇细胞与非耐药细胞混合接种在六孔板中，待细胞贴壁汇合度至70-80％时，吸出旧培养基，每孔加入2mL含有100μl 5-AF标记的PS-co-PAA纳米颗粒的培养基，在培养箱中继续培养2h。消化收集细胞，用磷酸盐缓冲液以500g，5min的条件离心洗涤2次，最后用500μL磷酸盐缓冲液重悬细胞，在流式细胞仪上检测。比较人子宫颈癌(Hela)耐紫杉醇细胞与非耐药细胞的荧光强度。人子宫颈癌(Hela)耐紫杉醇细胞的荧光强度小于非耐药细胞的荧光强度。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (10)

1.一种分析肿瘤细胞耐药性的方法，其特征在于，包括将二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒应用于肿瘤细胞的步骤。
2.如权利要求1所述的分析肿瘤细胞耐药性的方法，其特征在于，包括以下步骤：
(a)提供一种二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒；
(b)将所述二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒与肿瘤细胞共孵培养；
(c)检测肿瘤细胞对所述二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒的内吞性能。
3.如权利要求1或2所述的分析肿瘤细胞耐药性的方法，其特征在于，所述金属有机框架纳米颗粒包括ZIF-8纳米颗粒，ZIF-70纳米颗粒和ZIF-90纳米颗粒。
4.如权利要求2所述的分析肿瘤细胞耐药性的方法，其特征在于，在步骤(b)之前还包括步骤(d)，步骤(d)为对所述二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒进行荧光分子修饰。
5.如权利要求4所述的分析肿瘤细胞耐药性的方法，其特征在于，步骤(d)中所述荧光分子选自荧光素、罗丹明、菁染料、香豆素和荧光无机纳米晶中的一种或多种。
6.如权利要求4所述的分析肿瘤细胞耐药性的方法，其特征在于，所述二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒与所述荧光分子按照质量比20-2000:1进行反应。
7.如权利要求2所述的分析肿瘤细胞耐药性的方法，其特征在于，步骤(b)中将所述二氧化硅纳米颗粒、聚苯乙烯-co-聚丙烯酸纳米颗粒或金属有机框架纳米颗粒与肿瘤细胞共孵培养0.5-4h。
8.一种分析肿瘤细胞耐药性的试剂盒，其特征在于，包含经由荧光分子修饰的二氧化硅纳米颗粒、经由荧光分子修饰的聚苯乙烯-co-聚丙烯酸纳米颗粒或经由荧光分子修饰的金属有机框架纳米颗粒。
9.如权利要求8所述的分析肿瘤细胞耐药性的试剂盒，其特征在于，所述金属有机框架纳米颗粒包括ZIF-8纳米颗粒，ZIF-70纳米颗粒和ZIF-90纳米颗粒。
10.如权利要求8所述的分析肿瘤细胞耐药性的试剂盒，其特征在于，所述荧光分子选自荧光素、罗丹明、菁染料、香豆素和荧光无机纳米晶中的一种或多种。
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