CN113088493B - 人乳腺癌胸水转移细胞系及应用 - Google Patents
人乳腺癌胸水转移细胞系及应用 Download PDFInfo
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Abstract
本发明属于微生物动物细胞系领域,具体涉及人乳腺癌胸水转移细胞系及应用。所述人乳腺癌胸水转移细胞系包括:命名为人乳腺癌胸水转移细胞4Z‑B‑1,且保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:C202123的人乳腺癌胸水转移细胞系。本发明的人乳腺癌胸水转移细胞系细胞4Z‑B‑1是从中国人来源建立的,且建系时间较短,生物遗传性状稳定,以该人乳腺癌胸水转移细胞系作为研究模型,对于了解中国人群的原发性HER2+乳腺癌的发病机制有很大帮助。
Description
技术领域
本发明属于微生物动物细胞系领域,具体涉及人乳腺癌胸水转移细胞系及应用。
背景技术
乳腺癌是世界上女性最常见的恶性肿瘤。在我国,乳腺癌的发病率有逐步上升的趋势,在一些沿海经济发达地区,乳腺癌的发病率已占妇女恶性肿瘤的首位。乳腺癌是一类具有高度异质性的恶性肿瘤,经过数十年的科学研究技术的发展,通过采用基因微阵列技术可将其分为5个亚型:导管A型、导管B型、基底细胞样、HER2过表达型和正常乳腺样型,促进了临床乳腺癌患者的治疗效果显著提高,减少了患者在治疗疾病过程中所承受的身体和精神痛苦。
在过去的几十年间,数个病理和免疫组化亚分类的提出,有利于区分临床水平荷尔蒙受体和三阴性乳腺癌的广泛异质性特征。然而,这些分类不涉及对临床上HER2+乳腺癌的亚型分类。HER2+乳腺癌是指ERα阴性、PR阴性而HER2阳性的乳腺癌;其在所有乳腺癌中占15%~20%。和导管型乳腺癌相比,该类型组织学分级差,易复发和转移,预后差。尽管HER2靶向药物赫赛汀在一定程度上可以延长HER2+乳腺癌患者的生存时间,但是还是有大约15%的早期病人和70%以上的转移病人表现出对该药物的耐受。近年来,不断有研究指出,HER2+乳腺癌内部有很大的异质性,并根据其分子表达模式将临床上HER2+乳腺癌分为Luminal A/HER2+乳腺癌、Luminal B/HER2+乳腺癌、HER2 rich/HER2+乳腺癌和Basal like/HER2+乳腺癌,其中HER2 rich/HER2+乳腺癌对赫赛汀最敏感,而Basal like/HER2+乳腺癌则最耐受。
发明内容
本发明的第一个目的在于,针对目前国内缺乏中国人群来源的HER2 rich/HER2-positive乳腺癌细胞株,而提供了该类型的来源于中国人群的人乳腺癌胸水转移细胞系。
为此,本发明的上述目的通过以下技术方案来实现:
人乳腺癌胸水转移细胞系,其特征在于:所述人乳腺癌胸水转移细胞系包括:命名为人乳腺癌胸水转移细胞4Z-B-1,且保藏于中国典型培养物保藏中心,保藏编号为:CCTCCNO:C202123的人乳腺癌胸水转移细胞系。
在采用上述技术方案的同时,本发明还可以采用或者组合采用以下进一步的技术方案:
作为本发明的优选技术方案:所述人乳腺癌胸水转移细胞系还包括:命名为人乳腺癌胸水转移细胞4Z-B-1,且保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:C202123的人乳腺癌胸水转移细胞系的子代细胞系。
作为本发明的优选技术方案:所述人乳腺癌胸水转移细胞系在体外培养时,光镜下为圆形上皮样细胞,电镜下为典型的恶性上皮特征;核型分析结果为异倍体核型,主流染色体数目为61~72。
作为本发明的优选技术方案:所述人乳腺癌胸水转移细胞系在乳腺癌临床诊断时具有转移性乳腺癌特性,特征鉴定表型为HER2 rich/HER2-positive。
作为本发明的优选技术方案:所述人乳腺癌胸水转移细胞系在支原体检测试剂盒检测时,PCR结果为阴性,未受到支原体污染。
作为本发明的优选技术方案:所述人乳腺癌胸水转移细胞系的增殖能力较强,而迁移侵袭能力较弱。
作为本发明的优选技术方案:所述人乳腺癌胸水转移细胞系对HER2靶向性药物较敏感。
本发明的第二个目的在于,针对现有技术中存在的不足,提供一种根据前文所述的人乳腺癌胸水转移细胞系在HER2阳性乳腺癌发病以及耐药机制研究的细胞模型中的应用。由于本发明的人乳腺癌胸水转移细胞系的细胞4Z-B-1是从中国人来源建立的,且建系时间较短,生物遗传性状稳定,以该人乳腺癌胸水转移细胞系作为研究模型,对于了解中国人群的原发性HER2+乳腺癌的发病机制有很大帮助。
本发明还有一个目的在于,针对现有技术中存在的不足,提供一种人乳腺癌胸水转移细胞系的用途。
为此,本发明的上述目的通过以下技术方案来实现:
一种人乳腺癌胸水转移细胞系的用途,其特征在于:根据前文所述的人乳腺癌胸水转移细胞系用于制备在免疫缺陷哺乳动物中产生乳腺癌的模型,成瘤率为100%。
在采用上述技术方案的同时,本发明还可以采用或者组合采用以下进一步的技术方案:
作为本发明的优选技术方案:所述免疫缺陷哺乳动物为免疫缺陷裸小鼠。
本发明的人乳腺癌胸水转移细胞系取样自一例69岁的浸润性导管癌乳腺癌女性患者(原发灶ER+,PR+,HER2+)的转移性恶性胸水,经原代培养并建系成功后,命名为人乳腺癌胸水转移细胞4Z-B-1。
本发明的人乳腺癌胸水转移细胞系的细胞4Z-B-1生长旺盛,细胞多呈近圆形贴壁单层生长;CK-pan阳性率约在98.2%;细胞经过冻存复苏后仍具有良好的活力;上述细胞系目前已传代至70代左右,生长状态良好,已能永生化。
本发明的人乳腺癌胸水转移细胞系的细胞4Z-B-1的细胞倍增时间约为40h;染色体数目和结构均出现异常,染色体数目大多分布在61~72之间,结构异常包括染色体增加、缺失和易位等;电镜下有典型上皮特征,可见桥粒、微丝、丰富细胞器(线粒体、核糖体及粗面内质网)和核质比大。
本发明的人乳腺癌胸水转移细胞系采用目前最权威的检测方法即ATCC推荐的STR检测法,检测结果确定该人乳腺癌胸水转移细胞系为人源;与ATCC和DSMZ两个全球最著名的培养物保藏机构中的细胞遗传信息进行比对,发现均未受到其他细胞污染。
本发明的人乳腺癌胸水转移细胞系经免疫蛋白印迹法(Western Blot)鉴定乳腺癌表型相关蛋白表达分析,与现有已建立的细胞系进行对比,发现所建立的细胞系属于HER2rich/HER2-positive类别。
评估人乳腺癌胸水转移细胞系对HER2靶向药物的敏感性:将曲妥珠单抗、帕妥珠单抗、拉帕替尼以及吡咯替尼等HER2靶向药物用于细胞4Z-B-1,检测其对于上述药物的敏感性,并与其他HER阳性乳腺癌细胞系比较。人乳腺癌胸水转移细胞系的细胞4Z-B-1对曲妥珠单抗、曲多珠单抗联用帕妥珠单抗、拉帕替尼以及吡咯替尼均较敏感。
内分泌药物和化疗药处理细胞4Z-B-1:施用后显示他莫昔芬(Tamoxifen)、阿霉素(Adriamycin)、紫杉醇(Paclitaxel)、顺铂(Cisplatin)和奥沙利铂(Oxaliplatin)均能有效抑制细胞4Z-B-1;而5-氟尿嘧啶(5-Fu)则对细胞4Z-B-1则无显著抑制效果。
本发明的人乳腺癌胸水转移细胞系的成瘤实验结果发现,所建立的细胞系在裸鼠体内具有良好的成瘤性。通过将一定数量的上述人乳腺癌胸水转移细胞系细胞4Z-B-1接种于裸小鼠的皮下,建立了乳腺癌的动物模型。
本发明还提供了人乳腺癌胸水转移细胞系的细胞4Z-B-1的子代细胞系,子代细胞系基本或全部保留了亲代细胞系的特性。
再一方面,本发明提供了一种从患者恶性胸水中建立人乳腺癌胸水转移细胞系的制备方法,包括以下步骤:
(1)标本采集及保存的方法:按无菌规范操作对患者进行胸腔穿刺,引流胸水时用无菌的50ml离心管接取100ml胸水,然后迅速移至实验室进行离心。
(2)原代培养:胸水500g离心5分钟后弃去上清,沉淀组织用PBS重悬并离心,重复三次,然后用含10%胎牛血清的DMEM培养基重悬细胞,并接种于10cm培养皿中进行传代培养和观察培养。
(3)纯化细胞:采取有限稀释法,具体为待传代几次后,将细胞按照1:6或更高倍数传代至培养皿中培养,镜下观察并标记上皮特征的细胞团,无菌条件下刮除其周围细胞,PBS洗涤三次,加入新培养基继续培养至长出肉眼可见的细胞团。
(4)扩大培养:胰酶消化细胞团,离心收集细胞后接种至96孔板,随后依次接种至48孔板、24孔板、12孔板、6孔板和10cm培养皿中。
附图说明
图1为人乳腺癌胸水转移细胞系细胞4Z-B-1在光学显微镜和电子显微镜下的形态,SK-BR-3、ZJU-0725、ZJU-1127和ZJU-0327细胞系与之作为对比。
图2为人乳腺癌胸水转移细胞系细胞4Z-B-1的流式鉴定结果图和统计图(a和b)、支原体检测琼脂糖电泳图(c)、表型分析图(d)以及代表性单细胞的核型分析(e)。
图3为人乳腺癌胸水转移细胞系细胞4Z-B-1的增殖能力检测(a)、细胞的细胞周期分布图(b和c)、细胞周期相关蛋白表达(d)、迁移侵袭能力检测(e和f)、3D侵袭能力检测(g和h)和EMT相关蛋白表达(i)。
图4为人乳腺癌胸水转移细胞系细胞4Z-B-1对HER2靶向药物的敏感性检测(a-e)和HER2靶向药物敏感性相关基因表达分析图(f)。
图5为人乳腺癌胸水转移细胞系细胞4Z-B-1对内分泌治疗药物(a)和化疗药物敏感性检测(b-f)以及其基因表达模式检测(g)。
图6为人乳腺癌胸水转移细胞系细胞4Z-B-1的克隆形成实验结果(a-c)、干性标志物检测(d和e)、裸鼠成瘤试验结果(f-k)。
图7为移植瘤中乳腺癌相关指标的表达情况与患者术前穿刺标本和手术标本的对比。
具体实施方式
参照附图和具体实施例对本发明作进一步详细地描述。
实施例1
从一例浸润性导管癌乳腺癌69岁女性患者(原发灶ER+,PR+,HER2+)的转移性胸水中取样,离心获得其中的恶性细胞,用含双抗的磷酸盐缓冲液重悬并离心3次。将沉淀用含10%胎牛血清的DMEM培养基重悬,铺到无菌10cm培养皿中,置入37℃CO2培养箱培养,至少2天内不要移动培养皿,利于原代肿瘤细胞贴壁;待细胞在皿中长至70-80%时传代至6个10cm培养皿中,每日镜下观察,待观察到镜下细胞为卵圆形、片状分布细胞团时,刮除周围成纤维细胞,磷酸盐缓冲液洗3次,加入新的培养基继续培养;待细胞团块长至肉眼可见时,胰酶消化液消化细胞传至96孔板中,之后依次传至48孔板、24孔板、12孔板、6孔板和10cm培养皿中。建系成功后,命名为人乳腺癌胸水转移细胞4Z-B-1,并将其第60代细胞于2021年1月26日保藏于中国典型培养物保藏中心(CCTCC),保藏地址为:中国,武汉,武汉大学,保藏编号为:CCTCC NO:C202123。
实施例2
取传代培养的人乳腺癌胸水转移细胞系细胞4Z-B-1,在光学显微镜下(ZEISS倒置显微镜)观察细胞生长情况。
镜检:如图1中a所示,细胞学形态图片,细胞折射性好,细胞生长状态良好,背景清晰,细胞多呈近圆形。图1中a为第50代乳腺癌原代细胞形态。
实施例3
(1)样品准备:制备二维细胞爬片样品时,将5×104个培养的4Z-B-1细胞铺至无菌的细胞爬片上,置于培养箱中24h,等待细胞充分贴壁;制备三维细胞球样品时,将1×107个培养的4Z-B-1细胞铺至低吸附的培养皿中,72h后将形成的细胞球收集于15ml离心管中,然后再次洗涤离心收集;
(2)固定:向细胞中加入2.5%戊二醛固定液固定2h,0.1M磷酸缓冲液洗涤3次,加入1%饿酸固定液固定2h,0.1M磷酸缓冲液洗涤3次;
(3)脱水:在4℃冰箱内分别进行30%乙醇→→50%乙醇→→70%乙醇→→90%乙醇→→95%乙醇→→100%乙醇(2次),每道工序维持15-20min;
(4)干燥:将样品置于样品临界点干燥;
(5)镀膜:使用ACE200镀膜系统将样品用金镀膜;
(6)观察拍片:用扫描电镜(Thermo FEI,Nova Nano 450)观察及拍片。
SK-BR-3、ZJU-0725、ZJU-1127以及ZJU-0327细胞的样品制备过程同上,其结果作为对照;SK-BR-3细胞来源于ATCC,ZJU-0725、ZJU-1127以及ZJU-0327细胞的制备参照CN108034636A。结果如图1b-c和e-h所示,4Z-B-1细胞表面球状和棒状突起比例相当,而SK-BR-3表面主要为球状突起,ZJU-0725、ZJU-1127以及ZJU-0327表面则主要为棒状突起,三类有显著差异。
实施例4
(1)样品准备:将1×107个培养的4Z-B-1细胞铺至低吸附的培养皿中,72h后将形成的细胞球收集于15ml离心管中,然后再次洗涤离心收集;或将5×104个培养的4Z-B-1细胞铺至无菌的细胞爬片上,24h后进行下一步固定操作;
(2)固定:向细胞中加入2.5%戊二醛固定液固定2h,0.1M磷酸缓冲液洗涤3次,加入1%饿酸固定液固定2h,0.1M磷酸缓冲液洗涤3次;
(3)脱水:在4℃冰箱内分别进行50%乙醇→→70%乙醇→→90%乙醇→→100%乙醇→→100%丙酮(2次),每道工序维持15-20min;
(4)包埋:纯丙酮+包埋液(1:1)室温2h→→纯包埋液37℃2-3h;
(5)固化:37℃烘箱过夜→→45℃烘箱12h→→60℃烘箱24h;
(6)超薄切片制备:用超薄切片机切取50-60nm厚度切片;
(7)染色:3%醋酸铀-枸橼酸铅双重染色;
(8)观察拍片:用透射电镜(Thermo FEI,Tecnai G2 spirit)观察及拍片。
结果如图1d所示,胞浆内可见张力纤维,丰富细胞器(线粒体、核糖体及粗面内质网),细胞之间可见桥粒和微绒毛样突起。
实施例5
培养的4Z-B-1细胞胰酶消化后离心(300×g,7min)收集细胞,PBS洗3次;加入足量的4%多聚甲醛固定液室温固定10-15min,固定后细胞PBS洗3次;细胞在10%山羊血清+0.03%Triton-X100的PBS中固定和通透化40min;离心去除上清,FITC荧光标记的pan-CK流式抗体与细胞常温避光孵育30min,SK-BR-3作为阳性对照,人正常成纤维细胞作为阴性对照;PBS洗涤离心3次;30μm细胞滤网过滤后上机检测。结果如图2a和b所示,4Z-B-1纯度约为98.2%。
实施例6
对分离出的癌细胞需要同时进行支原体感染情况的检测,本研究采用的是PCR法,利用碧云天公司的支原体PCR检测试剂盒(Mycoplasma PCR Detection Kit),步骤如下:
(1)试剂盒组成:
①第一次PCR引物混合物(1μl/次,100次反应)
②第二次PCR引物混合物(1μl/次,100次反应)
③对照模板
④使用说明书1份
(2)样本收集及处理:
收集细胞培养液:将细胞接种后培养3~6天,然后收集培养上清液至15ml离心管中;
(3)第一次PCR反应:
①配制PCR反应液:从-20℃冰箱中取出PCR所需试剂,3个重复检测细胞培养上清液、阳性对照(对照模板)和阴性对照(无菌PBS)。每个反应体系加入5μl细胞培养上清液(或对照模板或无菌PBS)、第一次PCR引物混合物1μl、Easy-LoadTM PCR Master Mix 25μl,用双蒸水补齐至50μl。一次性配制所需反应体系,保持PCR反应的均一性。
表1
②PCR反应及参数设置:将PCR反应管放入PCR仪,按以下参数进行PCR反应:
(4)第二次PCR反应:
①配制PCR反应液:加入0.5μl第一次PCR产物、第二次PCR引物混合物1μl、Easy-LoadTM PCR Master Mix 25μl,用双蒸水补齐至50μl。一次性配制所需反应体系,保持PCR反应的均一性。
表2
②PCR反应及参数设置:将PCR反应管放入PCR仪,按以下参数进行PCR反应:
(5)PCR产物的电泳检测:
①1×TAE缓冲液配制:取l ml 50×TAE电泳缓冲液加入49ml去离子水;
②2.0%琼脂糖凝胶制备:称取1.09g琼脂糖于三角烧杯中,加入50ml l×TAE;
电泳缓冲液,微波炉溶化琼脂糖,稍冷后加入5μl GelRed核酸染料,倒入凝胶模中,冷却凝固,备用;
③上样:每个PCR管取10μl PCR扩增产物,加入琼脂胶板的胶孔中,扩增产物包含溴酚蓝指示剂;
④电泳:110V电泳20min后根据溴酚蓝指示剂停止电泳。
(6)凝胶成像分析:将电泳后的胶板取出放入Bio-Rad凝胶成像分析仪中观察并拍照保存,出现与阳性对照相同的条带即为支原体阳性样本。如图2中c所示,所建立的4Z-B-1未受支原体污染。
实施例7
(1)向指数生长期的人乳腺癌胸水转移细胞系细胞培养液中加入秋水仙素(终浓度为0.05mg/ml)作用6h;
(2)弃去培养液后洗涤并用胰酶消化收集细胞,PBS洗涤离心后将细胞收集于15ml离心管底部;
(3)低渗处理:加入已于37℃水浴锅中充分预热的低渗KCl溶液(0.075mol/L)4-5ml,于37℃水浴中作用20min,充分膨胀细胞;
(4)预固定:于上述低渗液中直接加入lml新鲜配制的固定液(甲醇:冰醋酸,3:l,V/V),轻轻吹打均匀,固定5min后离心(1500rpm,10min),弃上清;
(5)再固定:加入6-8ml新鲜配制的固定液,常温固定30min;
(6)吹片:将以上标本离心后再加5-6滴固定液,试取1滴,滴在4℃蒸馏水玻片上,在酒精灯火焰上迅速烘烤并吹片;
(7)Giemsa染色:将吹好的以上玻片表面滴加适量Giemsa工作液染色10-15min,自来水冲洗玻片后自然晾干即得到中期染色体标本。
图2e为4Z-B-1细胞代表性单细胞的核型分析。
实施例8
将培养的4Z-B-1细胞胰酶消化后,重悬于含10%FBS的DMEM培养基中并进行计数,经浓度调整后获得终浓度为1×104/ml的细胞悬液;将其接种在96孔板中,每孔200μl,每组设6个复孔,只加培养基的孔作为空白对照;将培养板放入37℃培养箱中培养,于0、24、48和72h时间点加入CCK-8孵育2h,随后450nm处测定吸光值。结果如图3a所示,4Z-B-1细胞的增殖能力强于SK-BR-3,其群体倍增时间约为40h。
实施例9
收集约1×106个处于指数分裂的4Z-B-1细胞和SK-BR-3细胞,加入0.3ml PBS缓冲液吹打成细胞悬液,再逐滴加入-20℃冰冻的乙醇0.7ml,边加边摇晃均匀,将标本置入4℃冰箱过夜;次日取出PBS洗1次;加入RNase A于37℃水浴消化30min,再加入400μl PI混匀,4℃避光反应30min;过30μm滤网获得单细胞悬液后上机检测。
图3b和c为人乳腺癌胸水转移细胞系细胞4Z-B-1的细胞周期分布图和统计图,SK-BR-3细胞作为对照。如图3b和c所示,4Z-B-1细胞约有一半位于G0/G1期,其次是S期,G2/M期最少;统计分析表明,与SK-BR-3细胞相比,4Z-B-1在G0/G1期比例更低,而在S期和G2/M期的细胞比例更高。
实施例10
将培养的4Z-B-1细胞胰酶消化后,用含10%胎牛血清的DMEM培养基中和,离心后用无菌PBS重悬并离心三次,最后重悬于不含血清的DMEM培养基中。计数,经浓度调整后获得终浓度为2.5×105/ml的细胞悬液;检测细胞的迁移能力时,将200ul细胞悬液铺至未铺matrigel的transwell上层小室中;检测细胞的侵袭能力时,将200ul细胞悬液铺至铺好matrigel的transwell上层小室中。将上层小室放入装有含20%胎牛血清DMEM的24孔板中,置于细胞培养箱。16h后,取出上层小室,置于4%多聚甲醛中固定20min,然后用棉签擦去小室上层未穿过transwell膜的细胞。用0.5%结晶紫溶液染色20min后,用PBS清洗干净,然后置于倒置显微镜(ZEISS)下观察并拍摄。
对SK-BR-3细胞迁移和侵袭能力的检测方法同上,其结果作为对照。如图3e所示,4Z-B-1细胞的迁移和侵袭能力弱,相比SK-BR-3,其穿过transwell膜的细胞数明显更少。图3f是穿过膜的细胞数的统计图。
实施例11
采用Cultrex 3D球体细胞侵袭分析试剂盒(Trevigen)对细胞的3D侵袭能力进行检测。将培养的4Z-B-1细胞胰酶消化后,用含10%胎牛血清的DMEM培养基中和,离心后用无菌PBS重悬并离心三次,最后重悬于试剂盒内促进细胞球形成的培养基中。计数,经浓度调整后获得终浓度为6×104/ml的细胞悬液;在低吸附的球形底面96孔板中打入50ul细胞悬液,室温下用摆臂离心机200×g离心3min,置于细胞培养箱中72h,促进细胞球体形成。将96孔板放在冰上,置于冰箱中15min,冷却孔板。冰上操作,每孔加入50ul侵袭基质胶。4℃下摆臂离心机300×g离心5min,以消除气泡,并将球囊置于孔的中央。将孔板转移至细胞培养箱放置1h,促进胶体形成。1h后,加入100μl含趋化和侵袭调节药物(如果可用的话)的培养基。37摄氏度培养箱内孵育3天,每24h在4×镜下拍照记录。
对SK-BR-3、MCF-7和MDA-MB-231细胞的3D侵袭能力的检测同上,其结果作为对照;SK-BR-3、MCF-7和MDA-MB-231细胞来源于ATCC。如图3g所示,4Z-B-1细胞的3D侵袭能力弱,与SK-BR-3和MCF-7相当,而MDA-MB-231则表现出明显更强的3D侵袭能力。图3h是上述细胞3D侵袭面积的统计图。
实施例12
(1)细胞准备:将培养的人乳腺癌胸水转移细胞系细胞4Z-B-1用胰酶消化后悬浮于DMEM培养液中并计数,经浓度调整后获得终浓度为2.5×104/ml的细胞悬液;
(2)细胞接种:将以上浓度的细胞悬液分别接种到96孔板中,每孔0.2ml(含5000个细胞),每组3个复孔,只加培养液不含细胞的孔作为空白对照;
待接种细胞贴壁后对每组分别加入适量的HER2靶向药物曲妥珠单抗(Trastuzumab)、帕妥珠单抗(Pertuzumab)、拉帕替尼(Lapatinib)、吡咯替尼(Pyrotinib)或内分泌治疗药他莫昔芬(Tamoxifen)或化疗药阿霉素(Adriamycin)、紫杉醇(Paclitaxel)、顺铂(Cisplatin)、奥沙利铂(Oxaliplatin)和5-氟尿嘧啶(5-Fu),设置不同浓度药物,放入培养箱中继续培养特定时间;
(3)CCK-8检测:在特定的处理时间后分别对以上每组细胞用CCK-8(1:10稀释,37℃孵化2h)进行活性检测,并记录每组细胞在450nm的吸光度值,利用Graphpad软件绘制曲线。结果如图4a-e和图5a-f所示,HER2靶向药物中,4Z-B-1对曲妥珠单抗、曲妥珠单抗联合帕妥珠单抗、拉帕替尼和吡咯替尼均敏感;化疗药物中,阿霉素、紫杉醇、顺铂和奥沙利铂均抑制4Z-B-1,而5-氟尿嘧啶则没有明显的抑制作用;内分泌治疗药物他莫昔芬对4Z-B-1也有明显的抑制效果。
实施例13
将生长接近汇合的4Z-B-1、ZJU-0725、ZJU-1127、ZJU-0327、SK-BR-3、MDA-MB-453和BT-474细胞,PBS洗3遍;加入1ml RIPA裂解液(含浓度为1nM的PMSF),冰上裂解30分钟,收集裂解物;4℃12000g离心5min,取上清,分装保存。BCA法测定各个裂解物浓度,调整浓度一致,加入4倍体积上样缓冲液,100℃煮5-10min。制备SDS-PAGE胶,上样20μg,80V电泳至分离胶和浓缩胶界面,110V电泳至溴酚蓝接近胶的底部;转膜仪转至PVDF膜;5%脱脂奶粉(TBST)室温缓慢摇动封闭2h;加入一抗抗体4℃孵育过夜,TBST洗涤3次,5min/次;加入稀释的二抗室温孵育2h;ECL发光液孵育,Western Blot成像分析系统成像显影。
图2d和图4f中,建立起来的4Z-B-1细胞系的分子表型为HER2-positive,且耐药相关基因表达和活性较低,这与其对药物较为敏感的表型一致。图3d中,4Z-B-1的CyclinE1表达水平较高,这与其生长速度较快的表型一致。图3i中,4Z-B-1E-Cadherin表达高,而Vimentin表达低,与其迁移侵袭能力弱的表型一致。图5g中,4Z-B-1的HER2表达高,而Luminal标志物和Basal标志物均为阴性,表现为HER2 rich/HER2+;大部分EMT标志物为阴性,而细胞周期标志物表达较高,这与其侵袭能力弱而增殖能力强的表型一致。
实施例14
(1)细胞准备:将培养的人乳腺癌胸水转移细胞系细胞4Z-B-1用胰酶消化后悬浮于DMEM培养液中并计数,经浓度调整后获得终浓度为1×103/ml的细胞悬液;
(2)细胞接种:将以上浓度的细胞悬液接种到6孔板中,每孔2ml(含2000个细胞),放入培养箱继续培养,毎3天更换一次培养基。
(3)观察拍照:1周后,将细胞置于倒置光学显微镜下观察并拍照。
(4)固定、染色及计数:弃去培养基,用PBS洗3次,4%多聚甲醛中固定20min,然后用0.5%结晶紫溶液染色20min,清水下清洗干净,然后置于倒置显微镜(ZEISS)下拍摄并计数。
SK-BR-3、MCF-7和MDA-MB-231细胞的克隆形成实验步骤同上,其结果作为对照。如图6a和b所示,4Z-B-1的克隆形成能力在4中细胞系中最弱。图6c中可见4Z-B-1所形成的克隆与其他三种不同,其细胞堆叠形成紧密的三维结构。
实施例15
(1)细胞准备:将处于对数生长期的人乳腺癌胸水转移细胞系细胞4Z-B-1用胰酶消化后用PBS清洗3次并计数,经浓度调整后获得终浓度为1×107/ml的细胞悬液;
(2)结合抗体:取100μl上述细胞悬液,加入10μl PE抗CD24抗体和10μl FITC抗CD44抗体,避光4℃孵育30min。
(3)流式检测:孵育完成后用PBS清洗2次,然后流式上机检测。
SK-BR-3、MCF-7和MDA-MB-231细胞的检测方法同上,其结果作为对照。图6d和e中,4Z-B-1、SK-BR-3和MCF-7细胞系几乎没有CD24-/CD44+细胞,而MDA-MB-231细胞系则几乎均为CD24-/CD44+细胞。
实施例16
(1)细胞准备:将指数生长期的人乳腺癌胸水转移细胞系细胞4Z-B-1胰酶消化后离心(800rpm,5min)后用PBS洗涤离心3次;
(2)将细胞重新悬浮于PBS缓冲液中,计数调整并按1:1加入matrigel,使其细胞密度为5×107/ml;
(3)将0.1ml的以上细胞悬液(含约4×106个细胞)注射到4周的BABL/c小鼠腹股沟部皮下(n=5),小鼠饲养于SPF级的层流间内;
(4)观察3周,每3天测量瘤体直径大小;
(5)3周后将小鼠安乐死,取出瘤体测量拍照,取部分瘤体浸泡于10%福尔马林溶液,进行常规石蜡包埋及切片制作,备用。
图6a-c为4Z-B-1细胞在无胸腺的裸鼠(nu/nu,BABL/C)小鼠皮下所成瘤体及瘤体生长曲线。图6d为HE染色病理图,提示该肿瘤为高级别浸润性乳腺癌。
实施例17
(1)烤片:将切片置于70℃1h。
(2)脱蜡:二甲苯I10min,二甲苯II 10min。
(3)水化:100%乙醇I1min、100%乙醇II 1min、95%乙醇1min、85%乙醇1min、75%乙醇1min(1min相当于上下焯几下),然后用净化水洗涤3遍。
(4)0.03%双氧水浸泡封闭5min。
(5)抗原修复:将切片浸入0.01M枸橼酸缓冲液,加热至沸腾,冷却至室温后PBS洗涤3次,每次5min;
(6)修复结束取出切片,净化水洗涤3遍。
(7)封闭:1%牛血清蛋白封闭10min。
(8)孵一抗:封闭液倒掉后放入湿盒加入对应一抗,室温孵育1h。
(9)孵二抗:PBS洗3次,每次2min,放到湿盒内,滴加二抗,室温孵育30min。
(10)DAB染色:PBS洗3次,每次2min。滴加DAB反应5min,然后转移到玻片架上,净化水洗涤15min。
(11)苏木素染色:苏木素染液中浸泡2min后用净化水冲洗。
(12)脱水:85%乙醇、95%乙醇、100%乙醇、100%乙醇各1min。
(13)透明:二甲苯I、二甲苯II各1min。
(14)封片:树胶封片。
图7是4Z-B-1细胞系来源患者术前穿刺标本、手术标本和裸鼠皮下瘤体的免疫组化结果。如图7所示,4Z-B-1细胞系裸鼠皮下瘤体的HER2、Ki-67、P120和E-Cadherin呈强阳性,EGFR呈弱阳性,而ERα、PR和CK5/6呈阴性。其ERα和PR的表达与患者穿刺标本和手术标本相比有所改变。
上述具体实施方式用来解释说明本发明,仅为本发明的优选实施例,而不是对本发明进行限制,在本发明的精神和权利要求的保护范围内,对本发明作出的任何修改、等同替换、改进等,都落入本发明的保护范围。
Claims (9)
1.一种人乳腺癌胸水转移细胞系,其特征在于:所述细胞系命名为人乳腺癌胸水转移细胞4Z-B-1,且保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO: C202123。
2.根据权利要求1所述的人乳腺癌胸水转移细胞系的子代细胞系。
3.根据权利要求1所述的人乳腺癌胸水转移细胞系或权利要求2所述的人乳腺癌胸水转移细胞系的子代细胞系,其特征在于:所述细胞系在体外培养时,光镜下为圆形上皮样细胞,电镜下为典型的恶性上皮特征;核型分析结果为异倍体核型,主流染色体数目为61~72。
4.根据权利要求1所述的人乳腺癌胸水转移细胞系或权利要求2所述的人乳腺癌胸水转移细胞系的子代细胞系,其特征在于:所述细胞系在乳腺癌临床诊断时具有转移性乳腺癌特性,特征鉴定表型为HER2 rich/HER2-positive。
5.根据权利要求1所述的人乳腺癌胸水转移细胞系或权利要求2所述的人乳腺癌胸水转移细胞系的子代细胞系,其特征在于:所述细胞系在支原体检测试剂盒检测时,PCR结果为阴性,未受到支原体污染。
6.根据权利要求1所述的人乳腺癌胸水转移细胞系或权利要求2所述的人乳腺癌胸水转移细胞系的子代细胞系,其特征在于:所述细胞系的增殖能力较强,而迁移侵袭能力较弱。
7.根据权利要求1所述的人乳腺癌胸水转移细胞系或权利要求2所述的人乳腺癌胸水转移细胞系的子代细胞系,其特征在于:所述细胞系对HER2靶向性药物较敏感。
8.根据权利要求1所述的人乳腺癌胸水转移细胞系或权利要求2所述的人乳腺癌胸水转移细胞系的子代细胞系在制备HER2阳性乳腺癌发病以及耐药机制研究的细胞模型中的应用。
9.根据权利要求1所述的人乳腺癌胸水转移细胞系或权利要求2所述的人乳腺癌胸水转移细胞系的子代细胞系在制备免疫缺陷裸小鼠乳腺癌模型中的用途。
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