CN110835623B - 一种原发性铂耐药的人卵巢癌细胞系fdovl、其制备方法和应用 - Google Patents
一种原发性铂耐药的人卵巢癌细胞系fdovl、其制备方法和应用 Download PDFInfo
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Abstract
本发明属于微生物动物细胞系领域，具体涉及一种原发性铂耐药的人卵巢癌细胞系、其建立方法及应用。本发明通过原发性铂耐药的卵巢高级别浆液性癌二次瘤体减灭术左盆腔转移淋巴结手术切除标本的原代培养，建立了原发性铂耐药人卵巢癌细胞系。该细胞系成瘤性好，可用于建立原发性铂耐药的上皮性卵巢癌动物模型。本发明的原发性铂耐药人卵巢癌细胞系可用于铂耐药的上皮性卵巢癌的发生发展及转移机制、新药筛选、耐药机理及临床前期研究。
Description
技术领域
本发明属微生物动物细胞系领域，具体涉及一种耐药的人卵巢癌细胞系的建立及其建立方法，具体涉及一种原发性铂耐药的人卵巢癌细胞系FDOVL、其制备方法和应用。
背景技术
现有技术公开了卵巢癌(Ovary Cancer，OC)是恶性程度最高的妇科恶性肿瘤。据报道，2015年，我国有5万余新发卵巢癌，同时有超过一半的患者死于卵巢癌复发与全身广泛转移[1]。上皮性卵巢癌(Epithelial Ovary Cancer，EOC) 是最常见的卵巢癌类型，约占90％，其中又以高级别浆液性癌(High grade serous carcinoma，HGSC)最为多见。因其发病隐匿，进展较快，75％患者发现时已为晚期(FIGO III、IV期)，并伴有腹腔内广泛转移，其5年整体生存率约为40％[2]。手术联合铂类为基础的化疗是国际标准的卵巢癌治疗策略，但大部分患者在治疗结束后18个月内复发[3]。铂类药物敏感性是评价预后的重要决定因素。根据铂类药物敏感程度将EOC分为三类：难治性、耐药性、敏感性。其中，铂类耐药性卵巢癌定义为接受以铂类为基础的一线化疗后6个月内复发的患者，这部分患者对后续化疗药物反应率也较差(<15％)，无进展生存期(Progression free survival,PFS)仅为3-4个月，随着进行性缩短的无病生存期(Disease-free Survival，DFS)，患者最终死于化疗耐药，其中位生存期通常小于1年[4]。因此，进一步探索铂类耐药EOC的生物学行为和分子机制尤为重要。
研究表明，人肿瘤细胞系的建立和鉴定，对肿瘤生物学行为及分子机制的研究尤为重要。尤其是构建原发性铂耐药卵巢癌细胞系可研究铂耐药卵巢癌的生物学行为及耐药机制，对延长铂耐药患者生存期，改善患者预后具有重要意义。
基于现有技术的现状，本申请的发明人拟一种原发性铂耐药的人卵巢癌细胞系、其制备方法和应用。
于本发明相关的现有技术有如下参考文献
1.Chen W,Zheng R,Baade PD,Zhang S,Zeng H,Bray F et al.Cancerstatistics in China,2015.CA:a cancer journal for clinicians 2016, 66(2):115-132.
2.Leamon CP,Lovejoy CD,Nguyen B.Patient selection and targetedtreatment in the management of platinum-resistant ovarian cancer.Pharmacogenomics and personalized medicine 2013,6:113-125.
3.Jayson GC,Kohn EC,Kitchener HC,Ledermann JA.Ovarian cancer. Lancet(London,England)2014,384(9951):1376-1388.
4.Davis A,Tinker AV,Friedlander M."Platinum resistant"ovarian cancer:what is it,who to treat and how to measure benefit？Gynecologic oncology 2014,133(3):624-631.。
发明内容
本发明的目的是基于现有技术的现状，建立一种原发性铂耐药的人卵巢癌细胞系，尤其是一种原发性铂耐药的人卵巢癌细胞系FDOVL。所述的细胞系其特征在于可稳定传代，成瘤性好，可适用于建立动物模型。该细胞系可用于研究原发性铂耐药卵巢癌的发生发展及转移机制、耐药原理及新药筛选等。
本发明的另一个目的提供上述细胞系的制备方法和应用。
本发明提供了一种人卵巢癌细胞系FDOVL，该细胞为原发性人卵巢癌细胞系并且铂耐药。
基于普通的卵巢细胞或者卵巢癌细胞并不具有铂耐药特性，本发明通过原发性铂耐药的卵巢高级别浆液性癌二次瘤体减灭术左盆腔转移淋巴结手术切除标本的原代培养，建立了原发性铂耐药人卵巢癌细胞系。
本发明所述的FDOVL细胞系来源于一个复发性EOC患者转移淋巴结的手术切除标本，该患者2016年10月行第一次卵巢癌减灭术，术后卡铂+紫杉醇化疗8 程后3月发现腹膜多发转移，腹膜后及髂血管旁淋巴结多发转移，于2017.8月行二次卵巢癌瘤体减灭术，术后白蛋白紫杉醇+顺铂化疗7程后4月再次发现腹膜后及双侧髂血管旁淋巴结多发转移。该患者两次复发均在铂类化疗后6月内，属于原发性铂耐药的卵巢癌，因而通过该患者手术切除标本构建原发性铂耐药卵巢癌细胞系可为研究铂耐药卵巢癌的生物学行为及耐药机制，对延长铂耐药患者生存期，改善患者预后提供有重要意义的基础。
本发明的原发性铂耐药的人卵巢癌细胞系，命名为FDOVL，分类命名为中国人卵巢癌细胞系，已于2018年3月7日由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏，地址：北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC NO.15484。
该细胞系具有癌细胞的生物学特征，单层贴壁生长，失去接触抑制，能够连续稳定传代50代以上。
较好的，该细胞TP53框内缺失及BRCA1多位点突变。
具体而言，该细胞的生物学特征如下：
单层贴壁生长，失去接触抑制，形态不一，呈圆形椭圆形或纺锤形扁平状，或不规则形；并且
体外培养生长良好，每4天传代一次，能够连续稳定传代50余代；并且
染色体核型分析提示细胞染色体数目多为58-62条，存在复杂的易位,错误拼接的染色体畸变；并且
该细胞与左盆腔转移淋巴结肿瘤组织基因谱分析结果高度相似，同时检测到TP53框内缺失及BRCA1多位点突变。
经试验，本发明的原发性铂耐药人卵巢癌细胞系种植裸鼠，两周成瘤，成瘤率100％。移植瘤HE染色呈上皮性来源，免疫组织化学染色结果与原始肿瘤免疫组织化学结果基本一致。
更具体的，所述的FDOVL具有以下生物学特性：
1.单层贴壁生长，失去接触抑制，形态不一，呈圆形椭圆形或纺锤形扁平状，或不规则形；
2.体外培养生长良好，每4天传代一次，能够连续稳定传代，目前已传至 50余代，倍增时间为87h；
3.染色体核型分析结果提示该细胞染色体数目58-80，存在复杂的易位,错误拼接等染色体畸变；
4.二代测序同时检测到FDOVL及肿瘤组织携带有TP53框内缺失及BRCA1 多位点突变；
5.将第32代FDOVL细胞按1×107数量级接种于NOD/SKID裸鼠，14天后移植瘤形成，病理切片提示移植瘤为卵巢上皮浆液性癌来源。
本发明中，采用离体的肿瘤组织，经取材、原代培养、传代培养和细胞纯化，建成原发性铂耐药的人卵巢癌细胞系；通过下述制备方法和步骤：
(1)取材：获得离体新鲜的复发性卵巢高级别浆液性癌左盆腔转移淋巴结手术切除标本，病灶组织浸入无菌PBS溶液清洗，并去除结缔组织和坏死组织；
(2)原代培养：将肿瘤组织切成米粒大小的组织数块，放入无菌漏网中研磨，过滤后的组织均匀接种于10cm大皿中，静置培养，每4天半量换液1次；
(3)传代培养：当细胞布满瓶底时，吸除培养液，PBS缓冲液漂洗，0.25％胰蛋白酶消化，按1:2的比例传代，目前已传至50余代；
(4)细胞纯化：细胞培养至第3代时开始分离成纤维细胞，胰酶消化后，将细胞悬液接种于培养皿中，静置约15～20分钟后于倒置相差显微镜下观察，见部分细胞贴壁，吸取细胞悬液入另一培养瓶中继续培养；按上述方法，重复2 次传代，使肿瘤细胞与成纤维细胞分开；当细胞培养6-7代后可观察到皿中剩余成纤维细胞逐渐死亡。
其中，细胞离体后的培养条件为：
含12％FBS、1％双抗、1％丙酮酸钠溶液、1％HEPES缓冲液、1％非必需氨基酸溶液的RPMI 1640细胞培养基；并且培养环境为37℃、5％CO2、饱和湿度。
经生物学特性鉴定获得一种原发性铂耐药的人卵巢癌细胞系。
本发明的细胞系可用于其发生发展、转移机制、新药筛选或者耐药机理的基础及临床前期研究。
所述原发性铂耐药的人卵巢癌细胞系可用于建立铂耐药的癌动物模型，为卵巢癌、尤其是其铂耐药性提供更好的研究模型。
本发明提供了一种原发性铂耐药的人卵巢癌细胞系FDOVL，该细胞系形态稳定，可以稳定多次传代，携带有TP53框内缺失及BRCA1多位点突变，可用于原发性铂耐药的上皮性卵巢癌生物学行为的研究；具有较强的致瘤性，可用于建立动物模型，所得的动物模型用于研究原发性铂耐药的上皮性卵巢癌发生发展的分子机制，探究其对传统化疗药物的耐药机理并筛选有效的靶向药物，是基础研究及临床前期应用的理想细胞系。
附图说明
图1.FDOVL细胞形态(倒置相差显微镜400×)。
图2.FDOVL细胞增殖曲线。
图3.FDOVL染色体核型分析。
图4.FDOVL及左盆腔淋巴结肿瘤组织已知驱动基因突变情况。
图5.FDOVL在NOD/SCID小鼠体内成瘤MRI成像(皮下注射24天后)。
图6.FDOVL移植瘤增殖曲线。
图7.肿瘤组织及FDOVL移植瘤HE染色及免疫组化分析。
具体实施方式
实施例1制备原发性铂耐药的人卵巢癌细胞系FDOVL
(1)取材
2017年8月9日从复旦大学附属肿瘤医院获得新鲜的复发性卵巢HGSC左盆腔转移淋巴结手术切除标本(女，53岁，复发性卵巢癌，病理诊断：高级别浆液性癌，取样经过患者知情同意)，取病灶组织1cm3浸入无菌PBS溶液清洗，并去除结缔组织和坏死组织；
(2)原代培养
将肿瘤组织剪成约1mm3大小的小块，放入无菌漏网中研磨，过滤后的组织均匀接种于10cm大皿中，静置培养，每4天半量换液1次；
(3)传代培养
接种约2周后传第1代，吸除培养液，PBS缓冲液漂洗，0.25％胰蛋白酶消化，按1:2的比例传代。目前已培养50余代；
(4)细胞纯化
细胞培养至第3代时开始分离成纤维细胞：胰酶消化后，将细胞悬液接种于培养皿中，静置约15～20分钟后于倒置相差显微镜下观察，见部分细胞贴壁，小心吸取细胞悬液入另一培养瓶中继续培养；按上述方法，重复2次传代，使肿瘤细胞与成纤维细胞分开；当细胞培养6-7代后可观察到肿瘤细胞皿中剩余成纤维细胞逐渐死亡；
制得一种原发性铂耐药的人卵巢癌细胞系，命名为FDOVL，分类命名为中国人卵巢癌细胞系，已于2018年3月7日由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏，地址：北京市朝阳区北辰西路1号院3号，保藏号为CGMCC NO.15484。
本实验体外常规培养条件为含12％FBS、1％双抗、1％丙酮酸钠溶液、1％HEPES 缓冲液、1％非必需氨基酸溶液的RPMI 1640细胞培养基，培养环境为37℃、5％CO2、饱和湿度。
实施例2生物学特性的鉴定
细胞形态：倒置相差显微镜下观察，FDOVL细胞单层贴壁生长，失去接触抑制，形态不一，呈圆形椭圆形或纺锤形扁平状，或不规则形(如图1所示)；
细胞生长曲线：取1×105个FDOVL细胞铺于6孔板中，设置3个复孔，分别在2天、4天、6天、8天、10天后同一时间使用500ul 0.25％Trypsin室温消化4～5min，收集细胞后计数，绘制细胞生长曲线(如图2所示)，计算细胞生长倍增时间为87小时；
染色体核型分析：取对数生长期细胞，用50ng/ml秋水仙素在37℃下处理2h；将细胞消化成单个细胞，1200rmp，5min离心收集，PBS重悬清洗一次；37℃预热低渗KCL，低渗KCL重悬细胞后转入15ml离心管中，加入低渗KCL补至10ml，37 ℃处理30min；加入固定液(甲醇:冰醋酸＝3:1)，1000rmp，10min离心，去上清；反复吹打悬液，缓慢加入固定液，直至离心管满，4℃静置过夜；次日弃上清，取出事先-20℃预冷载玻片，立即滴片；75℃，干燥1-2h；染片对玻片进行编号，再将玻片通过胰酶消化、生理盐水终止消化、吉姆萨染液(5ml吉姆萨+45ml PBS)染色，室温干燥；扫片、分析将玻片上载至徕卡扫片机扫片后再进行染色体核型分析。随机计数10个细胞，其中2n＝58-62条有9个(90％)，2n＝80条有1个 (10％)，染色体存在复杂的易位,错误拼接等染色体畸变，结果符合恶性肿瘤的遗传学特征(如图3所示)；
二代测序基因谱分析：按照Qiagen DNA提取试剂盒内实验步骤对病人左盆腔肿瘤组织、血及FDOVL DNA进行提取。经酶标仪检测DNA浓度，凝胶电泳检查DNA 完整程度。利用Covaris将gDNA随机打断成长度为180-280bp的片段，用sample purification beads进行纯化，产物的末端经过修补成平末端，接着用磁珠进行片段筛选，然后在3’末端加A。连接PCR反应接头，进行PCR扩增。然后与使用Sure Select capture library在杂交缓冲液中进行杂交，使用Dynal磁珠捕获杂交的目的片段并分离纯化。PCR扩增捕获的DNA片段并纯化PCR产物，然后进行捕获文库的质检，包括琼脂糖凝胶电泳质检、Qubit浓度测定和2100Bioanalyzer片段长度测定。将待测DNA文库浓度在cBot上完成cluster generation，然后将 Flowcell转移到测序系统上，按照Illumina的标准流程进行二代测序及CNV、SNV数据分析(肿瘤样本有效深度200X，血样本为100X)，驱动基因变异如图4所示。
实施例3细胞成瘤性
体外大规模扩增细胞，将第32代FDOVL细胞以1×107数量级皮下接种NOD/ SCID小鼠3只，14天后可观察到3只裸鼠全部移植瘤长出，继续生长10天后，小动物MRI成像显示移植瘤呈长椭圆形，外有包膜，边界清晰(如图5所示)。肿瘤继续生长至肿瘤体积为100mm3，绘制移植瘤生长曲线(如图6所示)。
实施例4肿瘤的病理学鉴定
待肿瘤体积增长至100mm3，处死小鼠，肿瘤组织经福尔马林固定，石蜡包埋切片进行HE染色及免疫组织化学染色，结果表明，移植瘤HE染色呈上皮性来源肿瘤，免疫组织化学染色跟原临床标本免疫组织化学染色结果基本一致，形成对应关系(如图7所示)。
Claims (8)
1.一种人卵巢癌细胞系，其特征在于,该细胞为原发性人卵巢癌细胞系并且铂耐药，该细胞由中国普通微生物菌种保藏管理中心(CGMCC)保藏，保藏号为NO.15484。
2.按权利要求1所述的人卵巢癌细胞系，其特征在于,该细胞单层贴壁生长，失去接触抑制，能够连续稳定传代50代以上。
3.按权利要求1或2所述的人卵巢癌细胞系，其特征在于,该细胞TP53框内缺失及BRCA1多位点突变。
4.按权利要求1所述的人卵巢癌细胞系，其特征在于,该细胞的生物学特征如下：
单层贴壁生长，失去接触抑制，形态不一，呈圆形椭圆形或纺锤形扁平状，或不规则形；并且
体外培养生长良好，每4天传代一次，能够连续稳定传代50余代；并且
染色体核型分析提示细胞染色体数目多为58-62条，存在复杂的易位,错误拼接的染色体畸变；并且
该细胞与左盆腔转移淋巴结肿瘤组织基因谱分析结果高度相似，同时检测到TP53框内缺失及BRCA1多位点突变。
5.按权利要求1所述的人卵巢癌细胞系，其特征在于,该细胞种植裸鼠，两周成瘤。
6.按权利要求5所述的人卵巢癌细胞系，其特征在于,其中，成瘤的移植瘤HE染色呈上皮性来源，免疫组织化学染色结果与原始肿瘤免疫组织化学结果一致。
7.权利要求1的人卵巢癌细胞系在用于建立所述卵巢癌发生发展、转移机制、新药筛选或者耐药机理的基础及临床前期研究平台中的用途。
8.权利要求1的人卵巢癌细胞系在用于建立铂耐药的癌动物模型中的用途。
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