CN106011067A - 一种食管癌细胞系及其应用 - Google Patents
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Abstract
本发明公开了一种食管癌细胞系及其应用,所述食管癌细胞系,命名为人食管鳞癌细胞系ZEC‑145,保藏号为:CCTCC NO:C201686。本发明人食管鳞癌细胞系ZEC‑145来自于一例中国的食管癌患者的手术切除后的肿瘤组织,STR检测结果证明其是唯一的,且在原代培养过程中未发生和其他细胞的交叉污染,克隆形成能力强,成瘤性差,可以作为食管癌研究及食管癌检测试剂盒开发、药物筛选等方面应用的理想细胞系。
Description
技术领域
本发明涉及生物学和肿瘤学技术领域,特别是涉及一种食管癌细胞系及其应用。
背景技术
食管癌是常见的消化道恶性肿瘤。全世界每年约有30万人死于食管癌。其发病率和死亡率各国差异很大。我国是世界上食管癌高发地区之一,每年平均病死约15万人。男多于女,发病年龄多在40岁以上。食管癌典型的症状为进行性咽下困难,先是难咽干的食物,继而是半流质食物,最后水和唾液也不能咽下。在我国,食管癌发病率居全身肿瘤第5位,其死亡率居第4位。食管癌的两种主要亚型:鳞癌和腺癌,其中鳞癌约占90%。
食管癌患者临床表现如下:
早期:症状常不明显,但在吞咽粗硬食物时可能有不同程度的不适感觉,包括咽下食物梗噎感,胸骨后烧灼样、针刺样或牵拉摩擦样疼痛。食物通过缓慢,并有停滞感或异物感。梗噎停滞感常通过吞咽水后缓解消失。症状时轻时重,进展缓慢。
中晚期:食管癌典型的症状为进行性咽下困难,先是难咽干的食物,继而是半流质食物,最后水和唾液也不能咽下。常吐黏液样痰,为下咽的唾液和食管的分泌物。患者逐渐消瘦、脱水、无力。持续胸痛或背痛表示为晚期症状,癌已侵犯食管外组织。当癌肿梗阻所引起的炎症水肿暂时消退,或部分癌肿脱落后,梗阻症状可暂时减轻,常误认为病情好转。若癌肿侵犯喉返神经,可出现声音嘶哑;若压迫颈交感神经节,可产生Horner综合征;若侵入气管、支气管,可形成食管、气管或支气管瘘,出现吞咽水或食物时剧烈呛咳,并发生呼吸系统感染。最后出现恶病质状态。若有肝、脑等脏器转移,可出现黄疸、腹腔积液、昏迷等状态。
治疗主要分外科治疗、放射治疗、化学治疗等,近年来,以手术为主的综合治疗已经达到了瓶颈,而食管癌化疗发展缓慢,尚无明确结论与标准方案。
为深入了解食管鳞癌的发病机理和药物及其他治疗方法的研究,必须建立合适的研究模型。目前国内外常用的食管癌细胞株主要来源于日本,包括KYSE系列和TE系列。这些细胞系长期在体外传代,一些已经丧失了原始肿瘤组织的特性。因为种族差异,生活习惯不同,环境因素等原因,这些细胞能否代表我国食管癌的类型和特点存在很大的疑问。国内少数实验室已建立的细胞株较少,部分广泛使用的细胞株受到其他细胞的污染,使得食管癌的研究不能深入开展。而且,随着肿瘤精准医学的治疗理念的深入临床实践,提供更多的食管癌细胞株有助于靶向药物的研发和应用。建立中国人来源的食管癌细胞株,为食管鳞癌的发病机制及治疗提供新的模型,具有重要的理论和实际应用价值。
发明内容
本发明针对目前国内缺乏中国人来源的食管癌细胞株,而提供了一种来源于中国人的食管癌细胞系。
本发明人食管鳞癌细胞系ZEC-145从一例食管癌患者(患者为66岁男性,肿瘤位于食管中段,术后病理显示中分化鳞状细胞癌,TNM分期T3N3M0,患者临床分期3C期。)的手术切除后的肿瘤组织取样,经原代培养并建系成功后,命名为人食管鳞癌细胞系ZEC-145,于2016年5月11日保藏于位于中国武汉武汉大学的中国典型培养物保藏中心,保藏号为:CCTCC NO:C201686。
本发明人食管鳞癌细胞系ZEC-145细胞生长旺盛,背景清晰,杂质少见,细胞呈扁平不规则多角形,细胞彼此紧密相连成单层膜,符合上皮样细胞的特点。贴壁后生长较快,具有良好的体外培养扩增性。染色体数目大多分布在50~71之间,众数为54,符合恶性肿瘤的特征。
本发明人食管鳞癌细胞系ZEC-145的STR测序结果与ATCC、DSMZ等细胞保存库的数据库进行查询对比,未发现相同STR检测结果,证明其是唯一的,且在原代培养过程中未发生和其他细胞的交叉污染。免疫组化实验发现,人食管鳞癌细胞系ZEC-145细胞呈CK5/6阳性,CK14阳性,p63阳性,CgA阴性,Sy阴性和CD56阴性。
克隆形成实验发现,人食管鳞癌细胞系ZEC-145细胞的克隆形成率与接种密度有关系,接种密度为4500个细胞时的细胞克隆形成能力较强。
裸鼠(裸小鼠)成瘤实验结果发现,人食管鳞癌细胞系ZEC-145成瘤性差。
本发明又提供了所述的食管癌细胞系的子代细胞。所述子代细胞基本或全部保留了亲代细胞的特性。
本发明又提供了所述的食管癌细胞系在作为食管癌发生机理研究的细胞模型中的应用。由于本发明人食管鳞癌细胞系ZEC-145是从中国人来源建立的,且建系时间较短,性状稳定,以该食管癌细胞系作为研究模型,对于了解中国人的原发性食管癌发生机理有很大帮助。
本发明还提供了所述的食管癌细胞系在建立哺乳动物食管癌模型中的应用。所述的哺乳动物为裸小鼠或裸大鼠。通过将一定细胞数量的所述人食管鳞癌细胞系ZEC-145细胞接种于裸小鼠或裸大鼠的皮下、肝脏、腹腔或者尾静脉等部位,获得食管癌的动物模型。
本发明还提供了所述的食管癌细胞系在提取食管癌特异性肿瘤标志物中的应用。通过与正常的食管细胞及其他种类的癌症细胞进行比较研究,可以发现食管癌的分子标记,针对该分子标记可以进行后续的疾病检测及药物开发等方面的应用。
本发明还提供了所述的食管癌细胞系在筛选或评估治疗食管癌药物中的应用。首先,通过向所述人食管鳞癌细胞系ZEC-145培养基中添加不同药物,观察细胞状态变化,获得初步有效的候选药物。然后,将候选药物施药于上述食管癌的动物模型,观察与未施药组动物的存活期、肿瘤大小、转移情况等,筛选获得潜在治疗食管癌的药物。
本发明还提供了所述的食管癌细胞系在开发食管癌检测试剂盒中的应用。发现食管癌特异性的肿瘤标志物后,可根据该肿瘤标志物开发出检测食管癌发生或发展情况的检测试剂盒。
本发明人食管鳞癌细胞系ZEC-145来自于一例中国的食管癌患者的手术切除后的肿瘤组织,STR检测结果证明其是唯一的,且在原代培养过程中未发生和其他细胞的交叉污染,克隆形成能力强,成瘤性差,可以作为食管癌研究及食管癌检测试剂盒开发、药物筛选等方面应用的理想细胞系。
附图说明
图1为人食管鳞癌细胞系ZEC-145细胞的形态检测图;
图2为人食管鳞癌细胞系ZEC-145的细胞生长曲线图;
图3为人食管鳞癌细胞系ZEC-145细胞的染色体核型分布图;
图4为STR分析结果图,其中,图A~F分别是不同等位基因的结果图;
图5为人食管鳞癌细胞系ZEC-145细胞免疫细胞化学分析结果图;
图6为与人食管鳞癌细胞系ZEC-145相同来源的肿瘤组织的免疫组织化学分析结果图;
图7为人食管鳞癌细胞系ZEC-145细胞克隆形成实验结果图,其中,图A、B和C接种细胞数分别为500、1500和4500个。
具体实施方式
实施例1
从一例食管癌患者(患者为66岁男性,肿瘤位于食管中段,术后病理显示中分化鳞状细胞癌,TNM分期T3N3M0,患者临床分期3C期。)的手术切除后的肿瘤组织取样。分离好的新鲜食管癌组织,在无菌超净台中用PBS洗净后,然后用眼科镊子、剪刀等器械在培养皿中剪碎分离成0.5~1mm3的小块平铺于皿底,并向培养皿中加入含10mL的10%FBS(GIBCO公司),1%双抗DMEM/F12细胞培养基(GIBCO公司),放入37℃、5%CO2的培养箱中进行培养。5~7天后换液,弃去组织块中坏死脱落的漂浮小块,进行传代培养。在传代培养过程中,利用成纤维细胞与肿瘤细胞消化能力的不同,不断去除成纤维细胞。多次传代后,培养皿中肉眼已无法观察到成纤维细胞,并能持续生长传代。建系成功后,命名为人食管鳞癌细胞系ZEC-145,于2016年5月11日保藏于位于中国武汉武汉大学的中国典型培养物保藏中心,保藏号为:CCTCC NO:C201686。
实施例2
取传代培养的人食管鳞癌细胞系ZEC-145细胞,在光学显微镜下(日本Olympus IMT-2倒置显微镜)观察活细胞生长情况。如图1所示,细胞光学形态图片,可见细胞生长旺盛,背景清晰,杂质少见,细胞呈扁平不规则多角形,细胞彼此紧密相连成单层膜,符合上皮样细胞的特点。
实施例3
细胞指数(Cell Index)是指活细胞与检测板孔中微电极相互作用,产生电阻抗的改变,xCELLigence细胞功能分析仪,将这些信号转化为特定的参数成为细胞指数。细胞指数很好地衡量了细胞的状态——生长、扩散、形状改变、死亡、应激等,细胞指数已经被多家杂志评论并采纳。
取生长状态良好的人食管鳞癌细胞系ZEC-145细胞,经胰酶消化,制成细胞悬液,并计数。xCELLigence细胞功能分析仪的E-Plate检测板中加入培养基并测定背景阻抗值,然后在E-Plate检测板中加入100μL细胞悬液(5000个),室温超净台内放置30min。将加入细胞的E-Plate检测板放入检测台上(检测台预先放入培养箱中),进行实时动态的细胞增殖检测即可获得细胞增殖曲线,持续计数4~6天。利用Graph Pad软件绘制生长曲线,并计算得到细胞的群体倍增时间约26小时。如图2所示,本发明人食管鳞癌细胞系ZEC-145贴壁后生长较快,具有良好的体外培养扩增性。
实施例4
取人食管鳞癌细胞系ZEC-145细胞,种植在六孔板中,培养48小时后,用0.01mg/mL的秋水仙素处理16h,当镜检观察到M期细胞比例为50%以上时,收集M期细胞。使用KCl低渗液低渗处理M期细胞15~20min,用甲醇/冰醋酸(体积比3∶1)作为固定液在室温下固定细胞,玻片上制片。将玻片置于0.02%胰酶溶液中消化30~60s,经PBS漂洗,Giemsa染色,晾干,完成制片。于显微镜下观察,计算每个细胞中染色体数目,随机选取20个细胞进行计算。如图3所示,染色体数目大多分布在50~71之间,众数为54,符合恶性肿瘤的特征。
实施例5
短串联重复序列(short tandem repeat,STR)又称为微卫星DNA。一般由一个长2~6bp的核心序列经多次串联重复排列而成,重复次数大多在10~60次之间。个体间核心序列的重复次数呈高度变异性,因而一组STR序列的重复次数在不同个体中几乎是唯一的,是细胞生物学对细胞身份和来源进行鉴定的主要方法。收集新鲜培养的人食管鳞癌细胞系ZEC-145细胞,用Qiagen基因组DNA小量提取试剂盒(购自Qiagen公司,货名QIAamp DNA Mini Kit,货号为51304)提取细胞基因组DNA,用5'端荧光标记的STR引物进行PCR扩增,对所得产物进行测序。其中STR位点的引物序列及拷贝数如表1和图4所示,其中标记D12S391和D8S1179出现三等位基因,可能是由于存在额外的染色体,因为肿瘤细胞的染色体数不正常;也有可能是部分STR区域的拷贝数较正常情况增加了所导致。上述序列和ATCC,DSMZ等细胞保存库的数据库进行查询对比,未发现相同STR检测结果,由此可以证明其是唯一的,且在原代培养过程中未发生和其他细胞的交叉污染。
表1 STR位点的拷贝数。
标记(Marker) | 等位基因1(Allele 1) | 等位基因2(Allele 2) | 等位基因3(Allele 3) |
TH01 | 7 | 9 | - |
D12S391 | 18 | 19 | 20 |
D7S820 | 12 | 12 | - |
CSF1PO | 12 | 12 | - |
FGA | 24 | 25 | - |
AMEL | X | Y | - |
D5S818 | 11 | 12 | - |
D2S1338 | 21 | 21 | - |
D21S11 | 32.2 | 32.2 | - |
D18S51 | 15 | 15 | - |
TPOX | 8 | 8 | - |
VWA | 14 | 18 | - |
D8S1179 | 13 | 14 | 15 |
D3S1358 | 16 | 16 | - |
D13S317 | 14 | 14 | - |
D6S1043 | 14 | 19 | - |
D16S539 | 9 | 10 | - |
PENTAE | 11 | 14 | - |
D19S433 | 14 | 14.2 | - |
PENTAD | 9 | 9 | - |
实施例6
免疫组化检测人食管鳞癌细胞系ZEC-145细胞标记蛋白。采用S-P法进行免疫组化染色,相关试剂盒均购自福州迈新生物技术开发有限公司。所用的抗体为CK5/6(货号MAB-0276,福州迈新公司),P63(货号ZM-04-06,北京中衫金桥公司),CK14(货号ZA-0540,北京中衫金桥公司),CgA(货号ZA-0507,北京中衫金桥公司),Sy(货号IR660,DAKO公司),CD56(I货号R628,DAKO公司)。
肿瘤组织免疫组化:组织(为实施例1中获取细胞的同一肿瘤样品)蜡块经常规脱蜡并水化,用3%H2O2去离子水浸泡10分钟,以阻断内源性过氧化物酶,然后放到0.01M pH6.0的拘橡酸缓冲液中,高压锅喷汽半分钟进行抗原修复。滴加一抗,室温下孵育60分钟,PBS缓冲液冲洗3次,每次2分钟。滴加Polymer Helper(聚合物辅助剂),室温下孵育20分钟,PBS缓冲液冲洗3次,每次2分钟。滴加Polyperoxidase-anti-mouse/rabbit IgG,室温下孵育30分钟,PBS缓冲液冲洗3次,每次2分钟。稀释配制DAB溶液,显色5~10分钟,镜下观察。清水冲洗,苏木素复染,常规脱水,透明、封片。
肿瘤细胞免疫组化:待检测细胞(人食管鳞癌细胞系ZEC-145细胞)提前24小时爬片于消毒载玻片上,24小时后,用PBS缓冲液冲洗2次,冷丙酮固定15分钟,浸泡于PBS中备用。取所需玻片加50μL过氧化物酶阻断溶液(试剂A)的室温下孵育10分钟,PBS缓冲液冲洗3次,每次3分钟,除去PBS液,滴加50μL正常非免疫动物血清(试剂B),室温下孵育10分钟;除去血清,滴加50μL一抗,室温下孵育60分钟,PBS冲洗三次,每次3~5分钟;滴加二抗,室温下孵育10分钟,PBS冲洗三次,每次3分钟。滴加Polyperoxidase-anti-mouse/rabbit IgG,室温下孵育30分钟,PBS缓冲液冲洗3次,每次2分钟。稀释配制DAB溶液,显色5~10分钟,镜下观察。清水冲洗,苏木素复染,常规脱水,透明、封片。
结果如图5和图6所示,人食管鳞癌细胞系ZEC-145细胞呈CK5/6阳性,CK14阳性,p63阳性,CgA阴性,Sy阴性和CD56阴性。细胞的和组织的免疫组织化学结果均说明人食管鳞癌细胞系ZEC-145是鳞癌来源的。
实施例7
取生长状态良好的人食管鳞癌细胞系ZEC-145细胞,经胰酶消化后制成细胞悬液,并计数。将细胞悬液以500,1500,4500个细胞每孔接种于6孔板中。将6孔板置于培养箱中,静置培养2周,每周换液2次。2周后,弃去细胞培养液,用PBS漂洗后,无水甲醇固定10min,然后用0.1%结晶紫染色10min,洗去染色液,室温干燥,并拍照记录。如图7所示,人食管鳞癌细胞系ZEC-145细胞的克隆形成率与接种密度有关系,接种密度为4500个细胞时的细胞克隆形成能力较强。
实施例8
取生长状态良好的人食管鳞癌细胞系ZEC-145细胞,经胰酶消化后制成细胞悬液,并计数。将细胞悬液以1000万个细胞分别接种到5只裸鼠(裸小鼠)腋下,连续观察。结果,8周后未成瘤。
Claims (8)
1.食管癌细胞系,其特征在于,命名为人食管鳞癌细胞系ZEC-145,保藏号为:CCTCCNO:C201686。
2.如权利要求1所述的食管癌细胞系的子代细胞。
3.如权利要求1所述的食管癌细胞系在作为食管癌发生机理研究的细胞模型中的应用。
4.如权利要求1所述的食管癌细胞系在建立哺乳动物食管癌模型中的应用。
5.如权利要求4所述的应用,其特征在于,所述的哺乳动物为裸小鼠或裸大鼠。
6.如权利要求1所述的食管癌细胞系在提取食管癌特异性肿瘤标志物中的应用。
7.如权利要求1所述的食管癌细胞系在筛选或评估治疗食管癌药物中的应用。
8.如权利要求1所述的食管癌细胞系在开发食管癌检测试剂盒中的应用。
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