CN113234678B - 一种对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞株及其建立方法和应用 - Google Patents
一种对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞株及其建立方法和应用 Download PDFInfo
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Abstract
本发明提供了一种对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞株及其制备方法和应用。所述人小细胞肺癌细胞株保藏在中国典型培养物保藏中心,保藏编号为CCTCC No:C202168。本发明的小细胞肺癌细胞株性状稳定,可稳定多次传代,可用于在哺乳动物中产生人小细胞肺癌,制备人小细胞肺癌模型,并且该细胞对小细胞肺癌一线治疗药物依托泊苷与卡铂联合用药耐受,可用于小细胞肺癌耐药分子机制研究,和筛选治疗耐药型人小细胞肺癌的候选药物,为小细胞肺癌研究提供新的更接近于临床肿瘤生物学特性的实验材料。
Description
技术领域
本发明属于生物技术领域,特别涉及一种具有依托泊苷与卡铂联合耐药性的人小细胞肺癌细胞株及其建立方法和应用。
背景技术
小细胞肺癌是肺癌的一种特殊病理类型,属于恶性度极高的神经内分泌肿瘤,占肺癌发病率的10%~15%。其特点是恶性程度高,生长迅速,倍增时间短,侵袭性强,极易发生转移,约60%的患者就诊时已发生远处转移,其中约30%左右的患者会发生骨转移,区域淋巴结和远处转移早期即可出现,中位总生存期只有8个月-13个月,而且预后差,五年生存率小于7%,大多数患者于诊断后1年内死亡。
在小细胞肺癌治疗当中,依托泊苷联合铂类制剂的化疗方案是目前小细胞肺癌化疗的一线治疗方案。依托泊苷能够通过与DNA拓扑异构酶II形成可逆性药-酶-DNA复合物,从而起到修复受损DNA,降低细胞毒作用,延长给药时间,提高药物抗肿瘤时间的作用,在小细胞肺癌的治疗中具有重要意义。卡铂为第二代铂类抗肿瘤药物,与顺铂相比其肝肾毒性和神经毒性明显降低。初次患者对化疗敏感,一线化疗缓解率高,中位生存期为9.4~12.8个月,2年总存活率为5.2%~19.5%,但大部分小细胞肺癌患者在化疗停止后3个月左右出现复发转移,其高耐药率和复发率仍是小细胞肺癌化疗中存在的最大问题,这使得许多接受依托泊苷联合铂类治疗的患者在遭受了化疗毒副作用的同时,却没有得到较好的治疗效果。但到目前为止卡铂耐药的确切分子机制还不明确。因此,建立对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞株对于研究小细胞肺癌耐药和筛选治疗耐药型人小细胞肺癌的候选药物具有必不可少的重要作用。
发明内容
本发明要解决的技术问题就是为小细胞肺癌耐药分子机制研究,和筛选治疗耐药型人小细胞肺癌的候选药物,提供一种具有对依托泊苷与卡铂联合耐药性的人小细胞肺癌细胞株及其建立方法和应用。
一种对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞,保藏编号为CCTCC No:C202168。
本发明还提供如上所述的人小细胞肺癌细胞的子代细胞。
所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的应用,用于小细胞肺癌依托泊苷与卡铂联合耐药机制研究提供实验材料。
所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的应用,所述的实验材料包括:小细胞肺癌依托泊苷与卡铂联合耐药细胞株和/或小细胞肺癌依托泊苷与卡铂联合耐药动物模型。
所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的应用,所述的动物模型是裸鼠。
所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的应用,用于构建体内或体外药物筛选平台,筛选治疗依托泊苷与卡铂联合耐药型人小细胞肺癌药物。
所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的应用,包括以下步骤:
(1)将所述的对依托泊苷与卡铂联合耐药的小细胞肺癌细胞的子代细胞制备成细胞悬液,接种于哺乳动物皮下,进行饲养,获得耐药型人小细胞肺癌动物模型;
(2)将测试药物施用于耐药型动物模型,施用后导致小细胞肺癌症状改善或治愈的测试药物即为治疗小细胞肺癌的候选药物。
在步骤中,将测试药物通过尾静脉注射、口服、腹腔注射或于肿瘤局部用药等方式施用于小细胞肺癌荷瘤动物。较佳的使用对照实验,一种优选方式是:同时还使用不含测试药物的溶剂施用于小细胞肺癌荷瘤动物作为对照。
所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的应用,所述的哺乳动物为裸小鼠;所述的裸小鼠为BALB/c裸小鼠;优选采用细胞悬液注射来建立动物模型。
所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的建立方法,包括以下步骤:
(1)将新鲜的对依托泊苷与卡铂联合耐药的小细胞肺癌肿瘤组织接种移植到免疫缺陷小鼠前肢或后肢背侧皮下,对于荷瘤小鼠,每周至少观察一次,将达到一定体积的肿瘤(400-1000mm3)及时传代并冻存;
(2)传代之后,选择肿瘤体积达到一定体积的小鼠,剥取瘤块进行单细胞分离,去除结缔组织和坏死组织,然后将肿瘤样本剪切成小块;
(3)将剪碎的组织转移到消化液中,孵育、过滤、离心;
(4)用培养基重悬细胞、培养;
(5)当细胞密度达到一定程度时,吸弃培养基,消化细胞并接种于新的培养瓶中,进行细胞传代。
所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的建立方法,
上述步骤(1)将肿瘤组织使用肿瘤接种针移植到免疫缺陷小鼠前肢或后肢背侧皮下,每块组织约30-50mm3;
上述步骤(2)传代4次-5次之后,选择肿瘤体积达到500-800mm3的小鼠,剥取瘤块进行单细胞分离,将肿瘤样本剪切成1-2mm3小块;
上述步骤(3)将剪碎的组织转移到消化液中,在37℃水浴中孵育,将孵育好的混合物用滤膜过滤,将滤液收集,离心,去掉上清;
上述步骤(4)当细胞密度达到80-90%时,吸弃培养基,消化细胞并接种于新的培养瓶中,进行细胞传代,传代至50代以上。
所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的建立方法,进一步具体包括以下步骤:
1)将新鲜的对依托泊苷与卡铂联合耐药的小细胞肺癌肿瘤组织,在无菌条件下放入无菌,4℃预冷的组织保护液中,转移到预灭菌的生物安全柜中,从离心管中取出组织,快速转移至10cm的培养皿中,并用含100U/mL青霉素和100μg/mL链霉素双抗的PBS清洗2遍,剔除坏死组织以及周围非肿瘤组织;
2)将肿瘤组织使用肿瘤接种针移植到免疫缺陷小鼠前肢或后肢背侧皮下,每块组织约30-50mm3,每只小鼠接种1~4个点;对于荷瘤小鼠,每周至少观察一次,将达到一定体积的肿瘤及时传代并冻存;
3)传代4次-5次之后,选择肿瘤体积达到500-800mm3的小鼠,安乐死并剥取瘤块进行单细胞分离,用含双抗的PBS清洗肿瘤组织,去除结缔组织和坏死组织,然后将组织转移至含有10mL不含胎牛血清的RPMI 1640培养基中,用无菌手术剪将肿瘤样本剪切成1-2mm3小块;
4)将剪碎的组织转移到15mL accumax消化液中,在37℃水浴中孵育1小时,将孵育好的混合物用70μm的滤膜过滤,将滤液收集在50mL离心管中,用30mL含10%胎牛血清的RPMI 1640培养基冲洗滤膜,合并滤液,将滤液用1300rpm离心5分钟,去掉上清;
5)用5mL含10%胎牛血清的RPMI 1640培养基重悬细胞,并转移到25mm3的培养皿中,分离出的肿瘤细胞在37℃培养箱中培养;5%CO2条件下培养;
6)当细胞密度达到80-90%时,吸弃培养基,0.5%胰酶消化细胞并接种于新的培养瓶中,进行细胞传代,传代至50代以上。
上述建立方法中采用新鲜的临床小细胞肺癌手术切除标本,优选的用哺乳动物细胞培养液或生理盐水漂洗后再进行接种。优选的用含100U/mL青霉素和100μg/mL链霉素双抗漂洗后,剔除坏死组织和非肿瘤组织,再进行皮下穿刺接种。
所述的接种方式可以是皮下穿刺接种,原位接种或者肾囊膜内接种。对于小细胞肺癌较佳的是进行皮下穿刺接种。
所述的原代培养方法可以是常规的哺乳动物细胞的原代培养方法。较佳的包括以下步骤:将肿瘤组织剪切成小块,将剪碎的组织转移到15mL accumax消化液中,在37℃水浴中孵育1小时。将孵育好的混合物用70μm的滤膜过滤,滤液收集于50mL离心管中,用30mL含10%胎牛血清的RPMI 1640培养液冲洗滤膜,合并滤液,将滤液用1300rpm离心5分钟,去掉上清。加入30mL含10%胎牛血清的RPMI 1640培养基重悬细胞。然后1300rpm离心5分钟,去掉上清。用5mL含10%胎牛血清的RPMI 1640培养基重悬细胞,并转移到25mm3的培养皿中。分离出的肿瘤细胞于37℃孵箱,5%CO2条件下培养。
所述的传代培养方法可以是常规的哺乳动物细胞的传代培养方法。较佳的包括以下步骤:吸弃旧培养液,向瓶内加入新鲜的0.05%胰蛋白酶溶液,待细胞脱落后,加入新鲜的RPMI 1640培养液,仔细吹打,使之脱离瓶壁形成细胞悬液:收集全部细胞,离心,分别接种于新的培养瓶。
本发明所用试剂和原料均市售可得。
相比于现有技术,本发明的有益效果如下:
本发明成功建立对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞株,细胞性状稳定,可稳定多次传代,丰富了小细胞肺癌细胞库;为小细胞肺癌对依托泊苷与卡铂联合耐药的机制研究提供一种新的实验材料,更能反应真实的耐药机制;利用本发明的细胞株可以成功制备对依托泊苷与卡铂联合耐药的人小细胞肺癌动物模型,用于基础研究及药物筛选,应用范围较广。
生物材料保藏信息
本发明的人小细胞肺癌细胞,于2021年4月28日保藏在中国典型培养物保藏中心(CCTCC)(武汉,中国),培养物名称为耐依托泊苷与卡铂的人小细胞肺癌细胞株SCLC2018,保藏编号为:CCTCC No:C202168。
附图说明
图1是本发明保藏的耐依托泊苷与卡铂的人小细胞肺癌细胞SCLC2018的形态学观察(10X);
图2是体外测试耐依托泊苷与卡铂的人小细胞肺癌细胞SCLC2018对依托泊苷和卡铂的反应性;
图3是耐依托泊苷与卡铂的人小细胞肺癌细胞SCLC2018倍增曲线。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1:人小细胞肺癌细胞SCLC2018的制备
NOD SCID小鼠,雌性,体重18-22g,鼠龄6-8周,饲养于SPF环境。小鼠由北京维通利华实验动物有限公司提供。
(1)从湖南肿瘤医院获得新鲜的临床小细胞肺癌手术切除样本(男,50岁,小细胞肺癌肿瘤,临床分期IV期,对依托泊苷与卡铂联合耐药,符合伦理学,以及经过患者同意),立即在无菌条件下将肿瘤组织放入无菌,4℃预冷的组织保护液中,转移到预灭菌的生物安全柜中,从离心管中取出组织,快速转移至10cm的培养皿中,并用含双抗(100U/mL青霉素和100μg/mL链霉素)的PBS清洗2遍,剔除坏死组织以及周围非肿瘤组织,尽可能确保坏死部分不被用于接种;
(2)将肿瘤组织使用肿瘤接种针移植到免疫缺陷小鼠前肢或后肢背侧皮下,每块组织约30-50mm3,根据病人肿瘤组织的总体大小决定接种数量,每只小鼠接种1~4个点;对于荷瘤小鼠,每周至少观察一次,将达到(400-1000mm3)及时传代并冻存;
(3)传代4次-5次之后,选择肿瘤体积达到500-800mm3的小鼠,安乐死并剥取瘤块进行单细胞分离,用含双抗的PBS清洗肿瘤组织,去除结缔组织和坏死组织,然后将组织转移至含有10mL不含胎牛血清的RPMI 1640培养基中,用无菌手术剪将肿瘤样本剪切成小块(1-2mm3大小);
(4)将剪碎的组织转移到15mL accumax消化液中,在37℃水浴中孵育1小时,将孵育好的混合物用70μm的滤膜过滤,将滤液收集在50mL离心管中,用30mL含10%胎牛血清的RPMI 1640培养基冲洗滤膜,合并滤液,将滤液用1300rpm离心5分钟,去掉上清;
(5)用5mL含10%胎牛血清的RPMI 1640培养基重悬细胞,并转移到25mm3的培养皿中,分离出的肿瘤细胞在37℃培养箱中,5%CO2条件下培养;
(6)当细胞密度达到80-90%时,吸弃培养基,0.5%胰酶消化细胞并接种于新的培养瓶中,进行细胞传代,传代至50代以上,细胞生长良好,形态较为均一。
在本发明中,来源于肿瘤组织的原代培养及传代培养细胞呈不规则多边形,细胞形态较为均一,该细胞株命名为SCLC2018,保藏编号为CCTCC No:C202168。
实施例2细胞的生物学特性
本发明使用RPMI 1640培养基培养SCLC2018细胞,使其能体外长期生长和稳定传代。当细胞传至30代以上,细胞性状逐渐稳定,进行相关的生物学、遗传学和组织来源鉴定,直至第50代都具有相同的稳定的性状。经实验观察和验证,体外生长的SCLC2018细胞具有典型的上皮样形态,失去接触生长抑制,呈恶性生长,该细胞能在裸小鼠体内形成肿瘤,具有致瘤性,该细胞株可以为研究体外和体内抗癌药物敏感性及耐药性,为人小细胞肺癌的发生、发展和转移提供新的试验材料。
形态学观察
将培养的SCLC2018细胞的培养瓶置于倒置显微镜下,在明视野下进行拍照,结果见图1(10X),可见SCLC2018细胞失去了接触抑制,呈恶性生长,具有上皮样细胞特点。
体外对依托泊苷和卡铂的反应性
体外测定该小细胞肺癌细胞SCLC2018对依托泊苷和卡铂的敏感性,取对数生长期的细胞用于铺板,调整细胞浓度,在培养板中每孔加入90μL细胞悬液,在空白对照孔中加入不含细胞的培养液;将培养板在37℃,5%CO2,及100%相对湿度的培养箱中培养过夜;分别取10μL的不同浓度的依托泊苷和卡铂工作液加入到上述的细胞培养板中,使依托泊苷终浓度依次为100、20、4、0.8、0.16、0.032、0.0064、0.00128和0.000256μM,卡铂的终浓度依次为100、20、4、0.8、0.16、0.032、0.0064、0.00128和0.000256μM,每组三个复孔,在溶媒对照(含有细胞和细胞培养液,不添加ALK抑制剂)和空白对照(含有细胞培养液,不含细胞,不添加ALK抑制剂)中加入10μL DMSO-细胞培养液混合液,DMSO终浓度为0.25%,将96孔细胞板放回培养箱中培养72h。然后每孔加入50μL(等于每孔中细胞培养液一半体积)PromegaCellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7573)的CellTiter-Glo工作液,用铝箔纸包裹细胞板以避光;将培养板在轨道摇床上振摇2分钟以诱导细胞裂解,培养板在室温放置10分钟以稳定发光信号,在2104EnVision读板器上检测发光信号;计算检测化合物的抑制率(Inhibition rate,IR):IR(%)=(1–(RLU化合物–RLU空白对照)/(RLU溶媒对照–RLU空白对照)*100%。在Excel中计算不同浓度化合物的抑制率,然后用GraphPad Prism软件作抑制曲线图和计算相关参数IC50,结果如表1和图2所示;在体外研究中,依托泊苷抑制小细胞肺癌细胞SCLC2018,IC50为4.79μM,卡铂抑制小细胞肺癌细胞SCLC2018,其IC50高于100μM。
表1.依托泊苷和卡铂对细胞的半数抑制浓度
细胞动力学
将SCLC2018细胞以3000/孔和6000/孔的密度接种在96孔板中,进行培养,分别在6小时,24小时,48小时,72小时,96小时,120小时,144小时,168小时,192小时,216小时和240小时使用CellTiter Glo试剂盒测定每孔中的活细胞数(见图3)。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
STR鉴定
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的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。收集新鲜培养的人小细胞肺癌细胞SCLC2018细胞,提取细胞的基因组的DNA,用5’端标记的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下表所示,上述序列和ATCC、DSMZ等细胞库的数据库进行比对,未发现相同STR检测结果,由此可证明其是唯一的,且在原代培养过程中未发生和其他细胞的交叉污染。
表2.STR位点拷贝数
Claims (4)
1.一种对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞,其特征在于,保藏编号为CCTCC No:C202168。
2.权利要求1所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的应用,其特征在于,用于小细胞肺癌依托泊苷与卡铂联合耐药机制研究提供实验材料。
3.根据权利要求2所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的应用,其特征在于,所述的实验材料包括:小细胞肺癌依托泊苷与卡铂联合耐药细胞株和/或小细胞肺癌依托泊苷与卡铂联合耐药动物模型。
4.根据权利要求3所述的对依托泊苷与卡铂联合耐药的人小细胞肺癌细胞的应用,其特征在于,所述的动物模型是裸鼠。
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