CN113322237B - 一种vav2基因缺失的肿瘤细胞系 - Google Patents
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Abstract
本发明提供了一种VAV2基因缺失的肿瘤细胞系及其在筛选和/或评价/制备肿瘤治疗药物,开发肿瘤药物靶点,制备肿瘤诊断试剂,开发肿瘤治疗新技术,开发检测肿瘤相关生物工程产品中的应用。所述VAV2基因缺失是通过Crispr/cas9敲除技术实现的,本发明的细胞系具有良好的细胞特性,并且其特性优于市售的细胞系。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种VAV2基因缺失的肿瘤细胞系及其构建方法。
背景技术
食管癌目前尚缺乏细胞模型,尤其是小鼠来源的肿瘤细胞系,本发明构建了一株小鼠来源的食管癌细胞系并命名为NCCE2,弥补该领域空白。
VAV2是一个重要的癌基因,目前有多项研究表明其影响肿瘤的增殖迁移等恶性表型,靶向VAV2可能是将来肿瘤治疗领域的一个重要方向。我们通过Crispr技术获得了VAV2基因缺失的小鼠,并且通过诱导成瘤而后继续培养癌组织得到了稳定表达的细胞系,该细胞系具有良好的细胞特性,并且其特性优于市售的细胞系。
CRISPR(Clustered regularly interspaced short palindromic repeats)是在细菌中发现的适应性免疫反应系统(Adaptive immune system),能有效抵抗噬菌体(Bacteriophage)等对细菌造成的损伤。在这套系统的基础上,科学家发展出一种新的CRISPR/Cas基因组编辑技术。现在所广泛使用的CRISPR/cas9系统是由II型CRISPR改造而来,并应用到分子生物学的相关研究中,成为一种可用于基因组编辑的分子生物学利器。CRISPR基因组编辑系统通过sgRNA上携带的靶点序列识别特定DNA序列,这种方式决定了CRISPR系统具有很强的特异性,由于其没有物种的限制,已经成功在多种动物和植物中实现了基因的精确编辑。
发明内容
细胞系
本发明提供了一种食管癌细胞系,所述细胞系命名为VAV2基因缺失小鼠食管癌细胞系NCCE2,于2021年3月3日由中国典型培养物保藏中心保藏,保藏号为CCTCC NO:C202140。
生物材料样品的保藏信息:
保藏单位:中国典型培养物保藏中心(CCTCC);
地址:中国武汉,武汉大学;
保藏日期:2021年3月3日;
保藏编号:CCTCC NO:C202140;
分类命名:VAV2基因缺失小鼠食管癌细胞系NCCE2。
方法
本发明提供了一种构建癌细胞系的方法,所述方法包括如下步骤:
1)构建包含VAV2完整或部分基因的载体;
2)使用载体敲除模型生物的VAV2基因,获得基因敲除的模型生物;
3)诱导基因敲除的模型生物成瘤;
4)继续培养肿瘤组织,获得癌细胞系。
优选地,所述癌细胞系是食管癌细胞系。
优选地,所述癌细胞系是前述保藏的NCCE2细胞系
优选地,所述VAV2完整或部分基因是gRNA,所述gRNA有引导基因编辑蛋白到特定位点进行基因编辑的功能;所述编辑包括敲除(KO)、插入、点突变。
优选地,所述载体上有Cas9的核酸序列。
优选地,所述载体上还有其他的调节元件和/或功能组分。
优选地,所述模型生物是可以是体内含有Cas9的mRNA或蛋白的模型生物。
优选地,所述调节元件包含启动子、增强子、翻译起始的核糖体结合位点、终止子、多聚腺苷酸序列。
优选地,所述基因敲除的模型生物指经过了基因敲除操作的模型生物,其体内的VAV2基因包括野生型VAV2基因和/或突变型VAV2基因。
优选地,所示野生型VAV2基因较突变型VAV2基因缺失的序列如SEQ ID NO:1所示。
优选地,所述基因敲除的模型生物可以是VAV2杂合的或VAV2纯合的。
优选地,所述模型生物包括哺乳动物或非哺乳动物。
优选地,所述模型生物是哺乳动物。
优选地,所述模型生物是小型哺乳动物。
优选地,所述模型生物是啮齿动物。
优选地,所述啮齿动物包括丽仓鼠科(例如小鼠样仓鼠)、仓鼠科(例如仓鼠、新世界大鼠和小鼠、田鼠)、鼠总科(真小鼠和大鼠、沙鼠、刺毛鼠、冠毛大鼠)、马岛鼠科(登山小鼠、岩小鼠、有尾大鼠、马达加斯加大鼠和小鼠)、刺睡鼠科(例如多刺睡鼠)和鼹形鼠科(例如摩尔大鼠、竹大鼠和鼢鼠)动物。
优选地,所述模型生物是小鼠。
优选地,所述模型生物包括BALB/c、A、A/He、A/J、A/WySN、AKR、AKR/A、AKR/J、AKR/N、TA1、TA2、RF、SWR、C3H、C57BR、SJL、C57L、DBA/2、KM、NIH、ICR、CFW、FACA、C57BL/A、C57BL/An、C57BL/GrFa、C57BL/KaLwN、C57BL/6、C57BL/6J、C57BL/6ByJ、C57BL/6NJ、C57BL/10、C57BL/10ScSn、C57BL/10Cr和C57BL/Ola的C57BL、C58、CBA/Br、CBA/Ca、CBA/J、CBA/st、CBA/H品系的小鼠。
优选地,所述模型生物是C57BL/6品系的小鼠。
优选地,所述诱导包括使用致癌剂对模型生物进行处理。
优选地,所述处理包括饲喂、注射。
优选地,所述处理是饲喂致癌剂。
优选地,所述致癌剂包括4NQO、N-甲基-N'-硝基-亚硝基胍、多环碳氢化合物、氨基偶氮化合物、芳香胺类、亚硝胺类霉菌毒素、部分金属元素。
优选地,所述致癌剂是4NQO。
优选地,所述肿瘤组织可以是模型生物身体任意部位的新生肿瘤。
优选地,所述肿瘤组织是食管处的新生肿瘤。
优选地,所述癌细胞系是经过实验验证具有高品质的细胞系。
优选地,所述实验验证包括增殖能力检测、克隆能力检测、细胞迁移能力检测。
优选地,所述高品质包括增殖能力强、克隆能力强、细胞迁移能力强。
应用
本发明提供了前述基因敲除的模型生物在制备VAV2缺失的细胞系中的应用。
本发明提供了前述gRNA或其所在的载体在敲除VAV2或构建VAV2敲除的细胞系中的应用。
本发明提供了前述细胞系的用途,其特征在于,所述用途包括以下任意一种:筛选和/或评价/制备肿瘤治疗药物,开发肿瘤药物靶点,制备肿瘤诊断试剂,开发肿瘤治疗新技术,开发检测肿瘤相关生物工程产品。
附图说明
图1为小鼠的基因鉴定电泳图。
图2为4NQO诱导的小鼠食管癌的实物图。
图3为NCCE2的镜下形态图。
图4为NCCE2与市售细胞系的增殖能力比较图。
图5为NCCE2的克隆形成实验的结果图。
图6为Transwell实验的显微镜下细胞形态图。
具体实施方式
下面结合实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化,均落在本发明的保护范围内。
实施例1、构建VAV2基因敲除的转基因小鼠
方案:使用利用Cas9/RNA system gene targeting技术,构建针对mouse VAV2基因的gRNA,指导Cas9蛋白在crRNA引导序列靶标的特定位点剪切DNA双链;双链断裂缺口通过NHEJ(non-homologous end joining)机制被修复,在断口造成不同长度序列的删除或插入,从而通过移码实现基因的敲除。
具体流程:
1)sgRNA设计及构建:设计sgRNA识别序列,构建sgRNA;
2)体外活性测试:体外测试sgRNA切割效率;
3)原核注射及移植:小鼠(C57BL/6小鼠)的超排;注射受精卵、移植;得到F0代小鼠;
4)F0代小鼠鉴定及繁育:通过PCR和测序验证阳性小鼠;将阳性F0代小鼠和C57BL/6N回交;
5)F1代小鼠鉴定:通过PCR和测序验证阳性小鼠。
表1.阳性F1代小鼠的基因型
ID | Gender | color | Gty | DOB | Gen | F/M |
25 | ♂ | B | -222bp/wt | 2015-8-31 | F1 | 15# |
27 | ♂ | B | -222bp/wt | 2015-8-31 | F1 | 15# |
对后代小鼠进行PCR验证,基因鉴定如图1所述,24#-27#是不同小鼠后代的PCR结果,B6为阴性对照,是B6基因组DNA;N为空白对照,无模板的对照。
25#和27#的基因型统计如表1所示,经PCR鉴定其缺失了ACGGTGTGCTTAGTTAACAGTTTCCGTGCTTGCAGAACCCTTGCTGTGGTTTGTGATGACTTCCTGATCATCATCTCTTACAGAACTCTCTCTTGTCTCCCCTAGGTCATCTCTGCAGTGTCCCGTCTGTCCCTGCACAGCATCGCACAGAGCAAAGGGATCAGGTGAGCTTTCGAGTGACAGTGACAGGTCACAGAGACACCCCTGCCTATGGGCCTGAAC(SEQ ID NO:1,222bp),Exon3全部删除,产生移码突变,选取25#、27#小鼠用于下一步实验。
实施例2、饲喂致癌剂4NQO,诱导小鼠产生食管癌
将1g 4NQO(4-nitroquinoline-N-oxide,购自SIGMA-ALDRCH,N8141-5G)和200ml1,2丙二醇配成浓度0.5g/100ml母液,震荡混匀,使用时1:50稀释,喂给6-8周VAV2缺失转基因小鼠水中,连用16周后停药,继续养小鼠12周后解剖小鼠食管查看成瘤情况,实物图如图2所示。
实施例3、细胞系构建成功并命名为NCCE2
1)将小鼠二氧化碳处死后立即浸泡入70%的酒精消毒液中全身消毒。
2)将小鼠的食管解剖下来,转移到无菌细胞操作台,用2%青霉素-链霉素混合液的PBS冲洗,剖开食管,暴露内壁,将食管壁内的新生肿物取出(食管癌)。
3)将食管癌组织块放入5ml离心管中,用小剪刀剪成1-3块。
4)将小块肿瘤组织贴于6孔板,倒置放入细胞培养箱30分钟,使组织贴壁。
5)轻轻加入培养基,注意避免组织飘起。
6)继续培养,避免触碰6孔板,72小时后可见肿瘤细胞沿组织爬出。
7)继续扩大培养细胞,两周后传代细胞继续扩大培养。
8)挑选单克隆,继续稳定培养细胞数月,使细胞表型稳定。将NCCE2的镜下观察,形态如图3所示。
9)NCCE2细胞系和对照NCCG1(野生型的小鼠肿瘤细胞),提取两株细胞系蛋白,使用Western–blot检测细胞的VAV2表达水平,GAPDH为内参,实验结果如图4所示,说明VAV2敲除成功。
实施例4、NCCE2的生物学特点检测
1、CCK8增殖能力检测
本研究细胞增殖能力检测使用CCK8试剂盒(Cell Counting Kit-8,CCK-8),CCK-8试剂是一种高度灵敏的可以间接反映活细胞数量的试剂。具体操作步骤按照说明书进行:
(1)将细胞胰酶消化,细胞计数2-4次,按照每孔相同的数量铺于96孔板,每组设置12个复孔,继续培养细胞。
(2)将CCK8与无血清培养基按照1:10混合,按照每孔总量100μl配置混合培养基,分别检测0、24、48、72、96、120小时的细胞数。
(3)将加入CCK8的96孔板放入细胞培养箱继续孵育90分钟,可见培养板成肉眼可见的黄色。
(4)上机前轻轻拍打混匀,酶标仪检测OD450波段信号值,绘制不同细胞增殖曲线。
将NCCE2细胞系和现有的食管癌细胞系CCK8增殖能力做比较,发现NCCE2具有较好的生长能力,高于目前市面上的细胞系,对比结果如图4所述。
2、克隆形成实验检测
(1)将待检测细胞胰酶消化,吹打混匀,使细胞处于单细胞状态,细胞计数。
(2)使用6孔板,每孔加入2ml完全培养基,将细胞按照每孔相同的数量加入,细胞培养箱继续培养。
(3)18-24小时后,根据实验具体内容给予不同的处理,继续培养细胞10-14天。
(4)细胞培养期间,每3天换液一次,注意观察克隆大小,待形成肉眼可见的白色克隆时即可停止培养。
(5)弃掉培养基,细胞清洗几遍去掉细胞残渣等,使用福尔马林固定15-25分钟,0.5%的结晶紫染色15-20分钟,自来水冲洗干净,通风厨晾干水分。
(6)拍照如图5所示,统计分析。六孔板上面三个是每孔铺设500个细胞,下面三个是每孔铺设250个细胞,说明NCCE2具有平板克隆形成能力。
以上实验证明,NCCE2具有较好的克隆形成能力,反应其可以作为较好的细胞模型用于研究。
3、Transwell实验检测细胞迁移能力
本研究使用Transwell实验检测肿瘤细胞的体外迁移能力,该实验以Transwell小室为主要实验材料。Transwell实验目前已经广泛应用于细胞侵袭、迁移、趋化性实验等。
本研究体外迁移实验的具体操作如下:
(1)将Transwell小室取出,放入24孔板中,每个处理组至少使用3个小室作为平行实验。
(2)将小室上层加入无血清培养基150μl,下室放入含血清的完全培养基500μl。
(3)胰酶消化细胞,收集细胞后PBS清洗3遍去除血清等成分。
(4)细胞计数,每个小室按照相同细胞数量接种细胞于上室,继续培养18-24小时,此时肿瘤细胞会营养趋化迁移至下室膜上。
(5)将小室取出,PBS清洗3遍去掉细胞残渣等,使用福尔马林固定15分钟,0.5%的结晶紫染色15分钟,使用棉棒将上室膜表面的肿瘤细胞擦净,自来水冲洗,通风厨晾干水分。
(6)使用显微镜观察并拍照(如图6所示),软件计算膜表面贴壁的肿瘤细胞,统计分析。
以上实验证明,NCCE2具有较强的迁移能力,侵袭能力是反应肿瘤恶性表型的重要特点。
序列表
<110> 中国医学科学院肿瘤医院
<120> 一种VAV2基因缺失的肿瘤细胞系
<141> 2021-06-16
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 222
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
acggtgtgct tagttaacag tttccgtgct tgcagaaccc ttgctgtggt ttgtgatgac 60
ttcctgatca tcatctctta cagaactctc tcttgtctcc cctaggtcat ctctgcagtg 120
tcccgtctgt ccctgcacag catcgcacag agcaaaggga tcaggtgagc tttcgagtga 180
cagtgacagg tcacagagac acccctgcct atgggcctga ac 222
Claims (11)
1.一种食管癌细胞系,所述食管癌细胞系命名为VAV2基因缺失小鼠食管癌细胞系NCCE2,于2021年3月3日由中国典型培养物保藏中心保藏,保藏号为CCTCC NO:C202140。
2.一种构建VAV2基因缺失的食管癌细胞系的方法,所述方法包括如下步骤:
1)利用Cas9/RNA system gene targeting技术,针对mouse VAV2基因设计gRNA,构建包含所述gRNA的载体;
2)使用1)中构建的载体敲除小鼠的VAV2基因,获得VAV2基因中缺失如SEQ ID NO:1所示序列的小鼠;
3)诱导2)获得的小鼠成瘤;
4)取3)的肿瘤组织,继续培养,获得食管癌细胞系。
3.如权利要求2所述的方法,其特征在于,所述小鼠是C57BL/6小鼠。
4.如权利要求2所述的方法,其特征在于,所述诱导包括使用致癌剂对小鼠进行处理。
5.如权利要求4所述的方法,其特征在于,所述处理是饲喂致癌剂。
6.如权利要求5所述的方法,其特征在于,所述致癌剂是4NQO。
7.如权利要求2所述的方法,其特征在于,所述肿瘤组织是食管处的新生肿瘤。
8.如权利要求2所述的方法,其特征在于,所述食管癌细胞系是经过实验验证的细胞系。
9.如权利要求8所述的方法,其特征在于,所述实验验证包括增殖能力检测、克隆能力检测、细胞迁移能力检测。
10.权利要求2所述的VAV2基因中缺失如SEQ ID NO:1所示序列的小鼠在制备VAV2缺失的细胞系中的应用。
11.权利要求1所述的细胞系的用途,其特征在于,所述用途包括以下任意一种:筛选和/或评价/制备肿瘤治疗药物,开发肿瘤药物靶点,制备肿瘤诊断试剂,开发肿瘤治疗新技术,开发检测肿瘤相关生物工程产品。
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