CN106967687A - Bancr过表达型人皮肤黑色素瘤稳转细胞株及其制备方法和应用 - Google Patents
Bancr过表达型人皮肤黑色素瘤稳转细胞株及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种BANCR过表达型人皮肤黑色素瘤稳转细胞株及其制备方法和应用,涉及分子生物学技术领域。本发明通过将BANCR基因***表达载体中,得到重组慢病毒载体,然后与包装质粒共转染包装细胞,获得重组慢病毒,并以该重组慢病毒感染人皮肤黑色素瘤细胞获得BANCR过表达型人皮肤黑色素瘤稳转细胞株。本发明提供的构建方法,通过慢病毒***将目的基因整合到宿主细胞中,并通过筛选和鉴定最终获得了高效、稳定的BANCR过表达型人皮肤黑色素瘤稳转细胞株。该细胞株可用于探究BANCR与肿瘤的相关性及其机理,为深入研究BANCR与人黑色素瘤发生、发展的可能机制提供一种可靠的研究基础。
Description
技术领域
本发明涉及分子生物学技术领域,尤其是涉及BANCR过表达型人皮肤黑色素瘤稳转细胞株及其制备方法和应用。
背景技术
遗传学的“中心法则”描述了遗传信息从DNA通过RNA到相应蛋白质的信息流途径。几十年来,传统观点认为DNA是遗传物质的储存载体,基因要通过其表达的相应蛋白质来发挥作用。随着人类基因组计划的顺利完成,人们发现仅仅一小部分基因为蛋白编码基因,而90%以上的人类基因都已经发生了转录,这表明众多数量的转录本是不编码蛋白质的,这部分转录本被称为“非编码RNA”(non-coding RNA,ncRNA)。起初,ncRNA被认为是转录“噪音”,然而近年来越来越多的研究表明ncRNA在维持正常细胞生理功能及疾病发生发展过程中均发挥着多样化的调节作用。基于ncRNA的主要生物学功能,可将其大致分为结构性和调节性两大类ncRNA。结构ncRNA在蛋白质翻译过程中发挥重要作用,包括转移RNA(transferRNA,tRNA),核糖体RNA(ribosomal RNA,rRNA),小核RNA(small nuclear RNA,snRNA)和核仁小RNA(small nucleolar RNA,snoRNA);调节ncRNA包括小干扰RNA(small interferingRNA,siRNA),微小RNA(micro RNA,miRNA),piwi蛋白相互作用的RNA(piwi-interactingRNAs,piRNAs)和长链非编码(long noncoding RNA,lncRNA)。lncRNA长度在200个核苷酸以上,大部分lncRNA被视为缺乏蛋白质编码的能力,极小部分lncRNA被证实可以编码一些小肽段。研究发现lncRNA可通过表观遗传、转录和转录后及代谢等多层面参与蛋白编码基因和非蛋白编码基因的表达调控。lncRNA在正常细胞分化、发育及人类疾病发生发展过程中发挥重要作用,包括恶性肿瘤。
恶性黑素瘤是来源于皮肤和其他器官黑色素细胞的肿瘤,恶性度高、转移发生早,死亡率高,中晚期患者的5年生存率仅10%左右。近年来,中国人群恶性黑色素瘤发病率上升较快,深入研究其致病机制,具有重要的临床意义和现实意义。黑色素瘤的发生是遗传和环境因素长期相互作用的结果,如皮肤长时间暴露在阳光下,又如癌基因的活化与抑癌基因的失活。已知40-70%的黑色素瘤与癌基因BRAF的活化有关,其中BRAF V600E(Exon 15的1799位T→A,撷氨酸→谷氨酸)在中国人群的黑色素瘤中突变率高达83.3%。研究发现,靶向应用BRAFV600E抑制剂,如Vemurafenib、Sorafenib、Trametinib和Dabrafenib对恶性黑色素瘤的治疗有较好疗效。尽管如此,恶性黑色素瘤的复发和耐药性的产生仍然是制约黑色素瘤临床治疗取得长足进步的两大瓶颈。只有深入研究黑色素瘤发生发展机制,才可能为临床治疗黑色素瘤提供有力的指导和理论依据。
BRAF活化的非编码RNA(BRAF-activated non-protein coding RNA,BANCR)是一种与黑色素瘤发生发展密切相关的lncRNA。该基因定位于染色体9q21.11,含4个外显子,BANCR是来自内含子的转录产物,长约693nt,在BRAFV600E突变的黑色素瘤细胞及黑色素瘤组织中高表达。体内外研究发现BANCR可通过激活ERK1/2和JNK MAPK信号转导通路参与黑色素瘤细胞的增殖调控,但是BANCR与MAPK信号转导通路之间的关系尚未完全阐明。此外,研究还发现BANCR可通过调控CXCL11基因的表达参与黑色素瘤细胞的迁移。鉴于BANCR在恶性黑色素瘤中的表达具有组织特异性及稳定性,可作为潜在的肿瘤诊断和肿瘤靶向治疗的生物学标记物。
然而,在目前的国内外关于BANCR和黑色素瘤相关性研究的报道中,均没有将BANCR过表达的人A375稳定转染细胞系作为进行黑色素瘤探索研究的实验体系。
因此,特提出本发明。
发明内容
本发明的目的在于提供一种BANCR过表达型人皮肤黑色素瘤稳转细胞株及其构建方法和应用,以弥补现有技术中的不足,为研究BANCR在人黑色素瘤发生、发展过程中的作用及机制提供研究基础。
本发明提供了一种BANCR过表达型人皮肤黑色素瘤稳转细胞株。
另外,本发明还提供了一种BANCR过表达型人皮肤黑色素瘤稳转细胞株的构建方法,所述构建方法包括以下步骤:
(1)将BANCR基因***慢病毒载体***中的表达载体中,得到携带有BANCR基因的重组慢病毒载体;
(2)将所述携带有BANCR基因的重组慢病毒载体与慢病毒载体***中的包装质粒混合,转染包装细胞,得到携带有BANCR基因的重组慢病毒;
(3)将所述携带有BANCR基因的重组慢病毒转染人皮肤黑色素瘤细胞,进行筛选和鉴定,得到所述BANCR过表达型人皮肤黑色素瘤稳转细胞株。
进一步的,步骤(3)中,所述筛选采用抗生素或者流式细胞仪。
进一步的,步骤(3)中,所述鉴定采用荧光显微镜观察和/或荧光定量PCR反应;所述荧光定量PCR反应包括提取待鉴定细胞的总RNA,反转录为cDNA,以所述cDNA为模板进行荧光定量PCR。
进一步的,所述荧光定量PCR的引物为:
BANCR-FO:5’-CTCGCTTTCACTTTATGGATTC-3’(SEQ ID NO:1);
BANCR-RE:5’-GGGTCAGGGGTCTCTTCAG-3’(SEQ ID NO:2)。
进一步的,
所述荧光定量PCR的反应体系为:2×Real-time PCR Master Mix 10μL,20μMBANCR-FO 0.1μL,20μM BANCR-RE 0.1μL,cDNA模板2μL,5U/μL rTaq DNA polymerase 0.4μL,ddH2O补充至20μL;
所述荧光定量PCR的反应条件为:95℃3min预变性;95℃30s变性,62℃40s退火,共40个循环。
另外,本发明还提供了按照所述的构建方法得到的BANCR过表达型人皮肤黑色素瘤稳转细胞株在建立黑色素瘤细胞模型、建立黑色素瘤动物模型、研究黑色素瘤发病机理以及研发治疗黑色素瘤药物中的应用。
本发明提供了BANCR过表达型人皮肤黑色素瘤稳转细胞株,其构建方法是通过慢病毒***将目的基因整合到宿主细胞中,并通过筛选和鉴定最终获得了能够高效、稳定的过表达BANCR基因的人皮肤黑色素瘤稳转细胞株。该细胞株可用于探究BANCR与肿瘤的相关性及其机理,为深入研究BANCR与人黑色素瘤发生、发展的可能机制提供一种可靠的研究基础。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为质粒LV5的物理图谱;
图2为PCR扩增的目的基因BANCR的测序结果图;
图3A为重组慢病毒感染A375细胞的光学图片(100×);
图3B为重组慢病毒感染A375细胞的荧光图片(100×)
图4为A375-NC-96h扩增曲线;
图5为A375-BANCR-OE-96h扩增曲线;
图6为基因GAPDH熔解曲线;
图7为基因BANCR熔解曲线;
图8为qRT-PCR检测瞬时转染96h后A375细胞中BANCR的表达结果图;
图9为空白对照组细胞图片(200×);
图10为加入1μg/ml Puromycin一周后的细胞图片(200×);
图11为加入2μg/ml Puromycin一周后的细胞图片(200×);
图12为加入4μg/ml Puromycin一周后的细胞图片(200×);
图13为qRT-PCR检测稳转细胞株中BANCR的表达结果图。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明提供了一种BANCR过表达型人皮肤黑色素瘤稳转细胞株,该细胞株是携带有BANCR基因的人皮肤黑色素瘤细胞(A375细胞,来自于购自中国科学院上海生命科学研究所细胞资源中心)。
本发明还提供了BANCR过表达型人皮肤黑色素瘤稳转细胞株的构建方法,包括以下步骤:
(1)将BANCR基因***慢病毒载体***中的表达载体中,得到携带有BANCR基因的重组慢病毒载体;
(2)将携带有BANCR基因的重组慢病毒载体与慢病毒载体***中的包装质粒混合,转染包装细胞,得到携带有BANCR基因的重组慢病毒;
(3)将携带有BANCR基因的重组慢病毒转染人皮肤黑色素瘤细胞,进行鉴定和筛选,得到BANCR过表达型人皮肤黑色素瘤稳转细胞株。
在一个优选的实施方式中,步骤(1)中的表达载体可以为LV5载体;步骤(2)中的包装质粒为pGag/Pol、pRev和pVSV-G;步骤(3)中的鉴定采用荧光定量PCR法;步骤(3)中的筛选为采用1μg/mL Puromycin筛选一周,能够正常存活的即为BANCR过表达型人皮肤黑色素瘤稳转细胞株。
为了有助于更清楚的理解本发明的内容,现结合具体的实施例,对本发明提供的BANCR过表达型人皮肤黑色素瘤稳转细胞株的构建方法进行详细介绍。如无特别说明,实施例中应用的试剂为市售常规试剂。
实施例1 BANCR过表达型人皮肤黑色素瘤稳转细胞株的构建方法
一、人LncRNA BANCR基因全序列合成
选取LV5(LV5质粒的物理图谱如图1所示)作为过表达载体,设计合成人LncRNABANCR基因序列(SEQ ID NO:7)所需的PCR引物,通过oligo在线软件设计(http://www.oligo.net/),上下游引物分别加上LV5载体上Not I和Nsi I两侧同源序列,用于载体的亚克隆(引物由上海吉玛制药技术有限公司合成),设计的引物如下:
BANCR-F:
5’-AGGGTTCCAAGCTTAAGCGGCCGCATTCCCTTACTTTCTTAATAAAC-3’(SEQ ID NO:3)
BANCR-R:
5’-GATCCATCCCTAGGTAGATGCATTTTTTTTTTTTTTAGGATTTTTTA-3’(SEQ ID NO:4)
将合成的引物稀释成50μM后,进行PCR反应,反应体系如下:人LncRNA BANCR模版1μL,10×Pfu Buffer(+Mg2+)5μL,dNTP 1μL,上下游引物各1μL,ddH2O 41μL,Pfu DNApolymerase 0.3μL。循环条件如下:95℃3min(1cycle);94℃30sec,55℃30sec,72℃30sec(30cycles);72℃5min(1cycle)。PCR反应完成后,利用Agarose电泳并切胶回收人LncRNABANCR基因片段。
二、重组慢病毒载体构建
LV5载体双酶切,在无菌的0.2mL EP反应管,取LV5载体15μL,用Not I和Nsi I双酶切,酶切体系按照说明书配制,混匀后,37℃反应2h。并通过Agarose电泳及DNA凝胶回收试剂盒回收载体LV5。利用Entry One Step Cloning Kit,将扩增回收好的片段,重组克隆到线性化的LV5载体中,反应体系如下:5×CE Entry Buffer 4μL,LV5 1μL,人LncRNA BANCR 2μL,Exnase Entry2μL,ddH2O 11μL。使用移液器上下吹打上述混合物数次,轻轻混匀各组分。置于37℃反应30min。反应完成后立即将反应管置于冰水浴中,冷却5min备用。
三、重组过表达载体转化DH5α感受态细胞并鉴定
重组连接产物的转化,将装有感受态细胞的离心管冰上放置4min,待感受态细胞解冻后,加入10μL重组连接产物,轻柔混匀内容物,在冰中放置30min。将离心管放到预加温到42℃的水浴锅中放好的试管架上,放置90s,不要摇动离心管。快速将离心管转移到冰浴中,使细胞冷却3min。向每支离心管加入800μL LB培养基(不含抗生素),然后将离心管转移到37℃摇床,250rpm,培育45min使细菌复苏。取200μL培育后的细胞均匀涂布于含50μg/mLAmpicillin LB平板上。等平板上液体被吸收后,将平板倒置于37℃培养箱中,培养16h。从培养好的平板上挑取4个单独、饱满的菌落,置于含有5mL(含50μg/mL Ampicillin)LB培养基的试管中,将上述试管置于细菌摇床中培养,37℃,250rpm,培养16h。将培养好的菌液,用质粒小提试剂盒(天根生化,DP104-02)抽提质粒,将抽提好的质粒进行双酶切鉴定。酶切37℃,1h后电泳,在目的条带大小对应区域有酶切得到的条带对应的克隆即为阳性克隆。取200μL阳性克隆对应的菌液送测序,并将剩余的菌液用甘油保存。将测序结果与目的基因序列进行比对,正确无误,显示重组载体构建成功,如图2所示。此外,用保存的甘油菌液接菌LB培养基,进行大量质粒抽提,得到足够量的重组过表达载体。
四、慢病毒包装、收集及滴度检测
病毒包装:将对数生长期的293T细胞接种至15cm培养皿,加入18mL含10%FBS的DMEM培养液,混匀后置于37℃,5%CO2培养箱培养过夜。次日,在一支无菌的5mL离心管中加入1.5mL无血清DMEM,按比例加入含目的序列的重组过表达载体和包装质粒(pGag/Pol、pRev、pVSV-G),混匀,取另一支无菌的5mL离心管,加入1.5mL无血清DMEM,再加入300μLRNAi-Mate,混匀,室温放置5min后将两管混合,室温放置20~25min。除去15cm培养皿中的培养液,加入8mL无血清的DMEM培养液。将转染混合物逐滴加入15cm培养皿中,轻轻地前后摇晃培养皿以混匀复合物,在37℃,5%CO2培养箱中温育4-6h。吸弃转染液,加入18mL含10%FBS的DMEM培养液,37℃,5%CO2培养箱继续培养72h。
病毒收集:将培养皿中细胞上清液吸到50mL离心管中,4℃,4000rpm,4min。低速离心后,将离心管上清液倒入50mL注射器内,用0.45μm过滤器过滤。滤液在离心机中进行超速离心,4℃,20000rpm,2h。将浓缩液收集分装至冻存管中。分装的病毒液贴上标签,-80℃冰箱保存。
病毒滴度检测:取对数生长期的293T细胞,按3×104个细胞/孔的浓度接种于96孔板,混匀后于37℃,5%CO2培养箱培养24h。次日,将慢病毒原液10μL,用10%FBS的DMEM培养液十倍稀释3-5个梯度。吸去96孔板中的培养液,每孔加入100μL稀释的病毒液,同时设立空白对照组,于37℃,5%CO2培养箱培养24h。吸弃96孔板中的稀释病毒液,每孔加入100μL10%FBS的DMEM培液,于37℃,5%CO2培养箱继续培养72h。通过荧光显微镜或FACS计数荧光细胞,结合稀释倍数计算病毒滴度。
五、慢病毒感染A375细胞
取对数生长期的A375细胞,按2.5×105个细胞/孔的浓度接种于24孔板中,加入500μL完全培养液,37℃,5%CO2培养箱培养过夜;次日,制备病毒稀释液(DMEM培养基400μL+终浓度5μLg/mL Polybrene),将慢病毒原液按1:9加入到稀释液中,配制三种不同浓度的病毒液,分别加入到各组对应的培养孔中,同时建立对照(blank,negative),37℃,5%CO2培养箱培养过夜。12-24h移去细胞侵染后的病毒液,加入500μL完全培养液,37℃,5%CO2培养箱继续培养24-48h,在荧光倒置显微镜下观察结果。培养48h后,荧光倒置显微镜下可见绝大多数细胞呈现较强的绿色荧光,提示重组慢病毒载体成功感染A375细胞,结果如图3B所示。另外,图3A为重组慢病毒感染A375细胞的非荧光的普通光学图片。
六、实时荧光定量PCR检测瞬时转染后细胞内目的基因的表达量
细胞总RNA提取:瞬时转染96h后的细胞用DEPC处理的PBS漂洗一次,加入400μLRNA裂解液重悬细胞;在裂解液中加入400μL异丙醇和30μL磁珠,颠倒混匀,室温放置10分钟,放置于磁力架上待磁珠完全吸附后吸弃液体,500μL RNA抽提去蛋白液洗涤磁珠;再向离心管中加入500μL 80%乙醇重悬磁珠,重复上述洗涤步骤1次,第二次洗涤待磁珠完全吸附后充分吸弃液体(不要有残留),室温晾干5-10min;在无RNA酶的1.5mL离心管用移液器配置以下DNase I反应液,将离心管从磁力架上取下,向离心管中加入75μL DNase I反应液,移液器吹打重悬磁珠,37℃孵育15min,每隔5分钟轻弹管底悬浮磁珠,以防磁珠成团。加入700μL RNA抽提去蛋白液重悬磁珠,再加500μL 80%乙醇重悬磁珠;待磁珠完全吸附后吸弃液体,室温晾干2min。向离心管中加入50-100μL洗脱液EB,56℃孵育5分钟,将离心管重新放置于磁力架上,待磁珠完全吸附后小心将总RNA溶液转移至新的离心管,放入-80℃保存备用。
逆转录合成cDNA:取总RNA 2ng-2μg,用Reverse Transcriptase M-MLV逆转录酶,按说明合成cDNA。具体步骤:先在总RNA中加入2μL随机引物,加水(DEPC处理)补足至15μL,混匀,70℃温育混合物5min后,冰上骤冷。再依次加入5×Reaction Buffer 4μL,10mMdNTPs mix 0.8μL,MMLV RTase RNaseH(100U)0.5μL,42℃温育45min,最后85℃加热10min灭活反转录酶终止反应后冰上冷却。反应液用作PCR模板。
荧光定量PCR:按说明书配置反应体系,上机进行PCR扩增和检测。反应总体系为20μL,分别是2×Real-time PCR Master Mix 10μL,上下游引物(20μM)各0.1μL,cDNA模板2μL,rTaq DNA polymerase(5U/μL)0.4μL,用灭菌水补足至20μL。
其中,BANCR基因荧光定量PCR引物序列如下:
BANCR-FO:5’-CTCGCTTTCACTTTATGGATTC-3’(SEQ ID NO:1)
BANCR-RE:5’-GGGTCAGGGGTCTCTTCAG-3’(SEQ ID NO:2)
另外,还设计了GAPDH基因的对照实验组,其中GAPDH基因荧光定量PCR引物序列如下:
GAPDH-FO:5’-CATGAGAAGTATGACAACAGCCT-3’(SEQ ID NO:5)
GAPDH-RE:5’-AGTCCTTCCACGATACCAAAGT-3’(SEQ ID NO:6)
反应条件95℃,3min预变性;循环内95℃,30s变性,62℃退火40s,共40个循环,并在每个循环延伸末收集荧光信号,绘制扩增曲线(图4、5),图4、5分别显示阴性对照病毒和过表达病毒瞬时转染A375细胞96h后目的基因BANCR的扩增曲线。40个循环后设置反应步骤(95℃15s,60℃30s,95℃15s),并且对60℃到95℃升温过程进行全程荧光信号收集,绘制熔解曲线(图6、7),图6、7分别显示基因GAPDH和BANCR的熔解曲线,可见上述两个基因的熔解曲线仅显示单一峰,说明设计的基因引物合格,无非特异扩增。用Bio-Rad CFX Managerversion 1.6软件进行数据分析,分析结果如图8所示。
七、人LncRNA BANCR过表达的A375-BANCR-OE稳转细胞株筛选及鉴定
最小致死浓度摸索:将处于对数生长期的A375细胞按1.5×105细胞/孔的浓度接种于24孔培养板中,混匀后于37℃,5%CO2培养箱培养24h。完全培养基稀释Puromycin至4μg/ml,两倍倍比稀释,最小浓度为0.25μg/mL。将上述不同浓度的药物换入24孔板中,同时设置Blank孔,一周后观察结果。用观察到的细胞全部死亡孔中的最小浓度作为稳筛株的筛选浓度。实验结果表明,与空白对照组细胞(图9)相比,在各细胞培养孔中分别加入浓度为1μg/ml、2μg/ml、4μg/ml的Puromycin一周后均可导致细胞全部死亡(如图10、11和12所示),故选择1μg/ml的药物浓度最为最小致死浓度。
A375-BANCR-OE稳转细胞株的筛选:经慢病毒侵染的A375细胞培养96h后,弃培养基,换含有1μg/mL Puromycin的抗性培养基培养细胞,持续筛选一周,然后用含0.5μg/mLPuromycin的抗性培养基继续筛选培养。筛选获得的细胞连续传代3次以上,在荧光倒置显微镜下可见90%以上的细胞均表达较强的绿色荧光。
A375-BANCR-OE稳转细胞株的鉴定:荧光定量PCR检测A375-BANCR-OE稳转细胞内BANCR mRNA表达水平,总RNA提取、逆转录合成cDNA和荧光定量PCR过程同前。鉴定结果如图13所示。
此外,图4和5中,横坐标为“Cycle Number”,纵坐标为“Delta Rn”;图6和图7中,横坐标为“Temperature”,纵坐标为“-(Df/Dt)”。
另外,本发明还提供了BANCR过表达型人皮肤黑色素瘤稳转细胞株的应用,该菌株能够稳定、高效的表达BANCR基因,能够作为黑色素瘤细胞模型,用于研究黑色素瘤的发病机理,或者用于筛选治疗黑色素瘤的药物。此外,用该细胞感染实验动物后,能够获得BANCR过表达的实验动物,得到黑色素瘤动物模型,同样能够用于研究黑色素瘤的发病机理,或者用于筛选治疗黑色素瘤的药物。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 昆明医科大学
<120> BANCR过表达型人皮肤黑色素瘤稳转细胞株及其制备方法和应用
<130> 2017
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Claims (10)
1.一种BANCR过表达型人皮肤黑色素瘤稳转细胞株。
2.权利要求1所述的BANCR过表达型人皮肤黑色素瘤稳转细胞株的构建方法,其特征在于,所述构建方法包括以下步骤:
(1)将BANCR基因***慢病毒载体***中的表达载体中,得到携带有BANCR基因的重组慢病毒载体;
(2)将所述携带有BANCR基因的重组慢病毒载体与慢病毒载体***中的包装质粒混合,转染包装细胞,得到携带有BANCR基因的重组慢病毒;
(3)将所述携带有BANCR基因的重组慢病毒转染人皮肤黑色素瘤细胞,进行筛选和鉴定,得到所述BANCR过表达型人皮肤黑色素瘤稳转细胞株。
3.根据权利要求2所述的构建方法,其特征在于,步骤(3)中,所述筛选采用抗生素或者流式细胞仪。
4.根据权利要求2所述的构建方法,其特征在于,步骤(3)中,所述鉴定采用荧光显微镜观察和/或荧光定量PCR反应;所述荧光定量PCR反应包括提取待鉴定细胞的总RNA,反转录为cDNA,以所述cDNA为模板进行荧光定量PCR。
5.根据权利要求4所述的构建方法,其特征在于,所述荧光定量PCR的引物为:
BANCR-FO:5’-CTCGCTTTCACTTTATGGATTC-3’(SEQ ID NO:1);
BANCR-RE:5’-GGGTCAGGGGTCTCTTCAG-3’(SEQ ID NO:2)。
6.根据权利要求5所述的构建方法,其特征在于,
所述荧光定量PCR的反应体系为:2×Real-time PCR Master Mix10μL,20μM BANCR-FO0.1μL,20μM BANCR-RE 0.1μL,cDNA模板2μL,5U/μL rTaq DNA polymerase 0.4μL,ddH2O补充至20μL;
所述荧光定量PCR的反应条件为:95℃ 3min预变性;95℃ 30s变性,62℃ 40s退火,共40个循环。
7.权利要求1所述的BANCR过表达型人皮肤黑色素瘤稳转细胞株在建立黑色素瘤细胞模型中的应用。
8.权利要求1所述的BANCR过表达型人皮肤黑色素瘤稳转细胞株在建立黑色素瘤动物模型中的应用。
9.权利要求1所述的BANCR过表达型人皮肤黑色素瘤稳转细胞株在研究黑色素瘤发病机理中的应用。
10.权利要求1所述的BANCR过表达型人皮肤黑色素瘤稳转细胞株在研发治疗黑色素瘤药物中的应用。
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