CN111484979A - 一种可体内示踪的肝肿瘤细胞株及其构建方法 - Google Patents
一种可体内示踪的肝肿瘤细胞株及其构建方法 Download PDFInfo
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Abstract
本发明提供了一种稳定携带萤火虫荧光素酶基因（FLuc）及杀稻瘟菌素‑s脱氨酶基因（BSD）的小鼠肝肿瘤细胞株hepa1‑6‑FLuc及其构建方法。采用逆转录病毒将Fluc基因导入小鼠肝肿瘤细胞hepa1‑6，稻瘟菌素筛选稳定表达FLuc基因的hepa1‑6‑FLuc细胞，该细胞具有与亲代hepa1‑6类似的形态及增殖、迁移、侵袭的生物学特性。细胞增殖后植入裸鼠体内，FLuc基因随着细胞增殖而复制，生物荧光活体成像技术检测到不断增强发光信号，可实际反映肝癌细胞的存活和增殖，hepa1‑6‑FLuc是一个建立肝癌动物模型的理想细胞，用于动态监测肿瘤的增殖、存活和转移。该方法可用于构建表达FLuc基因的其他细胞株，为细胞体内示踪提供了一种便捷的方法。
Description
技术领域
本发明涉及细胞生物学领域，公开了一种可用于体内示踪的小鼠肝肿瘤细胞株Hepa1-6-FLuc及其构建方法。
背景技术
肝癌原位移植瘤模型是研究肿瘤转移机制和肿瘤免疫的一个理想模型，进一步筛选抗肿瘤药物和判断疗效。传统的细胞追踪方法通常采用组织病理学技术观察活体组织或在实验终点活体标记细胞。这种方法虽然可以获得移植细胞在实验终点的信息，但无法获得细胞在体内定位、生存、迁移、激活的实时动态信息。体内细胞追踪技术可以实现对实验动物的无创监测，对肿瘤疾病的动态研究具有重要意义。常用的体内示踪技术包括放射性核素显像、磁共振成像、光学成像，活体成像等。其中，生物发光法是基于萤火虫荧光素酶（firefly luciferase, FLuc）能催化底物（D-Luciferin）化学发光的原理，将体外表达荧光素酶的细胞株植入动物体内，与后期注射入体内的底物发生反应，利用光学系统检测光强度，间接反映出细胞数量的变化或细胞的定位，是无创性监测细胞体内示踪的一个有价值的工具。生物荧光活体成像技术具有灵敏度高、定量准确、创伤小、操作简单、直观等优点。目前，在人类生物学和疾病的动物模型中，广泛应用于干细胞移植、肿瘤转移或疾病病理生理的生物学过程等研究领域，特别是生物发光成像技术可以用于体内深部肿瘤的检测。
腺病毒载体转导目的基因具有效率高、滴度高、瞬时转染在体内不与宿主细胞染色体整合等优点，使腺病毒载体在肿瘤的基因治疗研究与实践方面得以广泛运用，重组腺病毒可作为介导Fluc基因在不同动物细胞中的表达。一个成功的肝癌模型，移植的肝癌细胞应在体内快速增殖并形成肿块，然而，腺病毒介导的Fluc表达可能不会持续很长一段时间，导致Fluc-标记细胞的信号与肿瘤生长不一致，可能的原因是腺病毒不整合到宿主细胞基因组的基因，因此限制了来源于移植肿瘤细胞分裂的子代细胞在体内的追踪。
发明内容
本发明的目的是提供一种可体内示踪的肝肿瘤细胞株，本发明的目的是通过以下措施实现的：
一种小鼠肝肿瘤细胞株Hepa1-6-FLuc，其特征在于：稳定携带萤火虫荧光素酶基因（FLuc）及杀稻瘟菌素-s脱氨酶基因（BSD）。
上述小鼠肝肿瘤细胞株Hepa1-6-FLuc，其细胞增殖、克隆形成、迁移、侵袭能力不低于亲代Hepa1-6细胞，转代至20代以上FLuc基因表达水平下降低于5%，体内植入后随着肿瘤细胞的生长，FLuc基因稳定表达。
本发明的另一目的是提供一种稳定携带萤火虫荧光素酶基因（FLuc）及杀稻瘟菌素-s脱氨酶基因（BSD）细胞株的构建方法，本发明的目的是通过以下措施实现的：
一种稳定携带萤火虫荧光素酶基因（FLuc）及杀稻瘟菌素-s脱氨酶基因（BSD）细胞株的构建方法，采用逆转录病毒将萤火虫荧光素酶基因（FLuc）及杀稻瘟菌素-s脱氨酶基因（BSD）导入细胞，筛选具有稻瘟菌素抗性的细胞，检测细胞中FLuc的表达，获得稳定表达FLuc细胞株；将细胞株植入小鼠体内，活体生物发光成像技术动态检测细胞在体内的生物发光信号，用于细胞体内长时间示踪。
上述细胞株构建方法，包括以下步骤：
（1） 逆转录病毒构建：将Fluc基因克隆至pSEB-Flag质粒，获得携带FLuc基因的pSEB-FLuc质粒。将pSEB-FLuc与pAmpho质粒共转染293细胞，获得逆转录病毒；
（2）细胞构建：逆转录病毒感染目的细胞，经稻瘟菌素抗性加压筛选获得的抗稻瘟菌素的细胞；检测不同代数细胞的Luc活性；
（3）细胞鉴定：以亲代细胞株为对照，检测两种细胞的增殖、克隆形成、迁移、侵袭能力；
（4）细胞体内示踪：将亲代细胞、腺病毒Ad-Fluc感染细胞及FLuc稳定细胞，移植入裸鼠皮下成瘤， 分别于植入后第ld、7d、14d活体组织成像动态观察植入细胞的生长情况，比较荧光信号强度的变化趋势与同一视野白光下的细胞生长，优选的，细胞株为肿瘤细胞，测量瘤体大小并进行细胞体内成瘤模型的组织学鉴定。
上述细胞株构建方法，所述pSEB-Flag载体结构如图1所示，DNA序列为SEQ IDNO.1；所述pSEB-FLuc质粒结构如图2所示，DNA序列为SEQ ID NO.2，所述FLuc基因的DNA序列为SEQ ID NO.3，所述BSD的DNA序列为SEQ ID NO.4。
有益效果
1. 本发明提供了一种稳定表达Fluc基因的hepa1-6-FLuc小鼠肝癌细胞株，是一个建立肝癌动物模型的理想细胞，用于动态监测肿瘤的增殖、存活和转移，为肝癌基础研究及肝癌治疗中筛选新的抗癌药物等领域提供了一个重要的实验动物模型。
2. 本发明提供了一种稳定携带萤火虫荧光素酶基因（FLuc）及杀稻瘟菌素-s脱氨酶基因（BSD）细胞株的构建方法。将FLuc及BSD基因通过逆转录病毒的方式稳定导入细胞内，通过稻瘟菌素方便快捷地筛选出稳定表达FLuc基因的细胞，该细胞与亲代细胞的生物学性状无差异，并且体内生物荧光活体示踪可动态真实地反映细胞在体内的存活及增殖情况。该制备方法可用于构建表达FLuc基因的其他细胞株，为细胞体内示踪提供了一种便捷的方法。
附图说明
图1 是pSEB-Flag质粒结构图
图2 是pSEB-FLuc质粒结构图
图3 是稻瘟菌素筛选hepa1-6-FLuc细胞形态图 （a. hepa1-6细胞经稻瘟菌素筛选10天; b. hepa1-6-FLuc细胞经稻瘟菌素筛选10天; c.亲代hepa1-6细胞；d. 第10代hepa1-6-FLuc细胞)
图4是不同代数hepa1-6-FLuc细胞的Luc活性检测图
图5是hepa1-6-FLuc细胞及亲代hepa1-6细胞的增殖曲线图 （A. MTT检测增殖曲线；B.不同培养天数的结晶紫染色；C. B图孔板中结晶紫溶解后比色）
图6是细胞克隆形成及克隆形成率
图7是划痕实验检测细胞平行迁移能力
图8是 Transwell检测细胞垂直迁移能力(上图)及侵袭能力(下图)
图9是体内细胞示踪的生物发光信号图
图10是裸鼠皮下肿瘤体积
图11是肝癌组织病理学检测(H.E染色)
具体实施方式
下面通过实施例对本发明进行具体描述，有必要在此指出的是，以下实施例只用于对本发明进行进一步的说明，不能理解为对本发明保护范围的限制，该领域的技术熟练人员可以根据上述发明内容对本发明做出一些非本质的改进和调整。
一、逆转录病毒构建
将FLuc基因插入至pSEB-Flag逆转录病毒质粒（图1）的多克隆区域（BglⅡ和SalⅠ之间）获得携带FLuc基因及BSD基因的pSEB-FLuc质粒（图2）和pAmpho逆转录病毒包膜质粒共转染HEK293细胞获得逆转录病毒。
二、携带Fluc基因的小鼠肝癌细胞株hepa1-6-FLuc的构建，筛选及生物学性状鉴定
1. 细胞构建：逆转录病毒感染细胞，经稻瘟菌素3μg/ml抗性加压筛选，10天后有10%的细胞存活，而亲代hepa1-6细胞全部死亡（图3-a, b），维持稻瘟菌素3μg/ml至第3代。第10代的hepa1-6-FLuc细胞与亲代hepa1-6细胞的形态一致，均呈克隆样生长，细胞增殖活跃，呈上皮样细胞形态，细胞胞核大而圆，有多核，核浆比例大，有些细胞能见到明显的细胞分裂相（图3-c,d）。该方法能有效筛选出稻瘟菌素抗性的细胞，且与亲代细胞相比无差异。
2. hepa1-6-FLuc细胞鉴定：hepa1-6细胞Luc活性为正常本底，而hepa1-6-FLuc细胞的Luc活性显著增高，传代检测至第20代，FLuc活性没有降低（图4）。说明该方法构建的细胞能稳定表达FLuc基因。
3. hepa1-6-FLuc细胞的生物学性状鉴定：hepa1-6-FLuc的细胞生长曲线呈典型的S型（图5），细胞克隆形成率（图6），平行迁移（图7）及纵向迁移侵袭（图8）能力与亲代hepa1-6细胞相比无差异。
三、 hepa1-6-FLuc细胞体内示踪检测：
将亲代hepa1-6细胞株及腺病毒Ad-FLuc感染hepa1-6介导FLuc表达（hepa1-6-AdFLuc细胞）作为两个对照组，hepa1-6-FLuc细胞作为实验组，移植入裸鼠皮下成瘤，分别于植入后第ld、7d、14d活体组织成像动态观察植入细胞的生长情况，比较荧光信号强度的变化趋势与同一视野白光下的肿瘤大小趋势。如图9所示，三组肿瘤随着时间推移均在皮下形成肿块并逐渐增大，hepa1-6对照组无荧光信号，hepa1-6-AdFLuc组1d时的荧光信号强度与hepa1-6-FLuc组无显著差异，随着肿瘤增大，hepa1-6-FLuc组荧光信号逐渐增强，而hepa1-6-AdFLuc组荧光信号反而减弱。说明hepa1-6-FLuc细胞体内示踪信号与细胞的体内生长一致，而腺病毒感染的hepa1-6-AdFLuc细胞的FLuc信号随着肿瘤的生长会丢失。
细胞体内成瘤模型的组织学鉴定：各组细胞皮下成瘤的肿块体积无统计学差异（图10），H.E染色检测肝癌组织病理学结果（图11）显示hepa1-6和hepa1-6-Fluc细胞呈多边形或不规则形，细胞大小不一、排列紧密，胞核染色深，胞质少，一些细胞出现核固缩、核浓染或分裂期染色。hepa1-6-AdFLuc细胞形成的肿瘤具有相似的细胞形态，与其他两组相比，有大量的新生血管，血管壁浸润，血管腔出现肿瘤细胞和血液细胞。该方法构建hepa1-6-FLuc细胞能在体内稳定表达FLuc基因，用于体内示踪的同时，不会影响细胞的成瘤能力。
<110> 重庆医科大学附属儿童医院
<120> 一种可体内示踪的肝肿瘤细胞株及其构建方法
<160>
<210> 1
<211> 6151
<212> DNA
<213> Artificial(人工序列)
<400> 1
tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60
ggaaaataca taactgagaa tagagaagtt agatcaaggt taggaacaga gagacagcag 120
aatatgggcc aaacaggata tctgtggtaa gcagttcctc cccggctcag ggccaagaac 180
agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc 240
agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc 300
ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct 360
cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtat tcccaataaa 420
gcctcttgct gtttgcatcc gaatcgtgga ctcgctgatc cttgggaggg tctcctcaga 480
ttgattgact gcccacctcg ggggtctttc atttggaggt tccaccgaga tttggagacc 540
cctgcccagg gaccaccgac ccccccgccg ggaggtaagc tggccagcgg tcgtttcgtg 600
tctgtctctg tctttgtgcg tgtttgtgcc ggcatctaat gtttgcgcct gcgtctgtac 660
tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga gttctgaaca 720
cccggccgca accctgggag acgtcccagg gactttgggg gccgtttttg tggcccgacc 780
tgaggaaggg agtcgatgtg gaatccgacc ccgtcaggat atgtggttct ggtaggagac 840
gagaacctaa aacagttccc gcctccgtct gaatttttgc tttcggtttg gaaccgaagc 900
cgcgcgtctt gtctgctgca gcgctgcagc atcgttctgt gttgtctctg tctgactgtg 960
tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag tttgacctta 1020
ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga 1080
cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac 1140
ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc acctggcccg 1200
catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc 1260
cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc 1320
ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc 1380
ctcactcctt ctctaggcgc cggaattcac catggccaag cctttgtctc aagaagaatc 1440
caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg aagactacag 1500
cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca atgtatatca 1560
ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg ctgcggcagc 1620
tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct tgagcccctg 1680
cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca tagtgaagga 1740
cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg gttatgtgtg 1800
ggagggctaa gtttaaacat gcagcgctcc ggtgcccgtc agtgggcaga gcgcacatcg 1860
cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg 1920
tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt 1980
gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt 2040
gccgccagaa cacagctgaa gctagcttcg aggggctcgc atctctcctt cacgcgcccg 2100
ccgccctacc tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg 2160
tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg 2220
cctttgtccg gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct 2280
gaccctgctt gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc 2340
caagctgtga ccggcgccta catcgatccg cggagatcta agcttgcggc cgcgtcgacg 2400
gatccacgcg tgggcccgga ggcggcgatt acaaggatga cgacgataag atatgaattc 2460
ttaattaacg ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga 2520
ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaat 2580
acataactga gaatagagaa gttcagatca aggttaggaa cagagagaca gcagaatatg 2640
ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 2700
gtccccagat gcggtcccgc cctcagcagt ttctagagaa ccatcagatg tttccagggt 2760
gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc 2820
gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc 2880
ggcgcgccag tcctccgata gactgcgtcg cccgggtacc cgtgtatcca ataaaccctc 2940
ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt 3000
gactacccgt cagcgggggt ctttcatggg taacagtttc ttgaagttgg agaacaacat 3060
tctgagggta ggagtcgaat attaagtaat cctgactcaa ttagccactg ttttgaatcc 3120
acatactcca atactcctga aatagttcat tatggacagc gcagaagagc tggggagaat 3180
taattcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 3240
acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 3300
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 3360
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 3420
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 3480
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 3540
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 3600
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 3660
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 3720
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 3780
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 3840
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 3900
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 3960
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 4020
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 4080
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 4140
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 4200
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 4260
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 4320
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 4380
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 4440
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 4500
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 4560
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 4620
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 4680
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 4740
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 4800
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 4860
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 4920
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 4980
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 5040
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 5100
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 5160
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 5220
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 5280
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 5340
gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 5400
cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 5460
gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 5520
gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 5580
gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 5640
ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 5700
gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 5760
ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcgc aaggaatggt 5820
gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg 5880
ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg 5940
gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg 6000
gcgtagaggc gattagtcca atttgttaaa gacaggatat cagtggtcca ggctctagtt 6060
ttgactcaac aatatcacca gctgaagcct atagagtacg agccatagat aaaataaaag 6120
attttattta gtctccagaa aaagggggga a 6151
<210> 2
<211> 7775
<212> DNA
<213> Artificial(人工序列)
<400> 2
tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60
ggaaaataca taactgagaa tagagaagtt agatcaaggt taggaacaga gagacagcag 120
aatatgggcc aaacaggata tctgtggtaa gcagttcctc cccggctcag ggccaagaac 180
agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc 240
agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc 300
ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct 360
cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtat tcccaataaa 420
gcctcttgct gtttgcatcc gaatcgtgga ctcgctgatc cttgggaggg tctcctcaga 480
ttgattgact gcccacctcg ggggtctttc atttggaggt tccaccgaga tttggagacc 540
cctgcccagg gaccaccgac ccccccgccg ggaggtaagc tggccagcgg tcgtttcgtg 600
tctgtctctg tctttgtgcg tgtttgtgcc ggcatctaat gtttgcgcct gcgtctgtac 660
tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga gttctgaaca 720
cccggccgca accctgggag acgtcccagg gactttgggg gccgtttttg tggcccgacc 780
tgaggaaggg agtcgatgtg gaatccgacc ccgtcaggat atgtggttct ggtaggagac 840
gagaacctaa aacagttccc gcctccgtct gaatttttgc tttcggtttg gaaccgaagc 900
cgcgcgtctt gtctgctgca gcgctgcagc atcgttctgt gttgtctctg tctgactgtg 960
tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag tttgacctta 1020
ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga 1080
cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac 1140
ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc acctggcccg 1200
catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc 1260
cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc 1320
ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc 1380
ctcactcctt ctctaggcgc cggaattcac catggccaag cctttgtctc aagaagaatc 1440
caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg aagactacag 1500
cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca atgtatatca 1560
ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg ctgcggcagc 1620
tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct tgagcccctg 1680
cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca tagtgaagga 1740
cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg gttatgtgtg 1800
ggagggctaa gtttaaacat gcagcgctcc ggtgcccgtc agtgggcaga gcgcacatcg 1860
cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg 1920
tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt 1980
gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt 2040
gccgccagaa cacagctgaa gctagcttcg aggggctcgc atctctcctt cacgcgcccg 2100
ccgccctacc tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg 2160
tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg 2220
cctttgtccg gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct 2280
gaccctgctt gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc 2340
caagctgtga ccggcgccta catcgatccg cggagatcta ccatggatga aaatatttta 2400
acaggaccgg aattagatga taataatttt aaagagaaaa ccgttaccga atatttattc 2460
aattcattaa aatcacatcc gaaagattca attattcaga tcgatgcgag tacgggtgaa 2520
aaaataacgt gttggaatta tttgaaaaaa tgtttacaat tcggagaagg tttggtgaaa 2580
aatatgggag ttaaaaatgg cgacgttgtt tgtttggctt gtcaaaatag cataaatagt 2640
attattgctt ccgttggtac atttttttcg gggggcatag cagcacccat tgatcataat 2700
tccaaaccgg tcgaaatgca atatttactc aaccttttaa aaccaaaata cataatttgc 2760
gaaaatgaaa aagctcaaga gataaacaac aatttaaaac aaatcaattt ggattgtaaa 2820
ataatcgtct tagacacaaa agtatcggat tcgaattttt attctatgaa agacgtgtca 2880
attgaaaatg tcgatgtcga ttcatacgta cctagaaaag tcggcgataa taaaaaagat 2940
atttttgtca tttgttgttc cagcggatca actggatatc caaaaggtgt cgcattatcc 3000
catcattctc ttttgtataa cgtgcaaact ttaaagcaag tattccatca tccattccct 3060
ccgaaattcg gatcacaatt tttatcaaaa aaattattat tcacatctcc gttattttgg 3120
atatcaaatt tcattttaac gataatgtct tgcgtttttg gacaaactag aatagttcca 3180
aaatctacaa atacaataca ttatttggaa agcattgaaa aatatcagcc tgaattcacg 3240
gctttttcac cgtcttccct attattttta gtagatcatc cagattttga taaattcaat 3300
ttgaaaagtt taaaattaat ttttgtcggt ggttctcttt tgggatcgaa tttacttgat 3360
aaaattaagt ggaaaaatgt tcacatactc cagggttacg gtatgacaga atttggtgga 3420
ggaatcacaa gtccaaattt tggaaacgcc acgtcttccg ttggagaagt tttacccaat 3480
acttcaataa aaattgtgga tccatcatcg ggtaaaattc tcggtccgaa tgaaatcgga 3540
gaatgtagag ttaaagccga cagagttatg attggatatt acaacaatga aaaagcaacc 3600
aaagaatctt tcgacgatga tggttggttt aaaactggag atttgatgta ttacgatgat 3660
aataaattgt tatacgttgt cggaagaatc aaagaaaccg ttaaatatca aaattatcaa 3720
atatctttag cggaaataga aaattttata atatctttac cgggagtaaa agatgctgcg 3780
atagtaccca ttcctcatgg accaaatcat catttaaaag cggttgttgt taaaagttca 3840
ggttctaata tatctgaaaa tgacgtcatc gaagctgtaa aaaataattt ttctgaatac 3900
aaacatttaa gagaaggcgt tgaattttcc gattctcttc caagaacgga aacaggaaaa 3960
ttaagacgtt ggcaactttt gaagtcacat gatcctaatc tatctaaaac taattaagtc 4020
gacggatcca cgcgtgggcc cggaggcggc gattacaagg atgacgacga taagatatga 4080
attcttaatt aacgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga 4140
aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga 4200
aaatacataa ctgagaatag agaagttcag atcaaggtta ggaacagaga gacagcagaa 4260
tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca 4320
gatggtcccc agatgcggtc ccgccctcag cagtttctag agaaccatca gatgtttcca 4380
gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct 4440
tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc 4500
actcggcgcg ccagtcctcc gatagactgc gtcgcccggg tacccgtgta tccaataaac 4560
cctcttgcag ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt 4620
gattgactac ccgtcagcgg gggtctttca tgggtaacag tttcttgaag ttggagaaca 4680
acattctgag ggtaggagtc gaatattaag taatcctgac tcaattagcc actgttttga 4740
atccacatac tccaatactc ctgaaatagt tcattatgga cagcgcagaa gagctgggga 4800
gaattaattc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 4860
ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 4920
gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 4980
gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 5040
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 5100
cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 5160
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 5220
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 5280
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 5340
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 5400
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 5460
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 5520
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 5580
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 5640
ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta 5700
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 5760
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 5820
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 5880
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 5940
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 6000
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 6060
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 6120
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 6180
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 6240
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 6300
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 6360
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 6420
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 6480
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 6540
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 6600
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 6660
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 6720
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 6780
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 6840
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 6900
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 6960
aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 7020
gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 7080
tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 7140
gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 7200
agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga 7260
gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat 7320
cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat 7380
taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gcgcaaggaa 7440
tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc 7500
cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat 7560
gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg 7620
tccggcgtag aggcgattag tccaatttgt taaagacagg atatcagtgg tccaggctct 7680
agttttgact caacaatatc accagctgaa gcctatagag tacgagccat agataaaata 7740
aaagatttta tttagtctcc agaaaaaggg gggaa 7775
<210> 3
<211> 1635
<212> DNA
<213> Artificial(人工序列)
<400> 3
atggatgaaa atattttaac aggaccggaa ttagatgata ataattttaa agagaaaacc 60
gttaccgaat atttattcaa ttcattaaaa tcacatccga aagattcaat tattcagatc 120
gatgcgagta cgggtgaaaa aataacgtgt tggaattatt tgaaaaaatg tttacaattc 180
ggagaaggtt tggtgaaaaa tatgggagtt aaaaatggcg acgttgtttg tttggcttgt 240
caaaatagca taaatagtat tattgcttcc gttggtacat ttttttcggg gggcatagca 300
gcacccattg atcataattc caaaccggtc gaaatgcaat atttactcaa ccttttaaaa 360
ccaaaataca taatttgcga aaatgaaaaa gctcaagaga taaacaacaa tttaaaacaa 420
atcaatttgg attgtaaaat aatcgtctta gacacaaaag tatcggattc gaatttttat 480
tctatgaaag acgtgtcaat tgaaaatgtc gatgtcgatt catacgtacc tagaaaagtc 540
ggcgataata aaaaagatat ttttgtcatt tgttgttcca gcggatcaac tggatatcca 600
aaaggtgtcg cattatccca tcattctctt ttgtataacg tgcaaacttt aaagcaagta 660
ttccatcatc cattccctcc gaaattcgga tcacaatttt tatcaaaaaa attattattc 720
acatctccgt tattttggat atcaaatttc attttaacga taatgtcttg cgtttttgga 780
caaactagaa tagttccaaa atctacaaat acaatacatt atttggaaag cattgaaaaa 840
tatcagcctg aattcacggc tttttcaccg tcttccctat tatttttagt agatcatcca 900
gattttgata aattcaattt gaaaagttta aaattaattt ttgtcggtgg ttctcttttg 960
ggatcgaatt tacttgataa aattaagtgg aaaaatgttc acatactcca gggttacggt 1020
atgacagaat ttggtggagg aatcacaagt ccaaattttg gaaacgccac gtcttccgtt 1080
ggagaagttt tacccaatac ttcaataaaa attgtggatc catcatcggg taaaattctc 1140
ggtccgaatg aaatcggaga atgtagagtt aaagccgaca gagttatgat tggatattac 1200
aacaatgaaa aagcaaccaa agaatctttc gacgatgatg gttggtttaa aactggagat 1260
ttgatgtatt acgatgataa taaattgtta tacgttgtcg gaagaatcaa agaaaccgtt 1320
aaatatcaaa attatcaaat atctttagcg gaaatagaaa attttataat atctttaccg 1380
ggagtaaaag atgctgcgat agtacccatt cctcatggac caaatcatca tttaaaagcg 1440
gttgttgtta aaagttcagg ttctaatata tctgaaaatg acgtcatcga agctgtaaaa 1500
aataattttt ctgaatacaa acatttaaga gaaggcgttg aattttccga ttctcttcca 1560
agaacggaaa caggaaaatt aagacgttgg caacttttga agtcacatga tcctaatcta 1620
tctaaaacta attaa 1635
<210> 4
<211> 399
<212> DNA
<213> Artificial(人工序列)
<400> 4
atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc 60
aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc 120
cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg 180
gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga 240
aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg 300
catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt 360
cgtgaattgc tgccctctgg ttatgtgtgg gagggctaa 399
Claims (5)
1.一种小鼠肝肿瘤细胞株Hepa1-6-FLuc，其特征在于：稳定携带萤火虫荧光素酶基因（FLuc）及杀稻瘟菌素-s脱氨酶基因（BSD）。
2.如权利要求2所述的小鼠肝肿瘤细胞株Hepa1-6-FLuc，其特征还在于：细胞增殖、克隆形成、迁移、侵袭能力不低于亲代Hepa1-6细胞，转代至10代以上FLuc基因表达水平下降低于5%，体内植入后随着肿瘤细胞的生长Fluc稳定表达。
3.一种如权利要求1所述细胞株的构建方法，采用逆转录病毒将萤火虫荧光素酶基因（FLuc）及杀稻瘟菌素-s脱氨酶基因（BSD）导入细胞，筛选具有稻瘟菌素抗性的细胞，检测细胞中FLuc的表达，获得稳定表达FLuc细胞株；将细胞株植入小鼠体内，活体生物发光成像技术动态检测细胞在体内的生物发光信号，用于细胞体内长时间示踪。
4.如权利要求3所述的细胞株构建方法，包括以下步骤：
（1） 逆转录病毒构建：将Fluc基因克隆至pSEB-Flag质粒，获得携带FLuc基因的pSEB-FLuc质粒，将pSEB-FLuc与pAmpho质粒共转染293细胞，获得逆转录病毒；
（2）细胞构建：逆转录病毒感染目的细胞，经稻瘟菌素抗性加压筛选获得的抗稻瘟菌素的细胞；检测不同代数细胞的Luc活性；
（3）细胞鉴定：以亲代细胞株为对照，检测两种细胞的增殖、克隆形成、迁移、侵袭能力；
（4）细胞体内示踪：将亲代细胞、腺病毒Ad-Fluc感染细胞及FLuc稳定细胞，移植入裸鼠皮下成瘤， 分别于植入后第ld、7d、14d活体组织成像动态观察植入细胞的生长情况，比较荧光信号强度的变化趋势与同一视野白光下的细胞生长，优选的，细胞株为肿瘤细胞，测量瘤体大小并进行细胞体内成瘤模型的组织学鉴定。
5.如权利要求4所述的细胞株构建方法，所述pSEB-Flag质粒的DNA序列为SEQ IDNO.1；所述pSEB-FLuc质粒的DNA序列为SEQ ID NO.2，所述FLuc基因的DNA序列为SEQ IDNO.3，所述BSD基因的DNA序列为SEQ ID NO.4。
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| CN113621578A (zh) * | 2021-08-10 | 2021-11-09 | 上海南方模式生物科技股份有限公司 | 细胞毒性t细胞示踪系统以及动物模型的构建方法及应用 |
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| MAKOTO KIMURA等: "Blasticidin S deaminase gene from Aspergillus terreus (BSD):a new drug resistance gene for transfection of mammalian cells", 《BIOCHIMICA ET BIOPHYSICA ACTA》 * |
| YANG BI等: "Hepa1‑6‑FLuc cell line with the stable expression of firefly luciferase retains its primary properties with promising bioluminescence imaging ability", 《ONCOLOGY LETTERS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113621578A (zh) * | 2021-08-10 | 2021-11-09 | 上海南方模式生物科技股份有限公司 | 细胞毒性t细胞示踪系统以及动物模型的构建方法及应用 |
| CN113621578B (zh) * | 2021-08-10 | 2023-11-28 | 上海南方模式生物科技股份有限公司 | 细胞毒性t细胞示踪系统以及动物模型的构建方法及应用 |
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