CN111235120A - 一种构建荧光素酶高表达稳定细胞系的方法及细胞系 - Google Patents
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Abstract
本发明公开一种荧光素酶高表达的Huh7细胞系的构建方法。具体采用慢病毒载体获得荧光素酶高表达的Huh7细胞系。首先利用慢病毒侵染技术，将荧光素酶基因转入到Huh7细胞系中，使其在Huh7细胞里表达，其后采用挑取单克隆细胞的方法来筛选过表达荧光素酶的细胞，并进行大量扩增获得稳定过表达荧光素酶的Huh7细胞系。荧光素酶稳定高表达的Huh7细胞系能够作为一种特定的标记，检测细胞在动物体内的存在部位，用于肿瘤转移模型的研究。
Description
技术领域
本发明涉及到细胞生物学技术，具体涉及一种利用表达荧光素酶基因的慢病毒载体侵染肝癌细胞Huh7，单克隆筛选技术获得稳定高表达Luciferase的Huh7细胞系的方法。
背景技术
Luciferase的发现
荧光素酶Luciferase属于氧化还原酶的一类。在Mg2+、ATP和氧气存在的条件下，Luciferase催化底物荧光素(luciferin)氧化成oxyluciferin，发出可见的绿光。其反应式可以表示为：
Luciferin+ATP+Luciferase+O2+Mg2+—Oxyluciferin+AMP+PPi+CO2+光。
Luciferase的灵敏性高、半衰期短、特异性好、稳定性好、操作简便、反应迅速，不影响生物体正常生长繁殖并且对生物体无毒害作用。可以作为活体标记物用于活体成像，直接监测生物体内细胞活动和基因行为，尤其在肿瘤研究领域中，利用生物体发光成像技术对活体病灶的大小进行观察，能直接快速地测量肿瘤细胞的生长和转移，具有无损伤、高效灵敏、直观准确等特点。荧光标记的活体成像可对同一实验动物进行观察，排出了由于不同实验动物之间造成的差异对实验结果造成的影响。因此，利用荧光素酶标记的肿瘤细胞进行活体成像，被广泛用于肿瘤发生、转移的研究。
肝癌是常见的恶性肿瘤之一，在我国有着极高的发病率和死亡率。因其具有很高的转移性，使得肝癌的治疗成为难点。因此，萤光素酶过表达肝癌细胞系的构建，能够用于肝癌肿瘤小鼠成像研究，将有助于对肝癌细胞的转移进行观察和量化，对肝癌肿瘤转移的分子机制提供依据。
慢病毒是逆转录病毒，慢病毒载体是以人类免疫缺陷型I型病毒(HIV)为基础发展起来的基因治疗载体。慢病毒感染细胞后，在自身逆转录酶的作用下，其基因组经逆转录整合到宿主细胞染色体DNA上，作为细胞基因组的一部分，随着其感染的细胞基因组复制和分裂，进而继续传递下去，并随着宿主RNA聚合酶转录从而形成新的子代病毒。第二代慢病毒表达载体pWPXLd，包装质粒分别为psPAX，pMD2.G，具有侵染范围广，侵染效率高等特点。
发明内容
本发明的目的旨在提供一种荧光素酶稳定高表达的Huh7肝癌细胞系的构建方法。本发明采用慢病毒侵染技术构建Luciferase高表达的Huh7细胞系来建立肝癌成瘤及转移模型，可以更加方便的研究肝癌转移的分子机制。
首先利用慢病毒侵染技术，将荧光素酶基因转入到Huh7细胞系中，使其在Huh7细胞里表达，其后采用挑取单克隆细胞的方法来筛选过表达荧光素酶的细胞，并进行大量扩增获得稳定过表达荧光素酶的Huh7细胞系。荧光素酶稳定高表达的Huh7细胞系能够作为一种特定的标记，检测细胞在动物体内的存在部位，用于肿瘤转移模型的研究。
附图说明
图1：为不同单克隆细胞的荧光素酶表达情况。
图2：为Huh7-luciferase细胞系在mRNA水平上luciferase过表达效率的验证。
具体实施方式
现结合实例，对本发明做进一步说明。实例仅限于说明本发明，而非对本发明的限定。
实施例1 pWPXLd-luciferase病毒包装及侵染
所用的pWPXLd-luciferase质粒的构建方法参照硕士论文“稳定表达荧光素酶的MDA-MB-231乳腺癌细胞系的构建及其活性表征，周鑫，东北师范大学”。
1.第一天：将3x106个293T细胞种在10cm皿中，使用10ml添加胎牛血清的DMEM培养基培养，此培养基中不含抗生素，置于37℃，5％CO2(CO2体积含量5％的空气)培养箱中培养。
2.第二天：待293T细胞的覆盖率达到80％时，使用脂质体转染试剂lipofectamine2000做细胞转染。
转染试剂比例如下：
无菌EP管1：
无菌EP管2：
将管1，管2分别混合均匀后，吸取管2中的液体置管1中，轻轻摇晃混合均匀后，放置超净台中静置20min。
将反应体系加到293T细胞培养皿中，置于37℃，5％CO2培养箱中转染8h后，将培养基更换为5ml添加10％胎牛血清的DMEM培养基。
3.转染72h后，取病毒原液对Huh7细胞进行侵染。
4.病毒侵染72h后，消化细胞，对细胞进行计数，将1000个细胞铺到10cm培养皿中，第二天对培养皿里的单个细胞进行标记，待扩增到一定程度后，将细胞挑出来，置于新的培养皿中培养。
实施例2单克隆细胞中荧光素酶的表达情况
1.D-荧光素盐(YEASEN 40902ES03)用蒸馏水溶解，配置成30mg/ml的储存液(200x)。
2.用预热好的培养基1:200稀释储存液，配置成工作浓度(150μg/ml)。
3.一共17板细胞，弃掉培养皿中原有的培养基，正常的Huh7细胞不加荧光素工作液处理和正常的Huh7细胞加荧光素工作液处理当做对照，挑取单克隆的Huh7-luc细胞1-15加荧光素工作液处理当做实验组，37℃孵育10min，而后利用BioTek酶标仪检测荧光强度。其结果如图1所示，挑取的单克隆细胞7为luciferase过表达的细胞系。
实例3 Huh7-luciferase稳定过表达细胞系的验证
1.按照文献“Targeting KDM1A attenuates Wnt/β-catenin signaling pathwaytoeliminatesorafenib-resistant stem-like cells in hepatocellularcarcinoma,Huang M,Cancer Lett.2017Jul 10；398:12-21”提取Huh7和Huh7-luciferase细胞的RNA，而后反转录成cDNA，进行RT-PCR检测。
2.检测Luciferase基因所需的引物序列如下：
5'CTGAACAGCATGGGCATCA 3'(sense)
5'AAATGGGAAGTCACGAAGGT 3'(antisense)
3.反应体系如下：
| 体系 | 25μl |
| DNA模板(100ng/μl) | 5μl |
| 双引物(2μM) | 3μl |
| Es-tag酶(Takara RR902Q) | 12.5μl |
| ddH<sub>2</sub>O | to 25μl |
4.反应条件
结果如图2所示。
Claims (4)
1.一种稳定高表达荧光素酶的Huh7细胞系，其特征在于，Huh7细胞内含有LuciferaseCDS序列。
2.如权利要求1所述的稳定高表达Luciferase的Huh7细胞系，其特征在于，包括真核表达载体pWPXLd，及真核表达载体上的Luciferase CDS序列。
3.一种稳定高表达Luciferase的Huh7细胞系的构建方法，其特征在于，利用慢病毒包装体系包装成病毒后侵染Huh7细胞，首先利用慢病毒侵染技术，将荧光素酶基因转入到Huh7细胞系中，使其在Huh7细胞里表达。
4.如权利要求3所述的稳定高表达Luciferase的Huh7细胞系，其特征在于，利用极限稀释法将细胞铺到培养皿中，而后再利用单克隆筛选技术获得稳定高表达Luciferase的Huh7细胞系。
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| 周鑫: "稳定表达荧光素酶的MDA-MB-231乳腺癌细胞系的构建及其活性表征", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
| 李雪莹: "用于活体成像技术的表达荧光素酶人肝癌细胞系的建立及验证", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
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