CN108384784A - 一种利用CRISPR/Cas9技术敲除Endoglin基因的方法 - Google Patents

一种利用CRISPR/Cas9技术敲除Endoglin基因的方法 Download PDF

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CN108384784A
CN108384784A CN201810243301.5A CN201810243301A CN108384784A CN 108384784 A CN108384784 A CN 108384784A CN 201810243301 A CN201810243301 A CN 201810243301A CN 108384784 A CN108384784 A CN 108384784A
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endoglin
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赵永祥
钟莉娉
阳诺
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Guangxi Medical University
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Abstract

本发明公开一种利用CRISPR/Cas9技术敲除Endoglin基因的方法，属于基因工程、免疫学和肿瘤学技术领域。所述方法含有针对Endoglin基因敲除的CRISPR/Cas9的gRNA序列；所述序列如SEQ ID NO:1序列；所述针对Endoglin基因敲除的CRISPR/Cas9的gRNA序列的设计选择在第一外显子。本发明进一步研究了Endoglin在恶性肿瘤细胞发生、发展及转移过程中不可或缺的作用，并探讨利用CRISPR/Cas9基因编辑技术对小鼠肝癌模型进行治疗的效果，为临床的肿瘤治疗提供一种新的思路。

Description

一种利用CRISPR/Cas9技术敲除Endoglin基因的方法
技术领域
本发明属于基因工程、免疫学和肿瘤学技术领域，特别涉及一种利用CRISPR/Cas9技术敲除Endoglin基因的方法及其应用。
背景技术
肿瘤(Tumor) 是机体在各种致癌因素作用下，局部组织的某一个细胞在基因水平上失去对其生长的正常调控，导致其克隆性异常增生而形成的异常病变而产生的，20世纪后期近30年以来，癌症发病一直呈上升的趋势，据世界卫生组织(WHO)报告,1990年全球癌症新发病例数约807万，比1975年的517万增加了37.4%，而1997年全球的癌症死亡数约620万，并且按目前的趋势预测，至2020年随着世界人口达80亿，将有2000万新发癌症病例，其中死亡人数将达1200万，且其中绝大部分将发生在发展中国家；几十年来，由于生态环境的不断恶化、艾滋病等疾病的传播，恶性肿瘤的发病率呈上升趋势，并成为继心血管疾病之后的第二大致死病因，特别是肝癌，是威胁全世界人类健康及生命重要的因素之一，其中又以实体肿瘤最多见。实体肿瘤的发生、增长以及转移的基础是源于肿瘤内血管的形成和发展，是肿瘤细胞进行新陈代谢途径的关键。肿瘤血管的形成主要包括两个阶段，即肿瘤血管形成前期和血管形成后期。在肿瘤血管形成前期，肿瘤细胞仅仅依靠单纯的细胞增殖能力进行平稳的增殖，单纯扩散的方式进行营养物质的摄取与代谢产物的排放，形成无血管结构的细胞团，因此肿瘤的增殖能力较弱，肿瘤仅仅能够生长至几立方毫米左右，当这种简单的物质代谢途径不能满足肿瘤增长时，肿瘤即进入一个相对静止的增长期。在肿瘤血管形成后期，肿瘤细胞可以释放多种细胞因子，从而诱导肿瘤血管的形成，在机体本身的血管组织向肿瘤部位发出毛细血管幼芽，当形成肿瘤新生血管时，肿瘤细胞的营养供给充足，进入对指数生长阶段，并且通过血管对肿瘤细胞向向邻近组织进行输送，发生转移。由此可见，肿瘤中微血管形成越多，肿瘤的生长速度越快，转移及扩散越广泛，因此发现一种可靠的血管标志物来作为肿瘤临床诊断及治疗的依据。
基因编辑技术（Gene Editing Technology），作为人们研究基因功能的主要方法，近年来一直飞速发展，而CRISPR/Cas9基因编辑技术和转录激活因子样效应子核酸酶(Transcription activator-like effector nucleases, TALEN）技术作为近几年的热门技术，是众多学者研究和应用的对象。规律间隔的短回文重复序列（clustered regularlyinterspaced short palindromic repeats, CRISPR）简称CRISPR序列，其系统主要由两部分构成为重复序列和间隔序列交替排列所形成的CRISPR基因座和其上游的Cas蛋白家族的操纵子。其中，CRISPR基因座中的间隔序列(protospacers)源自于细菌对外源性DNA的拷贝，具有特异性识别外源性DNA的功能。
Endoglin是一种胞膜形成所必须的蛋白质，主要在人类内皮细胞表面和胎盘滋养层细胞表面表达。有研究表明，在增殖活跃的肿瘤细胞中可以检测到Endoglin的高表达，同时也有研究在临床样本检测及动物实验中得出结论，在新生血管内皮细胞表面Endoglin表达程度明显升高，尤其是在内皮细胞增至状态良好，呈对指数增长时高表达，因此通过以上研究表明，Endoglin可以作为新生血管内皮细胞的主要标志，而且Endoglin的表达程度的高低可以用来衡量血管内皮细胞的增至状态。
因此，提出一种利用CRISPR/Cas9技术敲除Endoglin基因的方法及其应用是本领域技术人员研究的新的思路。
发明内容
本发明的目的之一是利用CRISPR/Cas9基因编辑技术对小鼠肝癌模型进行治疗的效果，为临床的肿瘤治疗提供一种新的思路。
本发明的另一目的在于提供一种利用CRISPR/Cas9技术敲除人高转移性肝癌细胞株HCC-LM3的Endoglin（CD105）基因的方法。
本发明的再一目的是提供一种新型细胞并提供该新型细胞的应用。
为实现本发明目的所使用的技术方案为：
一种利用CRISPR/Cas9技术敲除Endoglin基因的方法，所述方法含有针对Endoglin基因敲除的CRISPR/Cas9的gRNA序列；所述序列如SEQ ID NO:1序列。
进一步地，所述针对Endoglin基因敲除的CRISPR/Cas9的gRNA序列的设计选择在第一外显子。
进一步地，所述序列将SEQ ID NO：1序列经过一个或几个核苷酸的取代或缺失或添加且与SEQ ID NO：1序列具有相同功能的DNA分子。
进一步地，所述的方法将设计的SEQ ID NO:1序列插入到CRISPR/Cas9载体中，得到如如SEQ ID NO:2序列，保种命名为Px458-gRNA。
进一步地，所述方法是利用CRISPR/Cas9技术敲除人高转移性肝癌细胞系HCC-LM3的Endoglin基因构建靶向CD105的新型细胞。
进一步地，所述Endoglin基因在肝癌细胞增殖迁移中的应用。
一种治疗或预防肝癌的药物，包括以上所述的靶向CD105的新型细胞
本发明突出的实质性特点和显著的进步是：
本发明主要是应用CRISPR/Cas9技术，敲除人高转移性肝癌细胞株HCC-LM3的Endoglin（CD105）基因，在体外通过迁移实验等评价Endoglin（CD105）基因对LM3细胞迁移和增殖能力的影响；并通过BalB/C裸小鼠体内成瘤实验观察小鼠生存期、肿瘤体积等指标并进行分析，进一步研究Endoglin在恶性肿瘤细胞发生、发展及转移过程中不可或缺的作用，并探讨利用CRISPR/Cas9基因编辑技术对小鼠肝癌模型进行治疗的效果，为临床的肿瘤治疗提供一种新的思路。
本发明利用CRISPR/Cas9基因编辑技术，对靶基因定点沉默，敲除人高转移性肝癌细胞株HCC-LM3（以下简称LM3）的Endoglin基因。通过设计靶向人Endoglin基因sgRNA，成功插入到CRISPR/Cas9系统载体Px458中后，在裸鼠荷瘤实验中对本研究的实验结果进行验证。在体外，经凋亡实验、迁移实验（Transwell assey）、流式细胞术等实验证明，敲除了Endoglin基因的LM3细胞增殖、转移及迁移能力明显下降，细胞凋亡率明显增高，同时体内实验结果发现与体外实验具有相同的结论：应用CRISPR技术构建的针对于人Endoglin基因的质粒对小鼠进行治疗后，肿瘤生长速度受到抑制，小鼠生存期明显延长，明显高于Px458-EGFP组和PBS，进入血管生成后期的时间明显延长，使肿瘤细胞的对指数生长阶段受到抑制甚至消除该阶段，在发现肿瘤的早期使肿瘤停留在血管形成前期。
综上所述，利用CRISPR/Cas9基因编辑技术，将人Endoglin基因作为靶向基因进行敲除，在肿瘤生血管形成过程中起到关键作用，能够有效干扰这一通路，使肿瘤的生长受到明显的抑制，因此，Endoglin在日后对于人类恶性肿瘤诊断、预后以及治疗方面势必会发展成为一个思路，CRISPR/Cas9基因编辑技术在肿瘤免疫学及治疗学势必成为一个全新且更加有力的武器。
附图说明
图1是实施例2临床收取的肿瘤组织标本和癌旁组织标本免疫组织化学方法示意图，其中A为肿瘤组织标本，B为癌旁组织标本，C为统计学分析。
图2是实施例2 sgRNA插入Px458载体的测序峰示意图。
图3是实施例2 sgRNA重组质粒转染LM3细胞的荧光示意图，其中A为转入细胞24小时，B为转入细胞48小时。
图4是实施例2野生型与Endoglin 敲除的LM3细胞进行T7E1酶切示意图，其中A为T7E1酶切结果，B为mRNA水平比较结果。
图5是实施例2野生型与Endoglin 敲除的LM3细胞全基因组作为模版扩增Endoglin片段的PCR产物测序峰图，其中A为野生型LM3基因的测序峰图，B为Px458-sgRNA重组质粒的测序峰图，C为T-A克隆比对结果。
图6是实施例2 野生型与Endoglin 敲除的LM3细胞Transwell迁移实验结果，其中A为迁移结果图，B为水平比较结果。
图7是实施例2野生型与Endoglin 敲除的LM3细胞细胞集落形成实验结果，其中A为集落形成结果，B为水平比较结果。
图8是实施例2野生型与Endoglin 敲除的LM3细胞流式细胞术检测结果，其中A为检测结果，B为水平比较结果。
图9是实施例2BalB/C裸鼠荷瘤实验结果，其中A为肿瘤生长速度，B为 肿瘤质量，C为小鼠生存率。
图10是实施例2流式检测小鼠肿瘤原代细胞Endoglin表达结果，A为检测结果，B为水平比较结果。
具体实施方式
下面结合实施例对本发明方案做进一步详细描述，下述说明仅是为了解释本发明，并不对其内容进行限定。
实施例1
1.1材料与方法
1.1.1 实验材料
从广西医科大学附属肿瘤医院肝胆外科收取肝癌组织及癌旁组织，通过免疫组织化学方法对肝细胞癌患者肿瘤组织及癌旁组织进行Endoglin表达量的检测；人高转移性肝癌细胞系HCCLM3购自于上海复祥生物有限公司；实验所用雌性BalB/C裸小鼠，出生（4-6）周龄，体重（18-20）克，购自于广西医科大学医学实验动物中心，饲养于SPF级小鼠饲养房。所有动物实验方案均遵循广西医科大学动物保护与使用管理条例，并且通过广西医科大学伦理委员会认证；CRISPR/Cas9 系统质粒载体Px458购自于普如汀生物技术（北京）有限公司；一切实验相关试剂、设备及耗材均由广西医科大学生物靶向诊治研究中心提供。
1.1.2 针对于CD105基因敲除的CRISPR/Cas9载体构建
通过Uniprot蛋白数据对CD105 蛋白保守功能区预测，gRNA的设计选择在第一外显子，通过CRISPR在线设计工具（http：//crispr.mit.edu/）设计针对于Endoglin基因的gRNA（一般设计3-5条），将所设计gRNA插入到CRISPR/Cas9载体Px458中，测序结果显示插入成功后保种命名为Px458-gRNA。
1.1.3 体外实验
将Px458-gRNA质粒转染到细胞中，48-72小时候提取细胞全基因组，通过T7E1酶切验证其对于CD105是否具有敲除作用后，以及mRNA水平的检测转染Px458-gRNA质粒后其mRNA水平是否降低；将具有敲除作用的Px458-gRNA选出进行细胞功能试验，主要分为：细胞迁移实验、集落形成实验、凋亡实验验证敲除CD105后的LM3细胞功能的变化。
1.1.4 体内实验
利用LM3细胞对于小鼠进行移植瘤成瘤实验。将BalB/C雌性裸鼠用随机数表法随机分成3组，即PBS组、无关对照组和治疗组。其中治疗组采用肿瘤局部注射Px458-gRNA质粒进行治疗，无关对照组利用针对于EGFP基因敲除的Px458质粒（以下简称Px458-EGFP）进行肿瘤局部注射治疗，PBS组则在肿瘤局部注射等体积PBS。治疗23天后观察各组小鼠肿瘤生长速度、肿瘤质量及小鼠生存期；将小鼠肿瘤取出后提取肿瘤原代细胞，检测各组小鼠肿瘤细胞中CD105的表达量。
1.1.5 统计学分析
每一项实验至少重复三次，所得数据均采用SPSS16.0软件及GraphPad Prism 6.0软件进行统计分析，绘制图片等。实验数据以均数±标准差（Mean±SD）表示，组间比较采用Student’s-t test进行分析。ns表示P ＞ 0.05，*表示P ＜ 0.05，**表示P ＜ 0.01。当P值小于0.05时认为差异有统计学意义。
实施例2
2 结果
2.1 CD105在临床患者肝癌组织中高表达
通过免疫组织化学的方法对临床收取的肝细胞癌患者标本进行检测分析，肿瘤组织标本及癌旁组织标本见图1A/B，可见CD105+细胞数在肿瘤组织中明显多于癌旁组织，在图1C的统计学分析中显示，在肿瘤组织中CD105+细胞数明显多于癌旁组织，**P<0.01。
2.2测序峰图结果显示sgRNA已经成功插入Px458载体
图2-A/B. 所设计sgRNA序列“GGACCGCGGCACGCTCCCTC”已经成功插入到Px458载体中，Px458-gRNA 1/2号单克隆质粒测序结果中目的序列已用黑色字体标出。
2.3 sgRNA重组质粒成功转染LM3细胞
Px458质粒载体本身带有增强型绿色荧光蛋白（Enhanced Green FluorescentProtein, EGFP）基因，因此在Lipo3000转染试剂介导下成功转入细胞（24～48）小时后，细胞则表达绿色荧光蛋白，在488纳米波长激发光EGFP发出绿色荧光（图3）
2.4 T7E1核酸内切酶验证实验结果显示敲除成功、EndoglinmRNA水平降低
转染了Px458-sgRNA重组质粒，并且表达绿色荧光蛋白的LM3细胞经过扩大培养后，收集细胞并提取LM3全基因组，同时提取野生型LM3细胞全基因组作为对照。分别使用Taq酶、Endoglin上下游引物，以野生型LM基因组作为模版扩增Endoglin目的基因后，升温至95度后利用梯度温度PCR仪进行退火，将退火后的PCR产物利用T7核酸内切酶进行酶切，将酶切产物注入C泳道，作为Px458-EGFP组；同样方法以转染了Px458-sgRNA重组质粒的LM3基因组作为模版扩增Endoglin目的基因后T7E1酶切产物注入K泳道；将Marker注入M泳道，所得T7E1酶切结果见图4-A.
为了进一步观察Endoglin在LM3细胞中是否被敲除，我们通过实时定量PCR技术对野生型和Endoglin基因敲除的细胞进行检测，同时用GAPDH做归一化处理，两组细胞中EndoglinmRNA水平比较结果见图4-B。
2.5 LM3-Px458细胞测序结果显示敲除成功
分别用野生型与Endoglin 敲除的LM3细胞全基因组作为模版扩增Endoglin片段的PCR产物测序结果如下图所示：野生型LM3基因组扩增Endoglin的PCR产物较为单一，峰图整齐（图5-A）；用转染了Px458-sgRNA重组质粒的细胞全基因组为模版的PCR产物测序峰图则较为杂乱，存在一部分矮小的杂峰，说明PCR产物中存在一些突变的Endoglin片段，证明CRISPR/Cas9系统在细胞中发挥作用，产生了敲除作用（图5-B）。
T-A克隆比对结果（图5-C）显示，Mutation-1、Mutation-2、Mutation-3分别在Endoglin基因靶位点处存在（5～7）个基因的缺失突变，说明敲除成功。
2.6细胞迁移实验表明敲除Endoglin基因后的LM3细胞迁移能力降低
Transwell迁移实验结果表明，转染了Px458质粒的细胞迁移能力明显比WT组细胞降低，细胞明显比WT组少，**P<0.01，差异有统计学意义（见图6）。
2.7细胞集落形成实验
通过细胞集落形成实验观察两组细胞在低密度下形成克隆的能力，结果见下图。KO组细胞形成克隆数明显比WT组减少，**P<0.01，差异有统计学意义（见图7）。
2.8流式细胞术检测Endoglin基因敲除后细胞凋亡率
将CRISPR/Cas9系统载体Px458-gRNA转染进入LM3细胞，培养72小时后进行7AAD-FITC和ANNEXIN-PE流式抗体孵育，同时采用野生型LM3细胞作为对照。检测结果显示，Endoglin基因敲除后的LM3细胞72小时后凋亡率达到20%以上，高于野生型LM3细胞（WT组）凋亡率，**P<0.01，差异有统计学意义（见图8）。
2.9 BalB/C裸鼠荷瘤实验
将野生型LM3细胞通过皮下注射种植到BalB/C裸小鼠腋下（n=18），如图9所示，通过体内肿瘤局部转染Px458-sgRNA重组质粒的处理方式与另外两组相比，可以有效抑制肿瘤的生长速度（P< 0.05），差异有统计学意义（图9-A）；在肿瘤生长至23天时将所有小鼠处死后称量肿瘤组织质量结果发现，Px458-sgRNA组肿瘤组织质量低于Px458-EGFP组、PBS组肿瘤质量（P< 0.05）（图9-B）。以同样的分组原则及处理方式重新建模，观察小鼠生存率结果如图9-C，其结果显示Px458-sgRNA组小鼠和生存期高于Px458-EGFP组和PBS组小鼠（P<0.05）。
2.10流式检测小鼠肿瘤原代细胞Endoglin表达情况
将三组小鼠处死后，取出皮下移植瘤制成肿瘤原代细胞悬液，与CD105-FITC共孵育后上机检测，结果见下图. PBS组肿瘤细胞中Endoglin表达率平均为0.9%，高于Px458-sgRNA组（P<0.05），而与Px458-EGFP组之间差异无统计学意义（P>0.05）。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等，均应包含在本发明的包含范围之内。
序列表
<110> 广西医科大学
<120> 一种利用CRISPR/Cas9技术敲除Endoglin基因的方法
<141> 2018-01-02
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence Latin
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gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 720
taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 780
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gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900
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cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg 2700
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cctggcccag tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 5640
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cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta 6720
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cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 6840
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aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc 7140
gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 7200
caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc 7260
atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 7320
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 7380
gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 7440
ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 7500
tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 7560
gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 7620
acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 7680
cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 7740
catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 7800
tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 7860
cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 7920
accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 7980
cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 8040
ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 8100
accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 8160
ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 8220
attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 8280
ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat 8340
tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 8400
tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 8460
gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 8520
tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 8580
ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 8640
ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 8700
agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 8760
cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 8820
caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 8880
tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 8940
ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 9000
ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 9060
gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 9120
gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 9180
tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 9240
cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 9290

Claims (7)

1.一种利用CRISPR/Cas9技术敲除Endoglin基因的方法，其特征在于，所述方法含有针对Endoglin基因敲除的CRISPR/Cas9的gRNA序列；所述序列如SEQ ID NO:1序列。
2.根据权利要求1所述利用CRISPR/Cas9技术敲除Endoglin基因的方法，其特征在于，所述针对Endoglin基因敲除的CRISPR/Cas9的gRNA序列的设计选择在第一外显子。
3.根据权利要求1所述利用CRISPR/Cas9技术敲除Endoglin基因的方法，其特征在于，所述序列将SEQ ID NO：1序列经过一个或几个核苷酸的取代或缺失或添加且与SEQ ID NO：1序列具有相同功能的DNA分子。
4.根据权利要求1所述利用CRISPR/Cas9技术敲除Endoglin基因的方法，其特征在于，所述的方法将设计的SEQ ID NO:1序列插入到CRISPR/Cas9载体中，得到如如SEQ ID NO:2序列，保种命名为Px458-gRNA。
5.根据权利要求1所述的利用CRISPR/Cas9技术敲除Endoglin基因的方法，其特征在于，所述方法是利用CRISPR/Cas9技术敲除人高转移性肝癌细胞系HCC-LM3的Endoglin基因构建靶向CD105的新型细胞。
6.根据权利要求4所述的利用CRISPR/Cas9技术敲除Endoglin基因的方法，其特征在于，所述Endoglin基因在肝癌细胞增殖迁移中的应用。
7.一种治疗或预防肝癌的药物，其特征在于，包括权利要求5所述的靶向CD105的新型细胞。
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