CN109321574B - 抑制ILT5表达的短发卡shRNA、慢病毒及其应用 - Google Patents
抑制ILT5表达的短发卡shRNA、慢病毒及其应用 Download PDFInfo
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Abstract
本发明公开了一种抑制ILT5表达的短发卡shRNA,其核苷酸序列如SEQ ID No.1或SEQ ID No.2所示;本发明还公开了一种ILT5基因shRNA敲低慢病毒表达载体,将抑制ILT5表达的短发卡shRNA插入慢病毒载体质粒pGLVH3/H1/GFP+Puro所得;以及一种ILT5病毒的制备方法制备得到ILT5病毒;本发明还公开了一种ILT5病毒的制备方法以及抑制ILT5表达的短发卡shRNA或慢病毒表达载体或ILT5病毒在制备抑制胶质瘤增殖、抑制胶质瘤迁移、抑制胶质瘤侵袭、抑制胶质瘤干细胞增殖的药物中的应用。
Description
技术领域
本发明属于基因工程技术领域,涉及一种抑制ILT5表达的短发卡shRNA,本发明还涉及一种ILT5基因shRNA敲低慢病毒表达载体、ILT5病毒及其应用。
背景技术
胶质瘤是临床上最常见的中枢神经系统恶性肿瘤,约占所有原发性颅内肿瘤的45%左右,手术、放化疗均不易根治,易复发,治愈率较低,病人存活期短,因此胶质瘤是疗效最差的恶性肿瘤之一。据统计,胶质瘤病人的一年总生存率低于30%,级别VI的病人中位生存期只有大概15个月。胶质瘤预后差的一个主要原因是胶质瘤多呈浸润性生长,肿瘤与正常脑组织无明显界限,手术往往不能将肿瘤组织全部切除从而导致术后复发率较高。
ILT5,也称为CD85a,LILRB3,是一种I型跨膜糖蛋白,由胞外四个免疫球蛋白样(IgG)结构域和胞内四个ITIMs组成。目前关于ILT5的生物学功能所知甚少,早期研究发现ILT5在树突状细胞、B细胞、粒细胞及肥大细胞等细胞表面高表达,认为其作为抑制性白细胞免疫球蛋白样受体,通过胞外段的IgG样结构域与胞外的配体结合,传导抑制信号介导机体的炎症和免疫反应。但是ILT5结合的配体及其调控的信号通路尚未阐明,其在肿瘤中的作用更是鲜有报道。
RNA干扰(RNA interference,RNAi)是指在进化过程中高度保守的、由双链RNA(double-stranded RNA,dsRNA)诱发的、同源mRNA高效特异性降解的现象,是一种转录后基因沉默的机制。目前RNAi的表达载体有很多中,慢病毒表达系统相比于其他表达系统,具有感染效率高、感染时间长、感染效果稳定,可容纳较大的基因片段,不易诱发宿主免疫反应等优点,已成为当前基因治疗和转基因动物中载体研究的热点。
目前尚无研究表明ILT5基因与胶质瘤发生发展相关,也无文献表明抑制ILT5的表达可以抑制胶质瘤细胞的增殖、迁移、侵袭及干细胞干性。
发明内容
本发明的目的是提供一种抑制ILT5表达的短发卡shRNA,能有效抑制ILT5在mRNA和蛋白水平的表达,进而抑制胶质瘤细胞的增殖、迁移、侵袭及干细胞增殖。
本发明的另一目的是提供一种ILT5基因shRNA敲低慢病毒表达载体;
本发明的第三个目的是提供一种ILT5病毒的制备方法。
本发明的第四个目的是提供抑制ILT5表达的短发卡shRNA、慢病毒表达载体以及ILT5病毒的应用。
本发明所采用的第一种技术方案是,抑制ILT5表达的短发卡shRNA,其核苷酸序列如SEQ ID No.1或SEQ ID No.2所示。
本发明所采用的第二种技术方案是,一种干扰质粒,该质粒含有上述核苷酸序列。
本发明所采用的第三种技术方案是,一种ILT5基因shRNA敲低慢病毒表达载体,将抑制ILT5表达的短发卡shRNA插入慢病毒载体质粒pGLVH3/H1/GFP+Puro所得;
以及重组ILT5慢病毒表达载体的构建方法,具体包括:合成用于编码ILT5的核苷酸片段;用限制性内切酶BamHI和EcoRI分别对核苷酸片段和pGLVH3/H1/GFP+Puro载体进行双酶切;将双酶切后的核苷酸片段和pGLVH3/H1/GFP+Puro进行连接,得到重组的ILT5慢病毒载体。
本发明的第四种技术方案是,一种ILT5病毒的制备方法,具体包括:用慢病毒包装质粒pGag/Pol、pRev和pVSV-G对上述的重组的ILT5慢病毒载体进行包装,转染293T细胞后,得到ILT5病毒,其中重组的ILT5慢病毒表达载体、pGag/Pol、pRev和pVSV-G的质量比为(1.5-2.5):(0.5-1.5):(0.6-1.5):(0.5-1.4)。
本发明的第五种技术方案是,抑制ILT5表达的短发卡shRNA或慢病毒表达载体或ILT5病毒在制备抑制胶质瘤增殖或抑制胶质瘤迁移或抑制胶质瘤侵袭或抑制胶质瘤干细胞增殖的药物中的应用。
本发明的有益效果是:
本发明的抑制ILT5表达的短发卡shRNA、慢病毒表达载体、ILT5病毒能有效抑制抑制胶质瘤增殖、迁移、侵袭及其干细胞增殖,为胶质瘤的治疗应用提供了一种新方法。
本发明为制备治疗人胶质瘤的药物提供了一种新的方法,可促进人胶质瘤新药的开发,获得稳定抑制ILT5表达的胶质瘤细胞株,为研究ILT5基因功能提供了实验材料,补充了ILT5基因在胶质瘤中的功能研究空白。
本发明所述的shRNA不仅可以用于制备人胶质瘤的药物,可以用于ILT5基因在人胶质瘤发生发展中的作用及机制研究。
附图说明
图1是本发明pGLVH3/H1/GFP+Puro质粒DNA图谱示意图;
图2是本发明shRNAs抑制U251MG ILT5mRNA表达的结果;
图3是本发明shRNAs抑制U251MG ILT5蛋白表达的结果;
图4是本发明抑制ILT5的表达后抑制U251MG增值的结果;
图5是抑制ILT5的表达抑制U251MG细胞迁移的结果;A:划痕实验不同时间点的拍照;B:不同时间点划痕愈合宽度的量化结果;
图6显示了抑制ILT5表达有效减少胶质瘤细胞的侵袭A:显微镜视野;B:A图的数据统计;
图7是抑制ILT5的表达后有效抑制U251MG干细胞增殖。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
本实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照常规条件,如分子克隆试验指南,第三版。北京:科学出版社2008中所述的条件。
一、胶质瘤细胞的培养:
胶质瘤细胞U251MG细胞系(购买自中国科学院上海生命科学院细胞资源中心)培养于37℃,5%CO2恒温培养箱。对数生长期的细胞用胰酶消化脱壁,之后加入适量培养液并反复吹打以混匀细胞;将得到的细胞悬液移入15ml离心管中,1000rPm离心3min;去上清,用4ml培养基将细胞悬起,吹打混匀;加约3ml细胞悬液于装有6ml新鲜培养基的10cm培养皿,放入37℃,5%CO2培养箱中培养,约3天左右传代1次(主要查看细胞有无铺满培养瓶)。
二、ILT5基因干扰慢病毒质粒的设计:设计对应于ILT5基因的shRNA的核苷酸序列:
ILT5-shRNA-1#GGAGTACCAACTGGATAAAGAGG
ILT5-shRNA-2#GGTGTTCCTCGGGATTCTGTTAT
各为67bp,并进行合成,该shRNAs的寡核苷酸序列为(sense是shRNA序列,anti是与其互补的序列):
ILT5-shRNA-1#
Sense:5'-GATCCGGAGTACCAACTGGATAAAGAGGTTCAAGAGACCTCTTTATCCAGTTGGTACTCCTTTTTTG-3'
Anti-sense:5'-AATTCAAAAAAGGAGTACCAACTGGATAAAGAGGTCTCTTGAACCTCTTTATCCAGTTGGTACTCCG-3';
ILT5-shRNA-2#
Sense:5'-GATCCGGTGTTCCTCGGGATTCTGTTATTTCAAGAGAATAACAGAATCCCGAGGAACACCTTTTTTG-3'
Anti-sense:5'-AATTCAAAAAAGGTGTTCCTCGGGATTCTGTTATTCTCTTGAAATAACAGAATCCCGAGGAACACCG-3';
主要步骤如下:本发明所用的慢病毒载体(pGLVH3/H1/GFP+Puro,见附图1)由吉玛基因公司合成,用BamHI和EcoRI双酶切穿梭质粒(上海吉玛制药技术有限公司,商品号:C06003),得到载体大片段,将上述模板序列(由ILT5shRNA的寡核苷酸sense与anti-sense序列退火形成)与载体大片段连接,得到重组穿梭质粒,测序正确后就构建成功ILT5基因shRNA敲除慢病毒表达载体(pGLVH3/H1-ILT5-shRNA)。
测序结果(包含插入片段的载体序列):
ILT5-shRNA-1#:
CAACGGTGGCGTGGGTGCACTGGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTCCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGATGTCAAGAATTGGAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTTGGATCCGGAGTACCAACTGGATAAAGAGGTTCAAGAGACCTC TTTATCCAGTTGGTACTCCTTTTTTGAATTCTTCGATTCTGCTTTTTGCTTCTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTCAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTGCAAAGAAA
ILT5-shRNA-2#:
CAACGGTGGCGTGGGTGCACTGGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACTGTCAGCTCCTTTCCGGGACTTTCGCTTTC CCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTCCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGATGTCAAGAATTGGAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTTGGATCCGGTGTTCCTCGGGATTCTGTTATTTCAAGAGAA TAACAGAATCCCGAGGAACACCTTTTTTGAATTCTTCGATTCTGCTTTTTGCTTCTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTCAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTGCAAAGAAA
三、慢病毒pGLVH3/H1-ILT5-shRNA的包装
293T细胞在10cm培养皿中培养至80-90%融合时,接种15cm培养皿;倾去培养液,用1ml D-Hank’s solution洗涤细胞两次;加入1ml Trypsin-EDTA solution,混匀后,37℃放置2-3分钟;小心吸去胰酶溶液,加入2ml含10%FBS的DMEM培养液,吹打使细胞形成单细胞悬液;将细胞悬液接种15cm培养皿,加入18ml含10%FBS的DMEM培养液,混匀后37℃、5%CO2培养过夜;在一支无菌的5ml离心管中加入1.5ml无血清DMEM,按比例加入ILT5shRNA的序列的穿梭质粒和包装质粒(pGag/Pol、pRev、pVSV-G),混匀质量比为(pGLVH3/H1-ILT5-shRNA:pGag/Pol:pRev:pVSV-G=2:1:1:1),取另一支无菌的5ml离心管,加入1.5ml无血清DMEM,再加入300ul RNAi-Mate,混匀,室温放置5分钟后将两管混合,室温放置20~25分钟;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4-6小时;吸弃转染液,加入18ml含10%FBS的DMEM培养液。37℃、5%CO2继续培养72小时;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0.45um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1.5mlEP管中,-80℃冰箱保存。
空载系统慢病毒制备同上。
四、获得一种稳定敲除ILT5基因的U251MG细胞株
采用含10%FBS的DMEM培养基培养U251MG(购自中国科学院上海细胞所),待细胞密度生长至约60%时进行靶细胞感染。采用MOI值为10的慢病毒感染U251MG细胞,用含有终浓度为3pg/ml嘌呤霉素的培养基传代筛选得到稳定敲除ILT5的U251细胞株。
五、利用定量PCR法和Western blot法检测shRNA对ILT5转录水平和蛋白表达的抑制作用
由primer premier设计出检测ILT5基因表达量的引物:
ILT5-qrtF:GTGGAGCTGGACAGTCAGAG
ILT5-qrtR:CAGACAGTGAGGAGGGAGGA
利用trizol法提取稳定敲除ILT5的U251细胞株总RNA,消化DNA后,利用反转录试剂盒反转录成cDNA;
定量PCR体系为:10μl的Premix Ex Taq(SYBR qPCR)(2×),10μmol/L的Bmi1上下游引物各0.5μl,待检cDNA模板或阴性对照1μl,加灭菌去离子水补足体积至20μl。
PCR反应条件为:95℃预变性30s;然后95℃15s,60℃20s,72℃20s,反应40个循环。
发现敲除ILT5的U251细胞株中ILT5转录水平的表达量明显下降,如图2所示。
收集细胞蛋白样品进行电泳。电泳时,按每孔50μg蛋白上样量等量上样。以400mA恒压转膜100min。将膜放入1%酪蛋白/TBS中于室温下振荡封闭1h。加入TBST稀释后的ILT5抗体(abcam)或对照α-tubulin(Santa Cruz Biotechnology)抗体一抗(ILT5抗体按1:2000稀释,抗α-tubulin抗体按1:2000稀释),4℃振荡孵育过夜。吸净一抗,TBST漂洗膜10min×3;加入稀释后的二抗(均为1:4000稀释),水平摇床室温孵育1h。吸净二抗,TBST漂洗膜,10min×4,混合ECL(A:B=100:1液),充分覆盖湿润NC膜表面,静置1min;于暗室中用保鲜膜将NC膜封好,将X光片平放于NC膜上显影并记录数据。结果可见,发现敲除ILT5的U251细胞株中ILT5蛋白水平的表达量明显下降,如图3所示。
六、胶质瘤增殖实验
取对数生长期的胶质瘤细胞接种于96孔板,每孔5000个细胞,每隔24h加入CCK-8在450nm处读数,根据吸光值分析细胞增殖的能力。发现与对照组相比,敲除ILT5的胶质瘤细胞增殖能力降低,如图4所示。
七、胶质瘤细胞的迁移实验
采用“划痕法”对胶质瘤细胞的迁移活性进行研究。敲除ILT5的U251细胞株细胞与对照细胞株先换无血清的胶质瘤细胞培养液继续培养细胞12h对其进行饥饿干预。此时细胞应布满培养板底,形成单层细胞。用200μl灭菌枪头匀速匀力的向一个方向划单层胶质瘤细胞,每孔平行的划1次,形成1条无细胞划痕区。PBS清洗细胞2次,加入无血清的胶质瘤细胞培养。在显微镜下用记号笔在培养板底部为细胞划痕边缘做记号并拍摄照片。在0小时,12小时和24小时镜下观察划痕两侧的距离变化并拍照。用Image-pro plus 6.0软件计算刺激前后划痕两侧的距离。结果发现,敲除ILT5的胶质瘤细胞迁移活性降低,划痕愈合为12小时是17%,24小时为23%。而对照组划痕愈合率为12小时是20%,24小时为29%,如图5所示。
八、胶质瘤细胞侵袭实验
利用transwell法对胶质瘤的侵袭活性进行研究。ILT5的U251细胞株细胞与对照细胞株细胞无血清饥饿24小时,用无血清培养基稀释,种5×105个细胞在transwell小室中(Chemicon公司的ECM550),下面加入含10%血清的胶质瘤细胞培养液,培养48小时后,用棉签擦去上层未侵袭的细胞,用100甲醇固定10min后空气中晾干,加入DAPI染核,在荧光显微镜下观察并拍照,随机数10个视野中的细胞数目,敲除ILT5的胶质瘤细胞侵袭活性降低,如图6所示。
九、胶质瘤干细胞增殖实验
ILT5的U251细胞株细胞与对照细胞株细胞经胰酶消化后,用PBS洗两次,每次3min,以5×105个/ml的浓度种植于6孔板中,每2天加入培养基0.5ml培养基,7天后收集神经球,经胰酶消化后一以1×105个/ml的浓度细胞种植于6孔板中,每2天加入培养基0.5ml培养基,七天后再次收集U251胶质瘤神经球,经胰酶消化后一以1×105个/ml的浓度细胞种植于6孔板中,每2天加入培养基0.5ml培养基,14天后拍照观察干细胞球的直径。结果发现抑制ILT5的表达可以限制抑制胶质瘤干细胞的增殖,如图7所示。
SEQUENCE LISTING
<110> 西安医学院
<120> 抑制ILT5表达的短发卡shRNA、慢病毒及其应用
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 1
ggagtaccaa ctggataaag agg 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial sequence)
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ggtgttcctc gggattctgt tat 23
Claims (7)
1.抑制ILT5表达的短发卡shRNA,其特征在于,其核苷酸序列如SEQ ID No.2所示。
2.一种干扰质粒,其特征在于,该质粒含有权利要求1所述的核苷酸序列。
3.一种ILT5基因shRNA敲低慢病毒表达载体,其特征在于,将权利要求1所述的抑制ILT5表达的短发卡shRNA插入慢病毒载体质粒pGLVH3/H1/GFP+Puro所得。
4.根据权利要求1或3所述的抑制ILT5表达的短发卡shRNA或慢病毒表达载体在制备抑制胶质瘤增殖的药物中的应用。
5.根据权利要求1或3所述的抑制ILT5表达的短发卡shRNA或慢病毒表达载体在制备抑制胶质瘤迁移的药物中的应用。
6.根据权利要求1或3所述的抑制ILT5表达的短发卡shRNA或慢病毒表达载体在制备抑制胶质瘤侵袭的药物中的应用。
7.根据权利要求1或3所述的抑制ILT5表达的短发卡shRNA或慢病毒表达载体在制备抑制胶质瘤干细胞增殖的药物中的应用。
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