CN105861504B - 短发夹shRNA及其在制备治疗胶质瘤的药物中的应用 - Google Patents
短发夹shRNA及其在制备治疗胶质瘤的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种短发卡shRNA，其核酸序列如SEQ ID No.1所示。短发夹shRNA敲除慢病毒表达载体，是将转录短发夹shRNA的核苷酸序列插入慢病毒载体质粒pGLVH3/H1/GFP+Puro得到；本发明还公开了该短发卡shRNA在制备抑制胶质瘤增殖或迁移的药物中的应用，为胶质瘤的治疗应用提供了一种新方法。
Description
技术领域
本发明属于基因工程领域，具体涉及一种短发夹shRNA，本发明还涉及该短发夹shRNA在制备治疗胶质瘤的药物中的应用。
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。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。目前已经发现很多基因在胶质瘤发生发展中起重要作用，但是至今还有许多与胶质瘤发生发展的基因没有被发现。
DISC1(Disrupted in scherophernia-1)基因最初是从一个具有遗传性精神分裂症的苏格家族中发现的，基因定位在1号染色体的1q42区域，有13个外显子，长约200kb，编码一种含有854个氨基酸残基的蛋白质。DISC1在神经系统中大量表达，参与调节神经发育，可以与细胞内的其他信号分子，比如NDEL1，GSK3β和PDE4B等共同调控神经突触的生长、神经细胞的迁移、神经发生、细胞内cAMP信号以及神经细胞的其他发育过程。
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DISC1基因与胶质瘤发生发展相关，也无文献表明抑制DISC1的表达可以抑制胶质瘤细胞的增殖和侵袭。
发明内容
本发明的目的是提供一种短发卡shRNA，解决了现有技术中存在的问题。
本发明的另一个目的是提供一种短发夹shRNA在制备治疗胶质瘤的药物中的应用。
本发明所采用的技术方案是，短发卡shRNA，其核苷酸序列如SEQ ID No.1所示。
本发明所采用的第二个技术方案是，短发夹shRNA敲除慢病毒表达载体，是将转录短发夹shRNA的核苷酸序列插入慢病毒载体质粒pGLVH3/H1/GFP+Puro所得。
本发明所采用的第三个技术方案是，短发卡shRNA在制备抑制胶质瘤增殖的药物中的应用。
本发明所采用的第四个技术方案是，短发卡shRNA在制备抑制胶质瘤迁移的药物中的应用。
本发明的有益效果是本发明设计DISC1基因的干扰慢病毒载体,并应用脑胶质瘤细胞模型研究其对细胞迁移和侵袭的抑制作用,为胶质瘤的治疗应用提供了一种新方法。
附图说明
图1是pGLVH3/H1/GFP+Puro质粒DNA图谱示意图；
图2是shRNA1抑制U251MG DISC1mRNA表达的结果；
图3是shRNA1抑制U251MG DISC1蛋白表达的结果；
图4是抑制DISC1的表达后抑制U251MG增值的结果；
图5是抑制DISC1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。
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。
实施例仅用于说明本发明，而非限制本发明的范围。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。
短发卡shRNA，其核苷酸序列如SEQ ID No.1所示。
本发明以GeneBank基因库中DISC1已知序列为基础，按照shRNA设计原则，设计shRNA序列，经细胞学干预、荧光定位和RT-PCR基因检测等试验，验证了该序列可以显著抑制DISC1基因的表达。本发明设计的shRNA序列委托genepharma生物公司合成。
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、DISC1基因干扰慢病毒质粒的设计：设计对应于DISC1基因GCAGTTGAGAATGATGATTAT的shRNA的寡核苷酸序列，各为63bp，并进行合成。该shRNA的寡核苷酸序列为：(sense是shRNA序列，anti是与其互补的序列)。
Sense:5'-GATCCGCAGTTGAGAATGATGATTATTTCAAGAGAATAATCATCATTCTCAACTGCTTTTTTG-3'
Anti-sense：5'-AATTCAAAAAAGCAGTTGAGAATGATGATTATTCTCTTGAAATAATCATCATTCTCAACTGCG-3'；
其中正向序列从5’至3’依次为BamHI酶切位点+靶序列+茎环结构+靶序列+RNApolyIII聚合酶转录终止位点+EcoRI酶切位点。
转录产物序列：
GCGTGACATGCATTCTTTACCTTCAAGAGAGGTAAAGAATGCATGTCACGCTT
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，主要步骤如下：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DISC1 shRNA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DISC1基因shRNA敲除慢病毒表达载体(pGLVH3/H1-DISC1-shRNA-1#)。
测序结果(包含插入片段的载体序列)：
TTTCTTTGCAGTTATAAATACTGAATAATAAGATGACATGAACTACTACTGCTAGAGATTTTCCACACTGACTGAAAGGGTCTGAGGGATCTCTAGTTACCAGAGTCACACAACAGACGGGCACACACTACTTGAAGCACTCAAGGCAAGCTTTATTGAGGCTTAAGCAGTGGGTTCCCTAGTTAGCCAGAGAGCTCCCAGGCTCAGATCTGGTCTAACCAGAGAGACCCAGTAGAAGCAAAAAGCAGAATCGAAGAATTCAAAAAAGCGTGACATGCATTCTTTACCTCTCTTGAAGGT AAAGAATGCATGTCACGCGGATCCAAGTGGTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCTCACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACTGGGCCCGCGATTCCTTGGAGCGGGTTGATGACGTCAGCGTTCCAATTCTTGACATCGTTGGGAGTGAATTAGCCCTTCCAGTCCCCCCTTTTCTTTTAAAAAGTGGCTAAGATCTACAGCTGCCTTGTAAGTCATTGGTCTTAAAGGTACCAGGCGGGGAGGCGGCCCAAAGGGAGATCCGACTCGTCTGAGGGCGAAGGCGGAGACGCGGAAGAGGCCGCAGAGCCGGCAGCAGGCCGCGGGAAGGAAGGTCCGCTGGATTGAGGGCCGAAGGGACGTAGCAGAAGGACGTCCCGCGCAGAATCCAGGTGGCAACACAGGCGAGCAGCCAAGGAAAGGACGATGATTTCCCCGACAACACCACGGAATTGTCAGTGCCCAACAGCCGAGCCCCTGTCCAGCAGCGGGCAAGGCAGGCGGCGATGAGTTCCGCCGTGGCAATAGGGAGGGGGAAAGCGAAAGTCCCGGAAAGGAGCTGACAGTGGTGGCAATGCCCCAACCAGTGGGGGTTGCGTCAGCAAACCAGTGCACCCACGCCACCGTTG
三、慢病毒pGLVH3/H1-DISC1-shRNA-1#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DISC1 shRNA的序列的穿梭质粒和包装质粒(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。
四、获得一种稳定敲除DISC1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DISC1的U251细胞株。
五、利用定量PCR法和Western blot法检测shRNA对DISC1转录水平和蛋白表达的抑制作用
利用trizol法提取稳定敲除DISC1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DISC1的U251细胞株中DISC1转录水平的表达量明显下降(见附图2)。
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DISC1抗体(abcam)或对照α-tubulin(Santa Cruz Biotechnology)抗体一抗(DISC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DISC1的U251细胞株中DISC1蛋白水平的表达量明显下降。(见附图3)
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DISC1的胶质瘤细胞增殖能力降低(见附图4)。
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DISC1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DISC1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％(见附图5)。
Claims (2)
1.短发卡shRNA在制备抑制胶质瘤增殖的药物中的应用，所述短发卡shRNA的核苷酸序列如SEQ ID No.1所示。
2.短发卡shRNA在制备抑制胶质瘤迁移的药物中的应用，所述短发卡shRNA的核苷酸序列如SEQ ID No.1所示。
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| Neuropeptide precursor VGF is genetically associated with social anhedonia and underrepresented in the brain of major mental illness: its downregulation by DISC1;Ramos et al;《Human Molecular Genetics》;20140616;Supplementary Table 1、第5863页右栏第5段 |
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