CN116377061B - 乳腺癌新辅助化疗耐药标志物及其应用 - Google Patents
乳腺癌新辅助化疗耐药标志物及其应用 Download PDFInfo
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Abstract
本发明公开了一种乳腺癌新辅助化疗耐药标志物及其应用。本发明通过检测CD96在肿瘤细胞中的表达判断乳腺癌患者化疗耐药及预后情况，并且能通过靶向CD96促进乳腺癌细胞对化疗药物的敏感性。本发明通过自身工作中临床乳腺癌组织标本、队列随访数据和体外细胞模型的分子生物学研究发现：本身在免疫细胞表面表达的CD96在部分患者乳腺癌细胞或乳腺癌细胞系中呈高表达，并与乳腺癌化疗后的不良预后呈正相关；经进一步免疫荧光、流式细胞实验显示CD96抑制乳腺癌细胞对新辅助化疗药物的敏感性，敲低CD96的配体CD155能起着与CD96的抑制相同的促化疗效果，突变CD96与CD155的结合位点能明显抑制CD96的耐药调控作用。
Description
技术领域
本发明涉及一种新辅助化疗耐药标志物及其应用，属于生物医学技术领域，特别是涉及乳腺癌新辅助化疗耐药标志物CD96及其在预测乳腺癌新辅助化疗耐药及预后情况的应用。
背景技术
乳腺癌是女性第一大恶性肿瘤，虽然目前的治疗方案能维持早期乳腺癌患者10年总生存率在80％以上，但大多患者诊断时肿瘤比较大或者出现转移等情况，给手术带来很大的难题，使治疗效果不理想，最终给家庭和社会带来沉重的经济负担。
新辅助化疗是乳腺癌治疗的主要方式之一，其优点有：第一，可以消除微小的转移病灶，减少术中播散的机会。其次，可以缩小肿瘤、降期，提高根治性手术的切除率。再次，可以判断肿瘤对化疗药物的敏感性。
但新辅助治疗的方案有多种，怎么预测患者是否对这些药物敏感目前并没有相应的方案，延误了患者的治疗时机和给肿瘤进展提供了更多的机会。
因此，尽早预测治疗耐药的患者、给予替代方案的干预显得尤为重要。
发明内容
本发明的目的是提供一种新的能预测乳腺癌化疗耐药的标记物CD96，通过检测CD96在肿瘤细胞中的表达判断乳腺癌患者化疗耐药及预后情况，并且能通过靶向CD96促进乳腺癌细胞对化疗药物的敏感性。
本发明的目的及解决其技术问题是采用以下技术方案来实现的。
本发明提供检测CD96表达水平的试剂在制备用于预测乳腺癌新辅助化疗耐药及预后情况的产品中的应用。
进一步而言，所述检测CD96表达水平的试剂包括检测CD96基因表达水平的引物。
更进一步而言，所述检测CD96基因表达水平的引物如SEQ ID NO.1～2所示。
本发明提供一种预测乳腺癌新辅助化疗耐药及预后情况的试剂盒，所述试剂盒包括检测CD96表达水平的试剂。
进一步而言，所述检测CD96表达水平的试剂包括检测CD96基因表达水平的引物的试剂；所述检测CD96基因表达水平的引物如SEQ ID NO.1～2所示。
进一步而言，所述试剂盒包括核酸提取试剂、PCR试剂、基因特异性引物或探针中的一种或多种。
本发明提供CD96抑制剂在制备提高乳腺癌新辅助化疗敏感性的产品中的应用。
进一步而言，所述CD96抑制剂选自CD96中和抗体，和/或基于CD96设计的shRNA，和/或基于其上游调控基因CD155设计的sgRNA，和/或突变CD155与CD96的第75个氨基酸的结合位点的试剂。
更进一步而言，所述基于CD96设计的shRNA如SEQ ID NO.3和/或SEQ ID NO.4所示；所述基因CD155的靶标序列如SEQ ID NO.5所示；所述基于其上游调控基因CD155设计的sgRNA如SEQ ID NO.6～7所示；所述突变CD155与CD96第75个氨基酸的结合位点的序列如SEQ ID NO.8所示。
本发明提供基于CD155设计的sgRNA的CRISPR CAS9在制备提高乳腺癌新辅助化疗敏感性的产品中的应用。
借由上述技术方案，本发明至少具有下列优点：
1)本发明的一种新辅助化疗耐药标志物，具体为CD96。通过自身工作中临床乳腺癌组织标本、队列随访数据和体外细胞模型的分子生物学研究发现，CD96在部分患者乳腺癌细胞或细胞系中呈高表达，并与乳腺癌化疗后的不良预后呈正相关；经进一步免疫荧光、流式细胞实验显示CD96抑制乳腺癌细胞对新辅助化疗药物的敏感性，敲低CD96的配体CD155能起着同CD96的抑制相同的促化疗效果，突变CD96与CD155的结合位点能明显抑制CD96的耐药调控作用。
2)CD96通过与CD155结合后激活肿瘤细胞对新辅助化疗药物治疗的耐药。该标志物的研究发现，一方面可通过深入研究鉴定CD96通路调控肿瘤耐药的关系，为靶向该通路治疗乳腺癌的新途径提供科学依据；另一方面还可对新辅助化疗耐药人群进行提示，在乳腺癌治疗或其他癌症治疗中起到个性化治疗、精准治疗的作用。
附图说明
图1为实施例1中CD96在乳腺癌患者组织切片中的表达结果；
图1a为组织免疫荧光技术检测CD96在乳腺癌术后肿瘤标本中肿瘤细胞的表达；
图1b为免疫组化技术检测CD96在乳腺癌术后肿瘤标本中肿瘤细胞的表达；
图1c为CD96高表达和CD96低表达的乳腺癌患者比例。
图2为实施例1中CD96在不同的乳腺癌细胞系中表达结果；
图2a为q-PCR技术检测CD96在不同乳腺癌细胞系中的表达情况；
图2b为Western blotting技术检测CD96在不同乳腺癌细胞系中的表达情况；
图2c为细胞免疫荧光技术检测CD96在MCF-7和MDA-MB-468乳腺癌细胞中的表达情况；
图2d为流式细胞技术检测CD96在不同乳腺癌细胞系中的表达情况。
图3为实施例1中CD96在乳腺癌细胞中的表达与患者生存率中的影响。
图4为实施例1中CD96对不同亚型乳腺癌患者的生存率影响。
图5为实施例2中CD96在癌细胞中的表达与患者新辅助化疗效果之间的关系分析。
图6为实施例2中新辅助化疗后的患者CD96和TUNEL在肿瘤细胞中的关系；
图6a为免疫荧光技术检测CD96和细胞凋亡标记物TUNEL在肿瘤细胞中的共定位；
图6b是20例乳腺癌术后患者的肿瘤细胞中CD96和TUNEL之间的相关性。
图7为实施例2中CD96中和抗体在体外化疗中的作用；
图8为实施例2中CD96基因敲除的效率检测；
图9为实施例2中CD96基因敲除后对体外化疗的影响；
图10为实施例3中CRISPR-CAS9敲除CD155的效率检测；
图11为实施例3中CD155敲除后与CD96中和抗体联合使用对体外化疗的影响；
图12为实施例3中突变CD96与CD155的结合位点后对肿瘤细胞的化疗影响。
具体实施方式
实施例1CD96在乳腺癌中表达水平及与乳腺癌患者预后关系研究
1、CD96在乳腺癌中表达的检测
1.1免疫荧光技术：随机选取616例经过化疗和手术的患者组织切片，首先60℃烘箱烤片20min脱蜡；再用二甲苯浸泡2次，10min/次。水化：100％酒精浸泡2次，10min/次；梯度酒精浸泡：依次按照95％、90％、80％、70％、60％、50％，每次5min。抗原修复：配制pH8.0EDTA，然后浸泡其中，放到高压锅煮沸修复2min。待液体降到常温后予以5％BSA封闭抗原20min，然后轻轻甩干，加入CD96一抗(Thermo公司，货号PA5-97568，479；浓度为1:150稀释)和肿瘤标记物Cytokeratin一抗(Abcam公司，货号MNF116，浓度为1:100稀释)，4℃过夜孵育。室温平衡30min，甩掉一抗；PBS浸泡2～3次，5min/次。组织切片相继与相应种属的免疫荧光二抗、DAPI孵育(室温1小时)；PBS浸泡2～3次，5min/次。用防荧光淬灭剂封片，蔡司800进行记录。最后对切片中肿瘤细胞表面CD96的表达情况进行记录；分析相关数据。
1.2免疫组化：切片脱蜡、抗原修复和一抗染色同免疫荧光；一抗清洗之后加通用二抗，常温孵育30min，PBS浸泡2～3次，5min/次。DAB染色(根据镜下显色效果)，甩掉染色液，PBS浸泡终止染色；苏木素染核浸泡，自来水洗掉苏木素，苏木素回收；缓水流慢慢冲洗冲完甩干37℃烘干；用中性树脂封片，通风橱中吹干，完成制片。
1.3构建CD96的PCR引物检测试剂盒及RT-PCR检测：合成CD96的上下游引物，序列为：上游(SEQ ID NO.1)：CATCCCCAATACGGCTTCTA；下游(SEQ ID NO.2)：TGCTGTTCCATTCATCTGC。将细胞通过0.25％胰酶消化成单细胞，然后PBS清洗2次，加入1mlTRIzol消化20分钟。每使用1ml TRIzol加入0.2ml氯仿，剧烈振荡15秒，室温放置3分钟。2-8℃以10000×g离心15分钟。
样品分为三层：底层为黄色有机相，上层为无色水相和一个中间层。RNA主要在水相中，水相体积约为所用TRIzol试剂的60％。把水相转移到无RNA酶EP管中，用异丙醇沉淀水相中的RNA(每使用1ml TRIzol加入0.5ml异丙醇)，室温放置10分钟。再次在2-8℃以10000×g离心10分钟。移去上清。用75％乙醇洗涤RNA沉淀(每使用1ml TRIzol至少加1ml75％乙醇)。2-8℃不超过7500×g离心5分钟，弃上清。室温放置干燥RNA沉淀。加入25-200μl无RNase的水使RNA溶解。使用Oligo(dT)18、反应条件为50℃进行逆转录反应45分钟，再85℃、5分钟灭活逆转录酶，将RNA逆转录为cDNA。
PCR反应检测CD96的表达情况，反应体系如下：
17.5ul DEPC水，2.5ul 10×Taq buffer，2.0ul MgCl2，0.5ul 10M dNTP Mix，0.5ul上游引物，0.5ul下游引物，0.5ul Tap酶(5u/ul)，1.0ul cDNA。通过DNA变性(90℃-96℃)，退火(25℃-65℃)和延伸，在qPCR仪进行扩增。
1.4蛋白印迹(western blotting)检测C96在SKBR3、BT-474、MCF-7、MDA-MB-361、BT-549、MDA-MB-468、MDA-MB-231乳腺癌细胞系中CD96的表达。
1.5细胞免疫荧光检测MCF-7、MDA-MB-468乳腺癌细胞系中CD96的表达。
1.6流式细胞术检测CD96在细胞系中的表达；0.25％胰酶消化细胞，PBS清洗2遍后重悬细胞并计数，用细胞洗液(含2％ BSA的PBS)重悬细胞封闭抗原，使细胞浓度为2×106/mL；CD96一抗孵育(Biolegend公司，货号338410 1:1000)：对照管加IgG1,κ同种通道抗体(Biolegend公司，货号400121 1:1000)，充分混匀，至4℃孵育30min，孵育期间每隔10min晃动一下反应管，使细胞和抗体充分反应；然后PBS行细胞清洗3次，每次1000rpm离心5min，弃上清后重悬细胞并上机检测(Cytoflex)。
2、CD96的表达水平与乳腺癌患者预后关系
邀请2名病理医生对乳腺癌切片CD96免疫组化结果进行评分，将表达面积(0-4分)、表达强度(0-3分)的乘积记作总分。使用imageJ分析切片中CD96阳性肿瘤细胞的比例，将阳性比例与免疫组化得分进行相乘，得到的数值进行X-tile分层，分为高表达和低表达组。生存分析采用Kaplan-Meier方法，使用GraphPad Prism7.0软件辅助进行。
结果：
1.1免疫组化和免疫荧光检测显示，CD96表达于部分肿瘤患者切片的肿瘤细胞表面。通过X-tile软件分析得出有38.3％的患者高表达CD96(图1)。
1.2RT-PCR、蛋白印迹、细胞免疫荧光和流式细胞术检测结果显示CD96在部分乳腺癌细胞系中高表达，特别是在恶性程度高、耐药性强的三阴乳腺癌细胞中的表达明显增强(图2)。这几个实验的结果一致，表明申请者构建的PCR引物是正确并得到证明的，是一个有效的试剂盒。
1.3对本实施例纳入616例样本中236例样本中的肿瘤细胞高表达CD96，CD96在肿瘤细胞中的高表达的乳腺癌患者具有明显较差的总生存率和无病生存率(图3)。在大部分乳腺癌的分子亚型和分期分级中，CD96在肿瘤细胞中的高表达也具有明显较差的预后(图4)。
上述结果表明，乳腺癌组织中CD96在癌细胞中的表达水平与患者病理分级、临床分期和恶劣预后呈正相关关系。
实施例2CD96与新辅助化疗耐药调控之间的关系研究
1、临床乳腺癌患者新辅助化疗与CD96在癌细胞中的表达关系
收集20例未经治疗的乳腺患者，并行空心针穿刺肿瘤组织确诊，进行含多西他赛的常规新辅助化疗4-6个疗程，手术切除残余病灶，将病灶用石蜡包埋固定，并切片(5-6μm)，再脱蜡、水化、抗原修复等免疫荧光染色CD96、Cytokeratin和凋亡标记物TUNEL(方法见上)。GraphPad Prism7.0和ImageJ分析CD96和Cytokeratin双阳细胞与TUNEL和Cytokeratin双阳细胞的比例和两者之间的关系。
通过磁共振结果评估患者化疗前后的肿瘤大小变化，按患者临床预后评估标准进行检测，完全缓解(Complete Response，CR)指肿瘤完全消失，部分缓解(PartialResponse，PR)为肿瘤最大直径缩小超过30％，疾病进展(Progressive Disease，PD)是指肿瘤直径增加20％以上，疾病稳定(Stable Disease，SD)是指介于部分缓解与疾病进展之间。CR和PR定义为化疗敏感，PD和SD定义为肿瘤化疗耐药。
2、CD96在肿瘤细胞中参与肿瘤化疗耐药的研究
2.1流式细胞术检测肿瘤细胞在干预CD96后的细胞凋亡变化，细胞凋亡检测使用Annexin V Apoptosis Detection Kit(BioLegend公司，货号640932)，具体操作步骤如下：
MDA-MB-231乳腺癌细胞培养到70％密度后，予以0.02μg/mL多西他赛联合50μg/mLCD96中和抗体予以处理24小时，把细胞培养液吸出至一合适离心管内，PBS洗涤贴壁细胞一次，加入适量不含EDTA胰酶细胞消化液消化细胞(EDTA可能会影响Annexin V与磷脂酰丝氨酸的结合)。室温孵育至轻轻吹打可以使贴壁细胞吹打下来时，吸除胰酶细胞消化液。需避免胰酶的过度消化。加入收集的细胞培养液，把细胞轻轻吹打下来，转移到离心管内，1000g离心5min，弃上清，收集细胞，用PBS轻轻重悬细胞并计数。取5-10万重悬的细胞，1000g离心5min，弃上清，加入195μl Annexin V结合液轻轻重悬细胞。加入5μl Annexin V，轻轻混匀15min。加入10μl碘化丙啶染色液，轻轻混匀，室温避光孵育10-20min，随后置于冰浴中，立即上机检测，1小时之内完成检测。
2.2构建CD96敲除的细胞株：
通过慢病毒转染系统构建CD96敲低乳腺癌细胞株(MDA-MB-231)，以无义序列和空载体作为阴性对照，细胞转染48小时后通过Western blotting检测CD96表达变化进行验证。慢病毒构建：将pLKO.1载体(Sigma公司)用限制性内切酶ageⅠ和EcoRⅠ行双酶切，将酶切产物用低熔点琼脂糖胶回收并和上述寡核苷酸退火后的产物混合，然后经T4 DNA连接酶连接过夜。
ShCD96序列为：Sh-CD96-1(SEQ ID NO.3)：CCAACGAAAGTGATCTGCC；Sh-CD96-2(SEQID NO.4)：AGTGGAAGGTACGAGTGTA；连接后的重组质粒转化感受态DH5α细菌(Takara公司)，经含氨苄青霉素培养基筛选阳性克隆后，挑取单菌落扩大培养。使用Omega试剂盒抽提质粒并通过DNA测序进一步鉴定。将shCD96质粒和包装质粒与脂质体3000(Thermo公司)共转染293T细胞。转染后24小时和48小时收集细胞上清，然后分别用于感染MDA-MB-231细胞。
2.3TUNEL荧光染色检测细胞凋亡，细胞接种到载有无菌盖玻片的24孔板，待细胞生长到60％时，予以0.02μg/mL多西他赛联合50μg/mL CD96中和抗体(Antibodies-online公司，货号AA321-519)予以处理24小时。细胞使用清洗2次，胰酶消化细胞后PBS清洗2次。TUNEL(罗氏公司，货号11684817910)以1:300浓度和DAPI(1:200)37℃染细胞30min，PBS清洗细胞2次，防荧光淬灭剂对盖玻片进行封片。蔡司800对细胞进行观察、拍摄。
结果：
1.1使用患者临床疗效评估标准对540例乳腺癌患者进行化疗后评估，结果发现CD96在肿瘤细胞中的高表达具有明显多的患者对含多西他赛药物的常规新辅助化疗方案的耐受，表明CD96抑制化疗药物对肿瘤的治疗效果(图5)。
1.2 20例新发乳腺癌患者化疗后的肿瘤标本，检测肿瘤细胞的凋亡时发现CD96与TUNEL在肿瘤细胞标记物Cytokeratin阳性的细胞中呈反比，进一步表明CD96抑制化疗药物对肿瘤细胞的杀伤作用(图6)。
1.3Annexin V流式细胞术和TUNEL染色检测发现，MDA-MB-231细胞通过CD96中和抗体抑制CD96通路之后，能明显促进新辅助化疗对肿瘤细胞的杀伤作用(图7)。
1.4CD96通过shRNA方式予以敲除后，能明显抑制CD96在细胞中的表达，证明敲除效率可靠(图8)。
1.5CD96敲除后促进MDA-MB-231细胞在新辅助化疗作用下对肿瘤细胞的杀伤(图9)，表明CD96是一个可靠的预测预后和肿瘤耐药的重要蛋白。
以上结果表明CD96在体内外参与肿瘤细胞的耐药调控。
实施例3CD96配体，CD155，参与CD96化疗耐药的调控研究
1、CRISPR-CAS9调控的CD155敲除
使用CRISPR direct设计CD155的靶标序列，本发明中使用5′-GTCACAGCTGACTTGGGCG-3′(SEQ ID NO.5)的CD155序列作为靶点。将该序列插入pCas9/gRNA载体中，构建成正义寡核苷酸(SEQ ID NO.6)：5’ACACCGGTCACAGCTGACTTGGGCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTT3’和反义寡核苷酸(SEQ ID NO.7)：3’TGTGGCCAGTGTCGACTGAACCCGCCAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAA5’,用水将寡核苷酸稀释为100μM。
按以下体系配制退火反应体系合成该片段：正义寡核苷酸5μl、反义寡核苷酸5μl、NaCl 100mM(终浓度)、Tris-Cl pH7.4 50mM(终浓度)、加水补足50μl。以90℃4min，70℃10min，55℃10min，40℃10min，25℃10min程序进行合成。
用EcoRV酶切2μg pCas9/gRNA载体(inovogen)。酶切后用琼脂糖凝胶回收线性化载体。将酶切产物用低熔点琼脂糖胶回收并和上述寡核苷酸退火后的产物混合，然后经T4DNA连接酶连接过夜。连接反应体系：T4 DNA连接酶5U、EcoRV 5U、线性化载体2μl、稀释100倍后双链寡核苷酸1μl、10×连接酶Buffer 1μl、50％ PEG40001μl、加水补足10μl，反应条件：22℃30min，37℃15min。连接后的重组质粒转化感受态DH5α细菌(Takara公司)，经嘌呤霉素培养基筛选阳性克隆后，挑取单菌落扩大培养。使用Omega试剂盒抽提质粒并通过DNA测序进一步鉴定并进一步转染到MDA-MB-231细胞。
2、CD155敲除的肿瘤细胞对化疗的作用
MDA-MB-231乳腺癌细胞经过CRISPR-CAS9技术敲除CD155的表达后，使用0.02μg/mL多西他赛单独或联合50μg/mL CD96中和抗体予以处理24小时。流式细胞术检测肿瘤细胞的凋亡情况(具体实验步骤同前)。
3、突变CD96与CD155结合位点后对肿瘤细胞化疗的变化
CD96的第一个Ig结构域是与CD155结合的主要基序，而第75个酪氨酸位点是两者结合最主要的氨基酸，是CD155激活CD96主要的位点。因此申请者构建了第75个酪氨酸的突变位点，将该位点氨基酸突变为丙氨酸(简称为T75A)，序列(SEQ ID NO.8)为：
GCTGTCTATCATCCCCAAGCTGGCTTCTACTGTGCCTAT；
突变的反应体系：10x pyrobest Buffer：5ul，dNTP Mixture(10mM)：1ul，模板DNA(5～50ng)：1ul，primer 1(125ng)：1ul，primer 2(125ng)：1ul，pyrobest DNA polymerase(TaKaRa)(5U/ul)：0.25ul，最后加无菌蒸馏水至50ul。总共循环数为15个。使用醋酸钠、75％乙醇清洗，进行DpnI酶切(30℃酶切1～4h)，然后65℃水浴15min终止反应。
将突变的CD96变体插入到pcDNA3.1-6his质粒的EcoRI/NotI位点，通过氨苄西林进行突变体的细菌筛选，提取大规模的质粒并转染到所示细胞中。TUNEL染色观察转染了突变体的MCF-7细胞(这些细胞正常时低表达CD96)对新辅助化疗的敏感性。
结果：
1.1CRISPR-CAS9技术成果敲除CD155在MDA-MB-231乳腺癌细胞中的表达，表明CD155 sgRNA的构建成功(图10)。
1.2敲除CD155后，MDA-MB-231乳腺癌细胞对化疗药物敏感性明显增强，肿瘤杀伤力同使用CD96中和抗体。但CD96中和抗体联合CD155的敲除，并不能相互促进，表明两者在发挥耐药调控中的通路是同一途径。综合CD96在免疫细胞中的功能需要配体CD155的作用，因此CD155-CD96信号途径是CD96发挥耐药的激活组合(图11)。
1.3突变CD96的第75个氨基酸位点抑制CD96与CD155结合后，能明显增强过表达CD96的MCF-7细胞对新辅助化疗药物的敏感性(图12)。
以上结果表明CD96发挥调控肿瘤细胞的耐药途径需要CD155的相互作用。
结论：
本发明的新辅助化疗耐药标志物，具体为CD96，本发明人通过自身工作中临床乳腺癌组织标本、队列随访数据和体外细胞模型的分子生物学研究发现：本身在免疫细胞表面表达的CD96在部分患者乳腺癌细胞或细胞系中呈高表达，并与乳腺癌化疗后的不良预后呈正相关；经进一步免疫荧光、流式细胞实验显示CD96抑制乳腺癌细胞对新辅助化疗药物的敏感性，敲低CD96的配体CD155的表达能起着同CD96的抑制相同的促化疗效果，突变CD96与CD155的结合位点能明显抑制CD96的耐药调控作用。
据此，我们推测CD155结合并激活CD96促使肿瘤细胞对新辅助化疗药物治疗的耐药。该标志物的研究发现，一方面可通过深入研究鉴定CD96通路调控肿瘤耐药的关系，为靶向该通路治疗乳腺癌的新途径提供科学依据。另一方面还可对新辅助化疗耐药人群进行提示，在乳腺癌治疗或其他癌症治疗中起到个性化治疗、精准治疗的作用。
以上所述，仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，故凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。
Claims (1)
1.CD96抑制剂在制备提高乳腺癌新辅助化疗敏感性的产品中的应用，其特征在于：
所述CD96抑制剂选自CD96中和抗体、基于CD96设计的shRNA、基于CD96上游调控基因CD155设计的sgRNA；
所述基于CD96设计的shRNA如SEQ ID NO.3或SEQ ID NO.4所示；
所述基于CD96上游调控基因CD155设计的sgRNA如SEQ ID NO.6～7所示。
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