CN116262120A - 特异性结合TRAF2的试剂在制备Wnt通路抑制剂中的应用 - Google Patents
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Abstract
本发明公开了特异性结合TRAF2的试剂在制备Wnt通路抑制剂中的应用和特异性结合TRAF2的试剂在制备治疗Wnt通路异常激活的疾病的药物中的应用。所述特异性结合TRAF2的试剂可以高亲和地集合TRAF2，并继而抑制Wnt通路，从而抑制Wnt异常相关的肿瘤的进展。
Description
技术领域
本发明涉及肿瘤治疗领域，具体涉及特异性结合TRAF2的试剂在制备Wnt通路抑制剂中的应用、特异性结合TRAF2的试剂在制备治疗Wnt通路异常激活的疾病中的应用、一种体外化疗耐药性的癌症模型及建立其的方法、一种试剂盒和一种药物组合物。
背景技术
大肠癌是发病率和死亡率最高的恶性肿瘤之一，迄今为止临床尚无相应的靶向药物。APC(adenomatous polyposis coli)突变导致Wnt/β-catenin信号通路异常激活是大肠癌最主要的驱动因子，但由于APC是截断失活性突变，相应的靶向药物开发面临诸多挑战，针对APC非依赖的Wnt/β-catenin信号通路发现抑制剂，有望开发新型的结肠癌靶向药物。
Wnt/β-catenin信号通路
Wnt信号通路是一个进化上非常保守的蛋白质作用网络。经典Wnt信号通路可简单概括为：1)Wnt分泌因子在细胞膜与受体(Frizzled家族及LRP家族)结合；2)信号通过Dishevelled蛋白将导致细胞质内的β-catenin被APC、Axin、CK1α和GSK3β组成的降解复合物磷酸化，使其能够被β-TRCP E3连接酶介导泛素化降解；3)β-catenin在细胞质内聚集并进入细胞核和TCF/LEF(T细胞因子)形成复合物启动靶基因(比如c-myc)的转录。
该通路最先在胚胎发育中发现，通路的异常抑制或激活将导致胚胎发育缺陷。Wnt信号通路不仅与肿瘤侵袭转移的相关事件如癌细胞的迁移黏附、细胞外基质的降解及肿瘤的血管生成密切相关，而且在调节肿瘤干细胞的自我更新、增殖与分化中发挥重要作用。此外，有研究证实Wnt/β-catenin信号通路与化疗耐药性有关。现在已有大量证据证实Wnt信号通路的异常激活和癌症尤其是大肠癌的发生和发展高度相关。
由于Wnt/β-catenin信号通路在大肠癌发生中的关键作用，过去20多年来世界上有众多研究机构和制药公司致力于Wnt抑制剂的开发研究。不过，迄今为止尚无靶向Wnt/β-catenin信号通路的药物应用于临床，大多数抑制剂处于临床前研究，很少数进入临床1期或2期。Wnt抑制剂开发进展缓慢的原因是大多数抑制剂是靶向Wnt信号通路的上游或中游，但是临床基因组测序表明81.9％的大肠癌组织有APC基因的失活性突变和4.7％β-catenin基因的激活性突变，理论上这些突变将导致β-catenin的降解受阻和异常积聚，Porcupine抑制剂的靶标Wnt蛋白或Tankyrase抑制剂的靶标Axin在通路上处于APC之前，因此对这些异常集聚的β-catenin不能有调控作用。
涉及Wnt/β-catenin信号通路的疾病包括癌症、神经系统疾病、骨病、皮肤病、心血管疾病、纤维化疾病及代谢综合征等疾病；涉及Wnt/β-catenin信号通路的癌症包括但不限于大肠癌、肺癌、前列腺癌、胰腺癌、胃癌、肝癌、乳腺癌及骨肉瘤；特别是大肠癌。
路路通酸(liquidambaric acid，LDA，结构式如下式I所示)是路路通的主要活性成分，最早是在1988年，从枫香树的干燥成熟果实路路通中提取鉴定出来的一种新的五环三萜类化合物。其研究较多的是抗病毒和抗炎作用，作用机制与毛细血管通透性有关。
TRAF2(tumor necrosis factor(TNF)receptor-associated factor2)
结构上，TRAF2属于TRAF家族，其家族具有有七个成员:TRAF1-TRAF7。除了TRAF7，TRAF家族成员通常共享一个共同的TRAF域。其中TRAF2是由一个环指结构域、几个锌指基元、一个包含N端卷曲螺旋结构域(TRAF-N)和一个C端β-三明治结构域(TRAF-C)组成。
功能上，TRAF2是一种促进k63连接多聚泛素化的E3泛素连接酶，同时也是一种适配蛋白。TRAF2可发挥多种受体特异性功能，但也介导TNFR1和TNFR2之间的交叉，决定TNF刺激的结果。当激活典型的NF-κB和JNK/MAP激酶信号通路，其中TNF可通过激活两种不同的受体TNFR1和TNFR2，TNFR1与TRADD招募TRAF2，TNFR2直接招募TRAF2进而激活。而TNFR2也可以激活非典型的NF-κB信号通路，导致大量基因表达的变化，驱动炎症，细胞增殖和细胞存活，区分TRAF2作为支架和E3泛素连接酶的功能，可能是其潜在的分子机制。
TRAF2作为一种适配蛋白，在肿瘤启动和进展中与多种受体特异性功能相关。目前已经在结肠癌、肺癌、乳腺癌和胰腺癌等多种癌症中都证实有TRAF2的异常激活，基因敲除或敲低TRAF2则显著抑制肿瘤的生长、侵袭，或增加肿瘤的化疗敏感性。
综上所述，TRAF2是个理想的肿瘤治疗靶标，急需发现靶向TRAF2的小分子抑制剂。
发明内容
针对现有技术中对Wnt通路研究的不足、缺少Wnt通路抑制剂和Wnt通路异常相关肿瘤的治疗药物的缺陷，本发明提供了特异性结合TRAF2的试剂在制备Wnt通路抑制剂中的应用、特异性结合TRAF2的试剂在制备治疗Wnt通路异常激活的疾病中的应用、一种体外化疗耐药性的癌症模型及建立其的方法、一种试剂盒和一种药物组合物。本发明的发明人意外发现TRAF2是Wnt通路的抑制蛋白，而LDA可特异性地结合TRAF2，从而抑制Wnt通路。本发明所述LDA的结构式如式I所示。
为解决上述技术问题，本发明提供的一个技术方案为：特异性结合TRAF2的试剂在制备Wnt通路抑制剂中的应用。
优选地，所述试剂为抗体或小分子化合物。更优选地，所述小分子化合物为LDA。
为解决上述技术问题，本发明提供的一个技术方案为：特异性结合TRAF2的试剂在制备治疗Wnt通路异常激活的疾病的药物中的应用，所述疾病包括癌症、神经系统疾病、骨病、皮肤病、心血管疾病、纤维化疾病和代谢综合征；所述癌症优选大肠癌、肺癌、前列腺癌、胰腺癌、胃癌、肝癌或骨肉瘤；所述大肠癌更优选为结肠癌。
优选地，所述试剂为抗体或小分子化合物。更优选地，所述小分子化合物为LDA。
为解决上述技术问题，本发明提供的一个技术方案为：LDA在制备TRAF2抑制剂中的应用。
为解决上述技术问题，本发明提供的一个技术方案为：LDA在制备治疗TRAF2相关疾病的药物中的应用。
为解决上述技术问题，本发明提供的一个技术方案为：一种体外化疗耐药性的癌症模型，所述癌症模型中缺失TRAF2基因；所述癌症优选大肠癌、肺癌、前列腺癌、胰腺癌、胃癌、肝癌或骨肉瘤；所述大肠癌更优选为结肠癌。
为解决上述技术问题，本发明提供的一个技术方案为：建立如本发明所述的癌症模型的方法，所述方法包括：将抑制TRAF2基因表达的试剂导入癌症细胞，得所述的癌症模型；所述癌症细胞优选为大肠癌细胞、肺癌细胞、前列腺癌细胞、胰腺癌细胞、胃癌细胞、肝癌细胞或骨肉瘤细胞；所述大肠癌细胞更优选为结肠癌细胞。
优选地，所述的导入为使用CRISPR/Cas 9技术导入。
优选地，所述抑制TRAF2基因表达的试剂为靶向TRAF2基因上游和下游的sgRNA，所述上游的sgRNA的核苷酸序列如SEQ ID NO:1和3所示，所述下游的sgRNA的核苷酸序列如SEQ ID NO:2和4所示。
为解决上述技术问题，本发明提供的一个技术方案为：一种试剂盒，所述试剂盒包括特异性结合TRAF2的试剂。
优选地，所述试剂盒还包括(i)施用特异性结合TRAF2的试剂的装置；和/或(ii)使用说明。
在本发明一具体实施方案中，所述特异性结合TRAF2的试剂为LDA。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明提供的特异性结合TRAF2的试剂可以有效抑制Wnt通路，进而减缓Wnt通路异常的肿瘤(优选为结肠癌)的进展。而LDA可以通过TRAF2有效抑制Wnt通路。
附图说明
图1展示了路路通酸是新型抑制Wnt通路抗肿瘤化合物；A部分显示了基于7TGC的Wnt抑制剂筛选系统；B部分显示了在携带有7TGC载体的EPT1细胞中，筛选可以抑制GSK3抑制剂6BIO激活的Wnt信号通路的化合物。
图2显示了TOPFlash测定LDA的Wnt信号通路活性；OA为结构类似的阴性对照小分子。
图3显示了斑马鱼胚胎的无眼拯救实验的代表图片(A部分)和定量分析(B部分)。
图4显示了LDA抑制多种肿瘤细胞克隆形成。
图5显示了HCT116细胞裸鼠移植瘤实验的代表图片(A部分)，移植瘤的生长曲线(图5B)。
图6显示了HCT116细胞裸鼠移植瘤实验，小鼠体重变化。
图7显示了非小细胞肺癌H1975裸鼠移植瘤实验的代表图片(A部分)，移植瘤的生长曲线(B部分)。
图8展示了路路通酸靶标蛋白TRAF2的鉴定；DARTS/MS鉴定LDA靶标蛋白的工作原理图(A部分)；DARTS/MS鉴定LDA在大肠癌SW480细胞中的直接结合蛋白(B部分)。
图9展示了MST测定LDA与TRAF2的结合亲和力，Pif1为对照蛋白。
图10展示了MST测定LDA与TRAF2的结合亲和力，OA为对照小分子。
图11展示了：针对TRAF2基因设计的sgRNA示意图以及针对敲除细胞基因组TRAF2位点的PCR产物DNA测序结果(A部分)；Western Blot分析敲除细胞克隆的TRAF2表达(B部分)。
图12展示了TOPFlash报告测定TRAF2敲除HCT116细胞克隆的Wnt信号通路活性。
图13展示了：TRAF2敲除HCT116细胞的裸鼠移植瘤实验的代表图片(A部分)，移植瘤的生长曲线(B部分和C部分)。
图14为LDA对TRAF2敲除HCT116细胞抑制作用的裸鼠移植瘤实验的代表图片(A部分)，移植瘤的生长曲线(B部分和C部分)。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1发现抑制结肠癌Wnt信号通路的中药活性分子路路通酸(LDA)
1.1基于7TGC的Wnt抑制剂筛选
将稳定整合Wnt双重荧光蛋白报告系统(7xTCF-GFP/SV40-Cherry，7TGC)的EPT2-TGC细胞(制备方法参见Qu,Y.,Gharbi,N.,Yuan,X.,Olsen,J.R.,Blicher,P.,Dalhus,B.,Brokstad,K.A.,Lin,B.,
A.M.,Zhang,W.,et al.(2016).Axitinib blocks Wnt/β-catenin signaling and directs asymmetric cell division in cancer.Proc NatlAcad Sci U S A 113,9339-9344.10.1073/pnas.1604520113.和Fuerer,C.,and Nusse,R.(2010).Lentiviral vectors to probe and manipulate the Wnt signalingpathway.PLoS One 5,e9370.10.1371/journal.pone.0009370.)，按照10000个/孔，每孔100μl接种于96孔板中，置于培养箱中培养12h后，给药加入LDA(APExBIO，N2227)，使得终浓度为10uM(完全培养基稀释)，孵育24h后，弃旧培养液，每孔加入100μl含有Hoechst 33342(B2261)(Sigma-Aldrich)的新鲜培养液，避光条件下染细胞核30min，PBS洗3次后，加100μl/孔PBS，高内涵细胞成像分析系统(MOLECULAR，ImageXpress Micro 4)检测。
1.2 Wnt信号通路荧光素酶(TOPFlash/Renila)报告系统筛选
细胞接种到96孔白板，贴壁后每孔同时转染TOPFlash和Renila质粒(Addgene12456，GB0056)，6小时后给予5、10μM的LDA及对照化合物OA，24小时后使用The Dual-
Reporter(DLRTM)Assay System检测荧光素酶的值。TOPFlash为荧光素酶表达载体，其中Flash的启动子上有8个串联的TCF结合位点(TOP)，因此荧光素酶的表达依赖于Wnt信号通路，细胞Wnt活性越强，荧光素酶的信号越强。Renilla荧光素酶表达载体作为内参，细胞的Wnt信号通路活性用TOPFlash与Renilla的比值表示。
结果如图1和图2所示，图1表明式I所示化合物(10μM)可以抑制Wnt信号通路双荧光7TGC(7xTCF-GFP/SV40-Cherry)报告系统，图2表明式I抑制细胞内β-catenin介导的转录活性。
1.3斑马鱼胚胎“眼缺失”实验步骤
AB系斑马鱼购自国家斑马鱼资源中心，当斑马鱼胚胎6hpf时，根据不同给药组进行分组同时给予相应化合物，其中，模型组给予(2’Z,3’E)-6-溴靛玉红-3′-肟(6BIO)1μM(Sigma-Aldrich，B1686)，化合物组在给予6BIO(1μM)的同时给予化合物(20μM)；随后在72hpf时观察胚胎状态。
结果如图3所示，式I化合物抑制Wnt信号通路的活性，其对斑马鱼胚胎无眼表型的拯救作用表明化合物对Wnt信号通路的抑制具有种属保守性。
1.4克隆形成实验
将以下细胞制成细胞悬液：大肠癌细胞HCT116(ATCC)、LS174T(ATCC)和SW480(中科院细胞库，TCHu172)，胃癌细胞NCL-N87(ATCC)，肺癌细胞HCC827(ATCC)、H1975(ATCC)和H460(ATCC)，骨肉瘤细胞U-2OS，胰腺癌ASPC1(ATCC)和CFPAC1(ATCC)，前列腺癌细胞PC-3(中科院细胞库，SCSP-532)，肝癌细胞HEPG2(中科院细胞库，TCHu72)。用细胞计数仪计数细胞密度，将细胞密度为1000个/ml，按照1ml/孔接种于12孔板中，使每孔含1000个细胞。细胞培养12h后，分别用不同浓度的LDA(0、2.5、5、10、20和40μM)给药处理，每三天更换新鲜培养基，并重新给予LDA以保持给药浓度，连续培养8天。
弃旧培养液，用PBS小心润洗贴壁细胞表面，除去培养基，弃PBS。12孔板每孔加入500μl 4％多聚甲醛固定液，将细胞固定30min，弃去固定液后，用PBS小心冲洗3次，弃净PBS后每孔加500μl结晶紫染色剂染色，10min后移除结晶紫染色剂，并用缓水流洗净结晶紫染色剂，将孔板置于阴凉处晾干后，拍摄照片观察结果。
结果如图4所示：式I化合物抑制大肠癌、肺癌、胰腺癌、胃癌、前列腺癌、肝癌细胞克隆形成。
1.5裸鼠移植瘤实验
BALB/c裸鼠，5-6周龄，雄性，SPF级，购自并饲养于上海西普尔-必凯实验动物有限公司(许可证号码SCXK(沪)2018-0006)，温度(20±2)℃，湿度(55±2)％，自由摄食和饮水，每日光照/黑暗各12h。将人结肠癌HCT116细胞(购自中科院细胞库)按照5×107细胞/ml重悬于PBS中，以200μl/只将人结肠癌HCT116细胞接种于5-6周龄的雄性裸鼠右腋下，使每只接种细胞数为1×106个，形成可触及的瘤块后，将裸鼠按照瘤体体积随机分组，LDA给药组以25、50和100mg/kg/day灌胃，每天给药一次，每周三次检测瘤体大小，并记录体重，连续给药14天，取出移植瘤组织，并对瘤体称重。
结果如图5-7所示：式I所示化合物在安全剂量下显著抑制HCT116细胞的移植瘤形成能力(图5的A部分和B部分，图6)；式I所示化合物抑制非小细胞肺癌细胞H1975皮下移植瘤形成能力(图7的A部分和B部分)。
实施例2鉴定了LDA抑制大肠癌的靶标蛋白TRAF2
2.1药物亲和反应的靶点稳定性技术(Drug Affinity Responsive TargetStability,DARTS)
DARTS流程如图8的A部分所示，利用小分子与蛋白结合后能提高靶蛋白的热力学稳定性，降低靶蛋白对蛋白酶降解的敏感性的特点，从而得到与小分子直接结合的蛋白。具体如下：取出培养箱中的HCT116细胞，弃去培养液，用PBS轻轻摇晃至覆盖细胞，将细胞用PBS洗三次以除去培养液，弃净PBS后置于冰上。将新鲜配制的M-PER裂解液(Thermoscientific，78501)加入细胞中，并用干净的刮刀快速刮下细胞，然后用移液枪将细胞裂解混合物移至1.5ml EP管(整个操作尽量在冰上进行)，充分混匀后，于冰上裂解30min。4℃12000rpm离心15min(提前开离心机预冷)，收集离心后的上清液于新EP管中，得细胞总蛋白。取200ml/管细胞总蛋白于EP管中，取4μl 100％DMSO或者10mM LDA混匀，室温孵育1h；同时加8μl/管4.2mg/ml链霉蛋白酶(Pronase)室温孵育，酶解30min；加16μl/管蛋白酶抑制剂(20×)(Roche，11836170001)，于冰上15min。超滤后得到DARTS超滤后样品。将全部超滤后的样品制成蛋白电泳样品，进行蛋白电泳，用考马斯亮蓝染色液染色，切下被染成蓝色的蛋白条带，用于蛋白质谱检测。
2.2蛋白质谱分析
将DARTS回收样品经过蛋白质谱上机前处理，上机检测。将得到的二级质谱数据使用Maxquant(v1.5.2.8)进行检索。检索参数设置：数据库为SwissProt Human(20130条序列)，添加了反库以计算随机匹配造成的假阳性率(FDR)，并且在数据库中加入了常见的污染库，用于消除鉴定结果中污染蛋白的影响；酶切方式设置为Trypsin/P；漏切位点数设为2；肽段最小长度设置为7个氨基酸残基；肽段最大修饰数设为5；First search和Mainsearch的一级母离子质量误差容忍度分别设为20ppm和5ppm，二级碎片离子的质量误差容忍度为0.02Da。
结果如图8的B部分所示，其显示了与LDA结合蛋白中，富集程度最高的是肿瘤坏死因子受体相关因子2(TNF receptor-associated factor 2，TRAF2)。
2.3微量热泳动技术(MST)
MST技术是一种分析生物分子相互作用的技术。这项技术基于nanotemper微量热泳动仪，使用红外激光进行局部加热导致分子定向移动，通过荧光分析温度梯度场中的分子分布比。构建靶标蛋白融合的GFP融合表达载体。在293FT细胞(Thermo scientific)中过表达靶标蛋白-GFP或者GFP表达载体24h后，裂解细胞制备总蛋白，直接用于MST(NanoTemper，Monolith NT.115)测定(nano red，80％)。以GFP荧光为影响信号，以改变蛋白在热力场中泳动模式为标准，验证齐墩果酸和路路通酸与差异蛋白的结合，确定结合解离常数(Kd)。
结果如图9所示：其显示了LDA与TRAF2蛋白的结合，其中以Pif1蛋白作为阴性对照，结合曲线的Kd值为6.02μM(n＝3)。
结果如图10所示：其显示了与蛋白TRAF2与LDA的结合，其中OA作为阴性对照，结合曲线的Kd值为6.02μM(n＝3)。
实施例3证实TRAF2是LDA抑制Wnt信号通路和结肠癌所必需靶标
3.1 CRISPR/Cas 9基因删除技术
设计靶向目标基因上游和下游的sgRNA序列：
TRAF2 sgRNA1-F：CACCGCAGGCGGAGCACAGGTACT(SEQ ID NO:1)；
TRAF2 sgRNA1-R：AAACAGTACCTGTGCTCCGCCTGC(SEQ ID NO:2)；
TRAF2 sgRNA2-F：CACCGATGGCATCGTCCCGCACGT(SEQ ID NO:3)；
TRAF2 sgRNA2-R：AAACACGTGCGGGACGATGCCATC(SEQ ID NO:4)；
分别克隆到表达Cas 9的pL-CRISPR.EFS.GFP(Addgene 57818)载体上。把pL-CRISPR.EFS.GFP-sgRNA 1和pL-CRISPR.EFS.RFP-sgRNA 2载体LIPO 3000(Thermo,#L3000-015,LipofectamineTM3000Transfection Reagent)转染进HCT116细胞中(各试剂用量如表1所示)。
表1 CRISPR敲除试剂用量
转染48h后，以单细胞/孔培养，荧光显微镜下观察挑出GFP荧光的单克隆细胞，通过WB和DNA测序验证阳性删除克隆。图11的A部分为针对TRAF2基因设计的sgRNA示意图以及针对敲除细胞基因组TRAF2位点的PCR产物DNA测序结果。
图11的B部分表明：利用CRISPR/Cas9技术在Wnt信号通路异常激活结肠癌HCT116细胞中建立了TRAF2的敲除细胞系，并用PCR-Seq和Western Blot实验分别在基因和蛋白水平验证了HCT116细胞TRAF2的敲除。
图12展示了将HCT116和TRAF2敲除HCT116细胞，分别接种到96孔白板，贴壁后每孔同时转染TOPFlash和Renila质粒，24小时后使用The Dual-
Reporte r(DLRTM)Assay System检测荧光素酶的值。其显示TRAF2敲除HCT116细胞的Wnt信号活性显著下降。
图13显示TRAF2敲除HCT116移植瘤在成瘤能力和成瘤时间上显著减少这进一步证实了TRAF2是Wnt信号通路的驱动因子和大肠癌的潜在治疗靶标。
图14显示式I化合物对TRAF2敲除HCT116细胞Wnt信号通路和移植瘤的均无明显抑制作用，表明TRAF2是式I化合物抑制Wnt信号通路和结肠癌所必需靶标。
SEQUENCE LISTING
<110> 上海中医药大学
<120> 特异性结合TRAF2的试剂在制备Wnt通路抑制剂中的应用
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Claims (10)
1.特异性结合TRAF2的试剂在制备Wnt通路抑制剂中的应用。
2.如权利要求1所述的应用，其特征在于，所述试剂为抗体或小分子化合物；优选地，所述小分子化合物为LDA。
3.特异性结合TRAF2的试剂在制备治疗Wnt通路异常激活的疾病的药物中的应用，所述疾病包括癌症、神经系统疾病、骨病、皮肤病、心血管疾病、纤维化疾病和代谢综合征；所述癌症优选大肠癌、肺癌、前列腺癌、胰腺癌、胃癌、肝癌或骨肉瘤；所述大肠癌更优选为结肠癌。
4.如权利要求3所述的应用，其特征在于，所述试剂为抗体或小分子化合物；优选地，所述小分子化合物为LDA。
5.LDA在制备TRAF2抑制剂中的应用。
6.LDA在制备治疗TRAF2相关疾病的药物中的应用。
7.一种体外化疗耐药性的癌症模型，所述癌症模型中缺失TRAF2基因；所述癌症优选大肠癌、肺癌、前列腺癌、胰腺癌、胃癌、肝癌或骨肉瘤；所述大肠癌更优选为结肠癌。
8.建立如权利要求7所述的癌症模型的方法，所述方法包括：将抑制TRAF2基因表达的试剂导入癌症细胞，得所述的癌症模型；所述癌症细胞优选为大肠癌细胞、肺癌细胞、前列腺癌细胞、胰腺癌细胞、胃癌细胞、肝癌细胞或骨肉瘤细胞；所述大肠癌细胞更优选为结肠癌细胞。
9.如权利要求8所述的方法，其特征在于，所述的导入为使用CRISPR/Cas 9技术导入；优选地，所述抑制TRAF2基因表达的试剂为靶向TRAF2基因上游和下游的sgRNA，所述上游的sgRNA的核苷酸序列如SEQ ID NO:1和3所示，所述下游的sgRNA的核苷酸序列如SEQ ID NO:2和4所示。
10.一种试剂盒，所述试剂盒包括特异性结合TRAF2的试剂；
优选地，所述试剂盒还包括(i)施用特异性结合TRAF2的试剂的装置；和/或(ii)使用说明；
和/或，所述特异性结合TRAF2的试剂为LDA。
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