CN113249476B - 一种影响肿瘤细胞干性的关键蛋白blt2及其应用 - Google Patents
一种影响肿瘤细胞干性的关键蛋白blt2及其应用 Download PDFInfo
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Abstract
本发明公开了一种影响肿瘤细胞干性的关键蛋白BLT2及其应用。本发明通过体外共培养实验发现5‑LO的上调导致LTB4分泌增加,还可以进一步通过ICC细胞的BLT2信号增强ICC细胞系的干性基因表达,且BLT2的表达水平与ICC病人的预后生存率显著负相关(P<0.001),BLT2低表达组病人的预后生存率要显著高于BLT2高表达组。在体外利用BLT2的抑制剂抑制BLT2信号可以显著降低5‑LO上调导致的ICC干性基因表达。而常规化疗与BLT2抑制剂联用可以显著抑制人源化肿瘤异体移植小鼠的肿瘤生长。
Description
技术领域
本发明涉及一种影响肿瘤细胞干性的关键蛋白BLT2及其应用,属于生物技术领域。
背景技术
人肝内胆管细胞癌(Intrahepatic cholangiocarcinoma,ICC)作为胆管上皮细胞恶性转化导致的原发性肝癌,其诊断的困难性和临床的不良预后严重影响病人的术后生存率,其发病率虽然只占肝癌数量的5%左右,然而其恶性程度非常高,病人死亡率很高,5年的生存率仅有25%,因此研究其相关发生发展的机制尤为重要。该疾病的特点是高度纤维化和结缔组织增生,此前大量的研究都认为肿瘤微环境其中特别是成纤维细胞对该疾病的发生发展都起到了重要的作用,与其不良的预后和高恶性程度密不可分。
肿瘤干细胞是肿瘤中具有自我更新能力并能产生异质性肿瘤细胞的细胞,其主要具有自我更新和无限繁殖能力。其对肿瘤的存活、增殖、转移及复发具有重要的意义,这类肿瘤干细胞既像干细胞一样处在不同的分化阶段,同时又具有极强的成瘤能力。肿瘤微环境作为恶性肿瘤无限增殖的温暖摇床,包括肿瘤细胞、免疫细胞以及细胞外基质中的各种细胞等都参与肿瘤微环境的形成过程。而这些细胞之间的相互作用是否参与调控肿瘤细胞的干性尚不可知。
肿瘤免疫治疗的过程中,疗效和患者体内的免疫抑制效应有着很大的关系,而在其中起关键作用的是一群具有免疫抑制效应的骨髓来源细胞即髓样抑制细胞(MDSCs)。现有的文章表明,MDSCs来源于未成熟分化的造血干细胞,又基于表达的Gr-1抗原不同表位分为了多核型粒细胞亚群(PMN-MDSCs)和单核型细胞亚群(Mo-MDSCs)。一些不成熟的髓样来源细胞的浸润是肿瘤发生的另一特征。肿瘤相关巨噬细胞(TAMs)和髓样抑制细胞是实体瘤中发现的最主要的两种细胞,它们有共同的特点功能,即抑制T细胞的杀伤肿瘤功能。在肿瘤的发生过程中,MDSCs受到肿瘤细胞分泌的一系列细胞因子影响,包括环氧合酶 2(COX-2)、前列腺素(prostaglandins)、巨噬细胞集落刺激因子(M-CSF)、白介素6(IL-6)、血管内皮生长因子(VEGF)等,最终活化,分泌精氨酸酶1 (Arg1)和诱导型一氧化氮合酶(iNOS)来发挥抑制T细胞的功能。除了他们的免疫抑制功能,TAMs和MDSCs通过各种途径促使肿瘤恶化,包括促肿瘤血管新生,促肿瘤细胞干性,以及上皮间质转化等等。CD163+TAMs具有M2型巨噬细胞的表型,有文献证明他们在ICC发病的连续性中表现出了不良预后的趋势。而MDSCs在多种类型的人类实体瘤中,比如卵巢癌、乳腺癌和肝细胞癌,都有检测到,但ICC病人当中MDSCs的作用尚不明确。
ICC相关的肿瘤微环境有大量MDSCs的存在。除了已经被研究充分的免疫抑制功能外,近期数据还表明,MDSCs能够通过旁分泌信号传导和诱导癌细胞的表观遗传程序来促进癌症干性和化疗耐药性。最近的研究表明,MDSCs的代谢变化与其功能状态即免疫抑制活性有关。Veglia F等人报道了肿瘤浸润性 PMN-MDSCs中花生四烯酸(AA)代谢增加,这通过下游PEG2影响了它们的免疫抑制功能。值得注意的是,一项较早的研究表明,5-脂氧合酶(5-LO)是 AA代谢通路下游白三烯(LT)合成的关键酶,介导嗜中性粒细胞支持高度转移性细胞的能力,从而导致小鼠乳腺癌模型中的肺转移。
发明内容
本发明所要解决的技术问题是:人肝内胆管细胞癌诊断困难、预后生存率较差以及常规化疗药物会产生耐药性而影响化疗效果等问题。
为了解决上述技术问题,本发明提供了一种检测BLT2的表达水平的试剂在制备至少一种如下产品中的应用:检测ICC肿瘤细胞干性的试剂和/或试剂盒、诊断ICC肿瘤的试剂和/或试剂盒以及预测ICC患者生存期的试剂和/或试剂盒。
优选地,所述的检测BLT2的表达水平的试剂包括BLT2的抗体。
本发明还提供了抑制BLT2的表达和/或活性的物质在制备抑制ICC肿瘤细胞干性的试剂中的应用和/或在制备抑制ICC肿瘤细胞干性相关特征分子的表达的试剂中的应用。
优选地,所述的抑制BLT2的表达和/或活性的物质包括BLT2的特异性干扰 RNA和/或BLT2抑制剂。
优选地,所述的BLT2的特异性干扰RNA包括序列为 5’-CCACGCAGUCAACCUUCUGUGTT-3’的siRNA,所述的BLT2的抑制剂包括 LY255283。
优选地,所述的ICC肿瘤细胞干性相关特征分子为基因NANOG、SOX2、 POU5F1和CTNNB1中的至少一种。
本发明还提供了BLT2抑制剂联合ICC化疗药物在制备至少一种如下产品中的应用:抑制ICC肿瘤细胞干性的试剂、治疗和/或预防ICC肿瘤的药物、抑制 ICC肿瘤进程的药物、抗ICC化疗药物耐药性的药物以及抑制ICC肿瘤细胞干性相关特征分子的表达的试剂。
优选地,所述的BLT2的抑制剂包括LY255283,所述的ICC化疗药物包括吉西他滨。
优选地,所述的ICC肿瘤细胞干性相关特征分子为基因NANOG、SOX2、 POU5F1和CTNNB1中的至少一种。
与现有技术相比,本发明的有益效果在于:
1.本发明验证了BLT2为影响ICC肿瘤细胞干性的关键蛋白,本发明采用 BLT2抑制剂与化疗药物吉西他滨(Gemcitabine)联用,可提高化疗药物的耐药性,体现在显著提高了在抑制ICC肿瘤细胞的生长、促进肿瘤细胞凋亡、抑制肿瘤细胞增殖和抑制肿瘤细胞的干性等方面的作用;
2.本发明的影响肿瘤细胞干性的关键蛋白BLT2为临床上开发新的诊断和治疗人肝内胆管细胞癌的手段和试剂/药物提供了新的分子靶标。
附图说明
图1A为ICC病人肿瘤组织及非肿瘤组织切片CD33免疫组化染色和阳性细胞计数结果,其中箭头所示为CD33染色细胞;
图1B为CD33高表达组和低表达组的ICC病人生存曲线图,图中圈出的是为CD33染色细胞;
图2A为ICC病人血液和肿瘤组织分离的CD33+MDSC的CD33中5-LO免疫荧光染色结果;
图2B为体外用ICC肿瘤相关的成纤维细胞培养上清刺激MDSC,5-LO的蛋白质免疫印迹检测结果;
图2C为ICC病人肿瘤局部和非肿瘤组织5-LO免疫组化染色结果和阳性细胞计数结果,图中圈出的是5-LO染色细胞;
图3A为CAF上清刺激后的MDSC上清培养三种ICC细胞系(QBC939, HCCC9810,RBE)以及加入不同LT受体抑制剂前后各自的成球百分比;
图3B为肿瘤细胞表面LT受体的表达情况;
图3C为肿瘤细胞表面BLT2受体的表达情况以及流式细胞术检测平均荧光强度指数的结果;
图3D为成球实验前后三种ICC细胞系表面BLT2表达情况;
图3E为不同刺激下5-LO下游产物分泌水平的检测结果;
图3F为LTB4刺激后的MDSC上清培养三种ICC细胞系(QBC939, HCCC9810,RBE)在不同LT受体抑制剂存在条件下的成球百分比;
图4为BLT2的特异性干扰RNA(siRNA)对MDSC上清处理的ICC细胞系成球率的影响;
图5A为肿瘤局部切片BLT2免疫组化染色结果以及BLT2低表达组与BLT2 高表达组病人的生存率曲线图;
图5B为BLT2表达水平与干性基因表达水平的相关性;
图6A为ICC病人来源肿瘤异体移植过程示意图;
图6B为PDX模型中肿瘤生长曲线以及终末肿瘤质量;
图6C为流式细胞检测肿瘤细胞凋亡比例结果;
图6D为肿瘤组织切片Ki67组化染色结果;
图6E为qPCR检测肿瘤组织干性基因表达情况的结果。
具体实施方式
为使本发明更明显易懂,兹以优选实施例,并配合附图作详细说明如下。
以下实施例中,所使用的实验方法如无特殊说明,均为常规方法;所用的材料、试剂等,如无特殊说明,均为常规市售产品;所涉及的定量试验,均设置三次重复实验,结果取平均值。
以下实施例中,部分实验材料与方法包括:
1.材料和试剂
细胞培养基(DMEM)、胎牛血清(FBS)购自赛默飞公司;琼脂糖购自上海生工生物公司;Taq酶和dNTPs购自TaKaRa公司;辣根过氧化物酶(HRP) 标记的驴抗鼠和抗兔IgG购自CST公司;胰岛素购自Roche公司;ECL检测试剂,Zileuton,U75302,LY255283,Zafirlukast,Bay-u9773购自Cayman化学;人重组EGF购自PeproTech公司;雷帕霉素和LY294002购自Selleck;寡聚核苷酸由上海生工公司合成。ICC细胞系HCCC9810,RBE,QBC939细胞系为复旦大学中山医院肝癌研究所赠予。
2.抗体
兔单抗5-LO,CD33抗体购自Abcam;鼠单抗BLT2抗体购自Sigma-Aldrich;兔多抗Ki67抗体购自R&D Systems。
3.组织学分析
用自动化平台(Aperio Technologies)ScanScope扫描系统将肿瘤组织芯片图像数字化,排除质量差或一抗染色阴性的样品。在200X HPF视野中计数CD33+和5-LO+细胞。BLT2阳性染色区域的密度通过Image J(v1.5.2)进行定量。根据五张随机选择的200X高分辨率视野中Ki67+细胞的数量来量化PDX模型中肿瘤的增殖指数。
4.细胞培养
将CAF(癌相关成纤细胞)在无血清DMEM-F12培养基中培养24h,再在含1%FBS的RPMI-1640持续培养24小时,收取CAF上清。使用CAF上清培养血液CD33+MDSC 6小时,然后再用新鲜的无血清培养基培养24小时,获得由CAF培养的CD33+MDSC的CM(条件培养基)。将所有上清过滤并用于进一步研究。
5.PDX模型建立
建立ICC患者异种移植(PDX)模型,从2017-2018年之间接受过肿瘤切除而未接受辅助治疗的两名独立ICC患者处获得新鲜肿瘤样品。将每个患者的癌组织小块,并植入NSG小鼠皮下进行扩增。直到肿瘤达到约1cm长为止,收集非坏死的皮下肿瘤组织并切成约1mm3的小块。植入6周龄的雄性NSG小鼠皮下。从肿瘤组织植入后第9天开始进行治疗,BLT2拮抗剂LY255283(20mg/kg) 腹腔注射,每天注射一次,和/或吉西他滨(50mg/kg)每周两次。
6.RNA提取以及荧光定量PCR
通过TRIzol(购自Invitrogen)从细胞中提取RNA,并通过PrimeScript RT MasterMix(购自TaKaRa)将RNA反转录为cDNA。使用Power SYBR Green Master试剂盒(#RR820A,TaKaRa)在Roche 480仪器进行qPCR。使用2-ΔΔC(t)方法计算靶基因的相对表达量。
7.LTB4测定
用LTB4 ELISA试剂盒(购自Cayman化学公司)测定LTB4的浓度。
8.免疫荧光
将处理好的细胞用4%多聚甲醛(Paraformaldehyde,PFA)在室温固定20 分钟,PBS洗两遍;0.1%Triton X-100室温处理5分钟;PBS洗三遍,用PBS 稀释的2.5%驴血清室温封闭60分钟;用封闭液稀释一抗,4℃孵育过夜;PBS 洗三遍;用封闭液稀释二抗,室温避光孵育1小时;PBS洗三遍;DAPI染色; PBS洗3遍,抗荧光淬灭封片剂封片。
9.细胞分选
使用70%/35%Percoll梯度液(GE Healthcare),将ICC患者的肿瘤和血液单细胞悬液进行密度梯度离心分离,然后使用CD33抗体包被的磁珠(购自 Miltenyi Biotec),通过磁珠细胞分选术进一步纯化。
10.RNA干扰(siRNA)
BLT2的特异性siRNA为双链siRNA,由Genepharma合成。BLT2的特异性siRNA序列是:5’-CCACGCAGUCAACCUUCUGUGTT-3’(SEQ ID NO:1),使用lipofectamine iMAX(#13778075,Invitrogen)将其转染到ICC细胞系中。
11.蛋白质印迹
将肿瘤组织或细胞在含有50mM Tris-HCl,2%SDS,蛋白酶抑制剂混合物和1mMPMSF的裂解缓冲液中匀浆。将PVDF膜的一抗在4℃孵育过夜,然后与HRP偶联的二抗孵育。使用ECL检测系统(Pierce/Thermo Fisher Scientific) 进行检测。
12.肿瘤细胞成球试验
将ICC肿瘤细胞悬浮在补充有B27(1:50),20ng/mL人重组EGF和4mg /mL胰岛素的无血清DMEM-F12(购自GIBCO)的球形成培养基中。然后以每孔500个细胞的量添加到超低吸附细胞培养板中。在不使用或同时使用以下每种抑制剂的情况下,将不同类型的上清液以1:1的体积比添加到球形成介质中。培养14天后,在显微镜下计算直径>75mm的肿瘤细胞团(球)。通过将球的数量除以接种的单个肿瘤细胞的原始数量,可以计算出球形成的效率。
13.细胞凋亡检测
为检测肿瘤细胞凋亡百分比,利用酶解法将来自ICC患者或荷瘤小鼠的肿瘤组织消化成单细胞悬液后,进行细胞凋亡的检测。FITC交联的Annexin-V染料购自BD生命科学,7-AAD染料购自赛默飞生物。将组织切成片并用胶原酶 IV(1mg/mL,购自Sigma),胶原酶II(0.5mg/mL,购自Sigma)和DNaseI (4U/mL,购自Sigma)在37℃下放置1小时,然后通过70μm的细胞滤网。收获单细胞悬液。之后按照说明书共染7-AAD和FITC交联的Annexin-V,流式细胞术分析凋亡细胞比例。
14.免疫组化染色实验和Ki67免疫组化检测细胞增殖
肿瘤组织切片进行IHC染色参考常规方法。石蜡切片经二甲苯脱蜡2次后,梯度酒精洗涤水化,3%过氧化氢耗竭内源性过氧化物酶,水洗,柠檬酸钠溶液抗原修复,PBS洗,0.1%Triton X-100室温处理5分钟,PBS洗三遍,用PBS 稀释的2.5%驴血清室温封闭60分钟,一抗4℃孵育过夜。第二天PBS洗3 次,加入二抗孵育1小时,PBS洗3次,加入ABC复合物,孵育30分钟,加入底物显色,镜下观察,自来水冲洗1min终止显色。苏木素复染核。所使用的一抗为CD33(1:100,#ab269456,购自Abcam),5-LO(1:100,#ab169755,购自Abcam),BLT2(1:100,#HPA029680,购自Sigma-Aldrich)或Ki67(1: 100,AF7617,购自R&D Systems)。底物和ABC复合物购自武汉塞维尔生物。利用自动化平台(Aperio Technologies)ScanScope扫描系统将TMA图像数字化,排除质量差或一抗染色阴性的样品。在200倍放大的视野中计数CD33+和5-LO+细胞。BLT2阳性染色区域的密度通过ImageJ(v1.5.2)进行定量。PDX肿瘤的增殖指数根据五张随机选择的200倍视野中Ki67+细胞的数量来相对定量。
15.生存曲线图
KM生存曲线图通过Graphpad Prism 7绘制。利用Log-rank检验来分析两组生存期间的差异显著性。
实施例1:5-LO在ICC肿瘤组织中表达上调
1.1将145名ICC肿瘤病人的癌组织样本和癌旁组织制成组织芯片,免疫组化染色标记CD33+的细胞,软件定量CD33+细胞。通过比较ICC病人肿瘤和非肿瘤组织的CD33+免疫抑制性髓样细胞数量发现,ICC病人肿瘤局部的CD33+细胞浸润水平显著高于非肿瘤组织,如图1A所示。根据肿瘤组织CD33+细胞的表达水平,将病人分成CD33高表达和CD33低表达组,随后通过比较高CD33 组病人和低CD33组病人预后水平,结果显示CD33+细胞水平与ICC病人术后生存率显著负相关(P=0.006),高CD33+细胞浸润的病人预后要显著差于低CD33+细胞浸润的病人,如图1B所示。
1.2通过密度梯度离心和磁珠分选分离ICC病人血液中的CD33+MDSC和肿瘤局部的CD33+MDSC,并使用免疫荧光染色标记CD33和5-LO,结果发现,肿瘤局部的CD33+MDSC上调表达5-LO,其结果显示肿瘤局部的CD33+MDSC 上调表达5-LO,如图2A所示。因此推测,肿瘤局部的微环境可以诱导MDSC 上调5-LO表达,为了验证这一猜想,随后收集了ICC肿瘤相关成纤维细胞的上清,用它来刺激血液来源的CD33+MDSC细胞,免疫印迹实验显示它能够刺激血液来源的CD33+MDSC细胞上调表达5-LO,如图2B所示。通过对ICC病人肿瘤局部和非肿瘤组织切片进行免疫组化染色,结果表明ICC病人肿瘤局部与非肿瘤组织相比,5-LO+细胞浸润得更多;此外,将ICC病人分为5-LO高表达组和5-LO低表达组,发现5-LO低表达组病人预后显著优于5-LO高表达组,如图 2C所示。
实施例2:BLT2对ICC肿瘤细胞干性的影响
由于5-LO是白三烯(LT)重要的催化酶,通过探究5-LO下游几种催化产物和他们的受体通路对ICC肿瘤细胞干性的影响,结果发现,5-LO下游催化产物LTB4通过结合其位于ICC肿瘤细胞表面的BLT2受体促进ICC肿瘤细胞干性:
2.1通过比较不同刺激下的ICC肿瘤细胞的成球效率,结果发现,MDSC上清对ICC肿瘤细胞有促进干性的作用,而5-LO抑制剂Zileuton和BLT2抑制剂 LY255283与MDSC细胞上清共刺激时,抑制了其对肿瘤细胞干性的促进作用,如图3A所示。通过逆转录荧光定量PCR,结果显示,ICC肿瘤细胞BLT2的基因水平较其它LT受体高,如图3B所示。且通过流式细胞术分析发现BLT2蛋白在ICC细胞表面的表达,如图3C所示,BLT2蛋白在ICC肿瘤细胞表面高表达。成球试验后,三种ICC肿瘤细胞系表面BLT2表达均显著升高,如图3D所示。此外,利用ICC肿瘤相关成纤维细胞上清刺激MDSC检测上清中LTB4的水平发现,肿瘤相关成纤维细胞分泌的上清能够上调MDSC表达LTB4,而5-LO 的抑制剂Zileuton能够阻断该效应,如图3E所示。相应的,利用BLT2的拮抗剂LY255283可以消除LTB4对ICC肿瘤细胞系的促干性效应,降低其成球率,如图3F所示。
2.2为了确定BLT2通过5-LO下游催化产物LTB4对ICC肿瘤细胞干性的影响,使用了BLT2特异性的干扰RNA(5’-CCACGCAGUCAACCUUCUGUGTT-3’) 来抑制BLT2的表达,结果发现,BLT2特异性siRNA(序列如SEQ ID NO:1所示)能够显著阻断MDSC上清对三种ICC细胞系干性的促进作用,如图4所示。
实施例3:BLT2的表达水平对ICC肿瘤病人预后以及肿瘤干性基因表达的影响
为了探究肿瘤局部BLT2表达水平与预后的关系,利用190个ICC病人的肿瘤组织芯片,免疫组化染色BLT2,并将病人按照BLT2表达水平分为BLT2高表达组和BLT2低表达组,进行生存分析,结果显示,BLT2的表达水平与ICC 病人的预后生存率显著负相关(P<0.001),BLT2低表达组病人的预后生存率要显著高于BLT2高表达组,如图5A所示。同时,BLT2基因的表达水平和其他肿瘤细胞干性基因NANOG、SOX2、POU5F1和CTNNB1的表达正相关,如图 5B所示。这些结果提示BLT2及其配体对ICC病人肿瘤干性和预后有显著影响。
实施例4:BLT2抑制剂联合化疗药物对ICC肿瘤的抑制作用
为了检验BLT2抑制剂的临床应用潜力,构建了人源化的异体肿瘤移植小鼠模型,并分别或者联用BLT2抑制剂LY255283和化疗药物吉西他滨 (Gemcitabine)来治疗肿瘤,如图6A所示。结果表明,BLT2抑制剂LY255283 和化疗药物吉西他滨联用组,肿瘤的生长速率以及给药8天后肿瘤终末质量明显低于其他对照组,如图6B所示,说明BLT2抑制剂能够进一步促进化疗药物吉西他滨对肿瘤生长的抑制作用。通过细胞凋亡实验检测给药8天后不同给药组肿瘤中细胞凋亡百分比,BLT2抑制剂LY255283和化疗药物吉西他滨联用组的细胞凋亡率最高,如图6C所示,说明BLT2抑制剂LY255283能够进一步促进肿瘤细胞凋亡;给药8天后通过Ki67免疫组化染色定量检测细胞增殖情况,结果发现BLT2抑制剂LY255283能够抑制肿瘤细胞增殖,如图6D所示;给药8天后,通过对肿瘤干性基因(POU5F1,CTNNB1,NANOG,SOX2)的荧光定量PCR 分析,结果发现单独的吉西他滨治疗会富集肿瘤干细胞浸润,但是这种效应可以被BLT2抑制剂消除,如图6E所示,说明BLT2抑制剂LY255283能够在人源化的肿瘤小鼠模型中抑制肿瘤的干性。
上述实施例仅为本发明的优选实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
序列表
<110> 复旦大学
<120> 一种影响肿瘤细胞干性的关键蛋白BLT2及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccacgcaguc aaccuucugu gtt 23
Claims (6)
1.检测BLT2的表达水平的试剂在制备至少一种如下产品中的应用:检测肝内胆管细胞癌ICC肿瘤细胞干性的试剂以及预测肝内胆管细胞癌ICC患者生存期的试剂。
2.如权利要求1所述的应用,其特征在于,所述的检测BLT2的表达水平的试剂包括BLT2的抗体。
3.抑制BLT2的表达水平的物质在制备抑制肝内胆管细胞癌ICC肿瘤细胞干性的试剂中的应用和/或在制备抑制肝内胆管细胞癌ICC肿瘤细胞干性相关特征分子的表达的试剂中的应用,其特征在于,所述的肝内胆管细胞癌ICC肿瘤细胞干性相关特征分子为基因NANOG、SOX2、POU5F1和CTNNB1中的至少一种。
4.如权利要求3所述的应用,其特征在于,所述的抑制BLT2的表达水平的物质包括BLT2的特异性干扰RNA或LY255283。
5.如权利要求4所述的应用,其特征在于,所述的BLT2的特异性干扰RNA包括序列为5’-CCACGCAGUCAACCUUCUGUGTT-3’的siRNA。
6.BLT2抑制剂联合肝内胆管细胞癌ICC化疗药物在制备至少一种如下产品中的应用:抑制肝内胆管细胞癌ICC肿瘤细胞干性的试剂、治疗肝内胆管细胞癌ICC肿瘤的药物、抑制肝内胆管细胞癌ICC肿瘤进程的药物以及抑制肝内胆管细胞癌ICC肿瘤细胞干性相关特征分子的表达的试剂;所述的BLT2抑制剂为LY255283,所述的肝内胆管细胞癌ICC化疗药物为吉西他滨;所述的肝内胆管细胞癌ICC肿瘤细胞干性相关特征分子为基因NANOG、SOX2、POU5F1和CTNNB1中的至少一种。
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