CN104062432B - ang2在制备ECTC血管类型肝癌诊断试剂及治疗药物中的应用 - Google Patents
ang2在制备ECTC血管类型肝癌诊断试剂及治疗药物中的应用 Download PDFInfo
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Abstract
本发明提供一种蛋白编码基因ang2及其应用。本发明进行了人肝癌标本组织免疫组化和血液标本Elisa检测,表明肝癌病人组织或血液标本中ang2的表达可以指示病人组织血管类型;另外,小鼠成瘤实验的结果表明体内敲低ang2的表达可显著抑制肝癌细胞的体内成瘤能力和ECTC类型血管的生成能力。因此,ang2可用于制备ECTC血管类型肝癌的指示剂以及治疗ECTC血管类型肝癌的药物。
Description
技术领域
本发明属于基因工程领域,具体涉及ang2及其在制备ECTC血管类型肝癌诊断试剂及治疗药物中的应用。
背景技术
原发性肝细胞肝癌(hepatocellularcarcinoma,HCC)是世界上最常见的恶性肿瘤之一,其发生率和死亡率均高居各种恶性肿瘤前列。众所周知,癌症难以根治的主要原因是局部复发和远处转移。一般认为,肝脏丰富的血管系统以及肝癌活跃的血管生成,是肝癌患者易于发生血行转移,尤其是早期肝内播散的重要因素。但是,肝癌中微血管的数量与肝癌复发转移的相关性尚存争议。而我们发现:肝癌中存在ECTC(endothelium-coatedtumorcellclusters,内皮细胞包绕肿瘤团块)和非ECTC(non-ECTC)两种血管结构,前者的血管呈网络状连接,将肿瘤块包绕分割;后者的血管则为分散的管状结构。更为重要的是,与non-ECTC肝癌相比,ECTC肝癌组织中镜下微小转移灶更多,行根治性切除术后复发率更高、生存时间更短。这提示:ECTC血管结构很可能为癌细胞生长及转移提供了更强大的便利条件,因此制备ECTC血管类型肝癌诊断试剂及治疗药物具有重要的应用价值。
Sugino等最早报道了肿瘤中类似ECTC的血管结构的存在。他们将低侵袭高转移的鼠乳腺癌细胞株接种小鼠乳房脂肪垫成瘤后,观察到肿瘤中血管发展成融合的血窦样结构,并包绕肿瘤团块;而且,该细胞株虽然促血管生成能力强,但分泌MMP和侵袭能力弱。此后,该课题组在人肝癌、肾癌和甲状腺癌中都观察到癌栓被内皮细胞包绕的现象。我们之前的报道首次证明了ECTC结构与肿瘤的转移复发密切相关,是独立的预后不良预测因素。至今,关于ECTC形成的潜在机制研究只有两篇报道,他们发现:过表达VEGF-A或分泌型白细胞蛋白酶抑制因子(SLPI)可促进肿瘤细胞株成瘤组织中ECTC结构血管的形成。
Angiopoietin/Tie介导了血管的成熟,是血管生成尤其是肿瘤血管生成的重要调控通路。Angiopoietin(血管生成素)家族主要包括Aang1,ang2和ang4,其受体tie家族主要包括tie-1和tie-2。ang1是tie-2的激活剂,而ang2则作为ang1的拮抗剂,可与ang1竞争性的结合tie-2。ang4目前报道不多,其功能可能与ang1相似。而tie-1的配体尚未鉴定,tie-1可能作为tie-2的负调节因子存在。Ang1/tie-2信号激活可以维持内皮细胞处在静息状态,同时ang1还可以刺激壁细胞(血管平滑肌细胞和周细胞)覆盖到内皮细胞表面以及基底膜的沉积,使血管连接更加紧密,表现出成熟血管的特征。肿瘤中,ang1可以促进内皮细胞存活和血管成熟帮助肿瘤生长,但同时促进血管的成熟抑制肿瘤细胞侵袭血管。而ang2可拮抗ang1,使内皮细胞由静息态变为激活状态,开始增殖和出芽,生成新的血管。同时ang2还可以降低壁细胞的覆盖,使血管通透性增加,并表现出不成熟的特征。由此可见,ang1在肿瘤发展中起到双面作用。而目前多数研究认为ang2主要起到促肿瘤的作用,是抗血管药物潜在的靶分子。
国内外文献中至今未见有关ang2与ECTC血管类型的相关性研究报道。因此,进一步研究ang2在ECTC类型血管生成中的作用及其在肝癌诊治中的应用,对于制备高效的抗肿瘤血管生成药物以及肿瘤个体化治疗意义重大。
发明内容
为了解决以上技术问题,本发明的一个目的是提供肝癌患者肝组织中的ang2表达水平作为ECTC血管类型肝癌的标记的应用。
本发明的另一个目的是提供肝癌患者血液标本中ang2的表达水平作为ECTC血管类型肝癌的标记的应用。
本发明的再一个目的是提供ang2在制备用于ECTC血管类型肝癌诊断及预后的试剂中的应用。其中患者肝癌组织或血液标本中ang2的高表达指示ECTC血管类型的可能性更高,从而预测患者更容易转移和复发。本发明还提供了检测ang2表达水平的试剂在制备用于ECTC血管类型肝癌诊断预测及预后的试剂中的应用。
此外,本发明还提供了检测ECTC血管类型肝癌的试剂盒,其包含用于检测肝癌组织或血液标本中ang2的表达水平的试剂。优选地,所述检测肝癌组织或血液标本中ang2表达水平的试剂,包括ang2特异抗体。
本发明的另一目的在于提供ang2在制备治疗ECTC血管类型肝癌的药物中的应用。
为证实ang2的表达水平在作为ECTC血管类型肝癌的标记中的应用,设置了A、B两个实验,具体如下:
实验A:免疫组化检测肝癌病人组织中CD34(血管内皮细胞标记分子)、ang2的表达。
实验B:Elisa法检测肝癌病人血液标本中ang2的表达。
对实验结果进行对比分析及相关性统计表明:实验A的140份肝癌病人组织中,经过CD34标记血管内皮细胞,以及软件得到的ang2阳性信号值,我们发现,在ECTC组织中ang2表达水平显著更高;实验B中的69份肝癌病人血液标本标本中,肝癌病人血液标本中的ang2的表达在组织血管类型为ECTC的标本中显著更高。以上结果均表明,ang2在肝癌病人肝组织中的表达或是血液标本的表达水平可以指示组织血管类型,从而预测肝癌病人的预后。
本发明通过小鼠成瘤实验研究ang2在体内表达对肝癌细胞生长和ECTC类型血管的影响。上述小鼠成瘤实验设置阴性对照组和实验组。阴性对照组的肿瘤细胞株中转染NC或感染control病毒,实验组的肿瘤细胞株中转染si-ang2或感染表达sh-mAng2的病毒。上述小鼠成瘤实验所采用的肿瘤细胞株为人原代肝癌细胞66#T和小鼠肝癌细胞株Hepa1-6。
对实验结果进行对比分析及相关性统计表明:与阴性对照组形成ECTC类型血管不同,实验组的肿瘤的血管形态转变为更多的no-ECTC血管,统计学分析具有显著差异。
小鼠成瘤实验结果表明,体内抑制ang2表达可以显著抑制肝癌细胞形成ECTC类型血管的能力,降低转移复发率,从而改善肝癌患者的预后。
因此,本发明还提供了抑制ang2表达的试剂在制备治疗ECTC血管类型肝癌的药物中的应用。优选的,所述抑制ang2表达的试剂包含抗ang2抗体、siRNA和/或shRNA。更优选地,所述siRNA为si-ang2,其RNA序列从5’端到3’端为:5’-GATGATAGAAATAGGGACAAAdTdT-3’。或者,所述shRNA为sh-mAng2,其引物序列如下,
shmAng2#1正向引物:5’-CATGGATCCTCGGGCAGGAAGAGGGCCTA-3’;
反向引物:
5’-CATCTCGAGGCGGCCGCCCGGGCTATCCGTAAAGAAGAGCAACTCGAGTTGCTCTTCTTTACGGATAGCTTTTTGGAATTCCGCGTCCTTTCCACAAG-3’;
shmAng2#2正向引物:5’-CATGGATCCTCGGGCAGGAAGAGGGCCTA-3’;
反向引物:
5’-CATCTCGAGGCGGCCGCCCGGCGTGCTTAAGATCCAGCTGAACTCGAGTTCAGCTGGATCTTAAGCACGTTTTTGGAATTCCGCGTCCTTTCCACAAG-3’。
附图说明
图1为免疫组化检测血管内皮细胞标记CD34和ang2表达代表图及统计图。图1是实施例1中的实验A的实验结果。
图2为Elisa检测肝癌病人血液标本ang2表达的统计图。图1是实施例1中的实验B的实验结果。
图3为66#T小鼠成瘤实验结果图。图3A为转染si-ang2后,66#Tang2表达检测图;图3B为血管类型及统计图。图3是实施例2的实验结果.
图4为Hepa1-6小鼠成瘤实验结果图。其中,图4A为免疫组化标记的小鼠肿瘤表达ang2的代表图和统计图;图4B免疫组化标记的小鼠肿瘤血管内皮细胞染色代表图和血管类型的统计图。图4是实施例2的实验结果.
具体实施方式
以下结合具体实施例,进一步说明本发明的技术方案。应理解,以下实施例只用于说明本发明而不以任何形式限制本发明。
ang2(Angiopoietin2)基因的序列如NCBI数据库所收录(NCBI.Annotation:Chromosome8,NC_000008.10)。
本发明所采用的ang2免疫组化特异抗体由Abcam公司(美国)购得;CD34免疫组化抗体由北京中衫公司(中国)购得;血液标本中ang2的Elisa检测试剂盒由R&D公司(美国)购得。
本发明所采用的si-ang2序列从NCBI数据库获得,其RNA序列从5’端到3’端为:5’-GATGATAGAAATAGGGACAAAdTdT-3’,由上海吉玛公司合成。阴性对照组的阴性对照RNA序列(也称NC)为上海吉玛公司合成的用于siRNA实验的标准阴性对照序列,其RNA序列从5’端到3’端为:5’-UUCUCCGAACGUGUCACGUdTdT-3’。
本发明所采用的sh-mAng2序列从NCBI数据库获得,引物序列如下,
shmAng2#1正向引物:5’-CATGGATCCTCGGGCAGGAAGAGGGCCTA-3’;
反向引物:
5’-CATCTCGAGGCGGCCGCCCGGGCTATCCGTAAAGAAGAGCAACTCGAGTTGCTCTTCTTTACGGATAGCTTTTTGGAATTCCGCGTCCTTTCCACAAG-3’;
shmAng2#2正向引物:5’-CATGGATCCTCGGGCAGGAAGAGGGCCTA-3’;
反向引物:
5’-CATCTCGAGGCGGCCGCCCGGCGTGCTTAAGATCCAGCTGAACTCGAGTTCAGCTGGATCTTAAGCACGTTTTTGGAATTCCGCGTCCTTTCCACAAG-3’。由上海英俊公司合成,通过pCDH慢病毒表达系统合成表达病毒。
实施例1:人组织标本ang2表达指示肝癌组织血管类型
1.1免疫组化检测
人肝癌组织的切片由中山大学附属肿瘤医院标本库提供。制成石蜡切片4℃保存。后续检测步骤为:
(1)预处理:取出55℃烤片1h,放于二甲苯中脱蜡两次,每次10min;依次用100%、95%、90%、80%、70%的乙醇洗一次,每步3min,后用双蒸水洗3min;0.3%的双氧水处理10min,双蒸水再洗3次,每次3min;浸泡于修复液中,用微波炉进行热修复,室温冷却半小时;用1×PBS洗三次,每次3min;
(2)杂交染色:用CD34或ang2抗体进行孵育,4℃过夜;恢复室温,弃去抗体液,用1×PBS洗三次,每次5min;加入二抗,37℃半小时;用1×PBS洗三次,每次5min;加入DAB显色液进行显色,然后用苏木精复染。
(3)计数:CD34染色判断血管类型;ang2染色后400倍镜下随机拍10个视野,通过ImagineProPlus软件计数ang2阳性面积。
1.2Elisa检测
人肝癌病人的血液标本由中山大学附属肿瘤医院提供,相关实验获得中山大学伦理委员会的批准。-80℃保存。后续检测步骤为试剂盒提供,具体如下
(1)加样:加一定稀释的待检样品0.1ml于上述已包被之反应孔中,置37℃孵育1小时。然后洗涤。
空白对照组:空白孔,仅加入显色液;
阴性对照组:样品为样品稀释液;
实验组:经样品稀释液稀释的样品检测液;
(2)加酶标抗体:于各反应孔中,加入新鲜稀释的一抗0.1ml。37℃孵育2小时,洗涤。加入二抗工作液0.1ml。37℃孵育1小时,洗涤。
(3)加底物液显色:于各反应孔中加入新鲜配制的TMB底物溶液0.1ml,室温15-30分钟。
(4)结果判定:在ELISA检测仪上,于450nm处测定各个样品孔的OD值。
1.3结果分析
实验A,如图1所示,我们共检测了140份肝癌组织标本,通过CD34染色血管内皮细胞,根据血管形成分型,发现所检测的肝癌组织中有53份(38%)为ECTC血管类型,87(62%)为no-ECTC血管类型。通过ImagineProPlus软件计数ang2阳性值,与血管类型进行相关性分析,我们发现形成ECTC类型血管的肝癌组织ang2的平均表达值0.073,而形成no-ECTC类型血管的肝癌组织其平均表达值为0.022,ECTC类型组织显著高表达ang2(P<0.001,T检验)。
实验B,如图2所示,我们通过Elisa共检测了69份肝癌病人的血液标本,在所检测血液标本对应的肝癌组织中,通过CD34染色血管内皮细胞,进行血管形态分型。发现所检测血液标本对应的肝癌组织中有32份(46%)为ECTC血管类型,37(54%)为no-ECTC血管类型。通过酶标仪读取ang2的光密度值,与血管类型进行相关性分析,我们发现形成ECTC类型血管的肝癌组织对应的血液标本中ang2的平均光密度值为0.22,而形成no-ECTC类型血管的肝癌组织对应的血液标本中,ang2平均光密度值为0.14,ECTC类型的肝癌病人的血液标本中显著高表达ang2(P<0.01,T检验)。
上述结果显示,肝癌病人组织或血液标本中ang2的表达水平可以作为ECTC血管类型肝癌的指示分子。
实施例2:小鼠成瘤实验
2.1细胞转染或感染
将目的RNA或病毒导入细胞。具体实验步骤为:
(1)转染:取胰蛋白酶消化细胞重悬于含有10%FBS的无双抗DMEM培养基(购自Invitrogen公司)中,使得细胞密度为3×104个细胞/ml;将30pmol目的RNA稀释于100μlopti-MEM(购自Invitrogen公司)中,轻敲管壁混匀;每管加入1μlLipofectamineRNAiMAX,轻敲管壁混匀,室温放置15min;得到RNAiMAX/siRNA稀释液;每孔加入100μlRNAiMAX/siRNA稀释液,再加入500μl细胞稀释液,该细胞稀释液可使细胞密度在转染24h后大约为30-50%汇合。上述目的RNA可为si-ang2、阴性对照RNA(也称NC)。48h后,选取部分细胞进行RT-PCR检测敲低效率。
上述实施例为24孔板,其他孔板可根据孔的大小按比例放大或者缩小试剂用量。
(2)感染:包括病毒包装和感染靶细胞两个步骤。
A.包装:以24孔板为例,加入0.3μg包装混合质粒和0.3μg表达质粒以及50μl的opti-MEM,轻柔混匀,室温孵育5min。取1μlLipofectamine2000(购自Invitrogen公司)溶于50μlopti-MEM中。轻柔混匀,室温孵育5min。将上述两管试剂混合,轻柔混匀,放置15min后加入预先贴好的293T包装细胞中。转染后48-72h收获含病毒的上清。0.45um孔径的滤器过滤获得病毒上清,于-80°C贮存。
B.感染靶细胞:在24孔板中种植适当密度的靶细胞,加入病毒感染,24h后更换培养基,换入新鲜的培养基。48h后使用含有抗生素的新鲜培养基进行阳性细胞的筛选或是通过流式分选GFP阳性的细胞。筛选到阳性细胞后进行RT-PCR检测敲低效率。
2.2小鼠成瘤
选用5只5周龄的BALB/c裸鼠,在其两只后腿分别皮下注射1×106的表达si-ang2或NC的66#T细胞。或是选用5只5周龄的C57小鼠,在其后腿分别皮下注射1×106表达sh-mAng2或control的hepa1-6细胞。指定时间后,完整剥离肿瘤组织,以备后续实验。
2.3免疫组化
小鼠的肿瘤组织取自步骤2.2,制成石蜡切片4℃保存。两者后续检测步骤为:
(1)预处理:取出55℃烤片1h,放于二甲苯中脱蜡两次,每次10min;依次用100%、95%、90%、80%、70%的乙醇洗一次,每步3min,后用双蒸水洗3min;0.3%的双氧水处理10min,双蒸水再洗3次,每次3min;浸泡于修复液中,用微波炉进行热修复,室温冷却半小时;用1×PBS洗三次,每次3min;
(2)杂交染色:用鼠特异性的CD34抗体和ang2抗体进行孵育,4℃过夜;恢复室温,弃去抗体液,用1×PBS洗三次,每次5min;加入二抗,37℃半小时;用1×PBS洗三次,每次5min;加入DAB显色液进行显色,然后用苏木精复染。
(3)计数:CD34染色判断血管类型,并计数形成血管内皮细胞包绕肿瘤细胞团的ECTC血管个数;ang2染色后400倍镜下随机拍5个视野,通过ImagineProPlus软件计数ang2阳性面积。
2.4结果分析
如图3所示,A图显示我们所设计合成的si-ang2能够显著地敲低人原代肝癌细胞66#T的ang2表达。而如B图所示,敲低ang2表达的人肝癌原代细胞66#T在小鼠体内形成的肿瘤,其组织血管类型发生了显著的改变,通过计数ECTC类型血管的数量,我们发现敲低ang2的肝癌细胞肿瘤形成ECTC血管的能力显著减弱(NCvs.Si-ang2=11.14vs.4.22;p<0.01)。
如图4所示,利用shRNA敲低ang2表达的鼠肝癌细胞株Hepa1-6在小鼠体内形成的肿瘤,其ang2的表达显著降低(平均表达值为control:sh-mAng2#1:sh-mAng2#2=0.15:0.06:0.06,p<0.05),而且计数ECTC类型血管的数量,敲低ang2表达的小鼠肝癌细胞肿瘤形成ECTC血管的能力显著减弱(平均ECTC个数为control:sh-mAng2#1:sh-mAng2#2=11.7:1.4:2.3,p<0.05)。
综上所述,敲低内源ang2表达可以显著抑制人或鼠肝癌细胞于小鼠体内形成ECTC类型血管的能力。这表明降低Ang2的表达以及抑制ang2表达的试剂可以用于制备治疗ECTC血管类型肝癌的药物。
Claims (6)
1.ang2在制备用于ECTC血管类型肝癌诊断预测及预后的试剂中的应用。
2.检测ang2表达水平的试剂在制备用于ECTC血管类型肝癌诊断预测及预后的试剂中的应用。
3.抑制ang2表达的试剂在制备治疗ECTC血管类型肝癌的药物中的应用。
4.根据权利要求3的应用,其中所述抑制ang2表达的试剂包括抗ang2抗体、siRNA和/或shRNA。
5.根据权利要求4的应用,其中所述siRNA为si-ang2,其RNA序列从5’端到3’端为:5’-GATGATAGAAATAGGGACAAAdTdT-3’。
6.根据权利要求4的应用,其中所述shRNA为sh-mAng2,其引物序列如下,
shmAng2#1正向引物:5’-CATGGATCCTCGGGCAGGAAGAGGGCCTA-3’,反向引物:5’-CATCTCGAGGCGGCCGCCCGGGCTATCCGTAAAGAAGAGCAACTCGAGTTGCTCTTCTTTACGGATAGCTTTTTGGAATTCCGCGTCCTTTCCACAAG-3’;
shmAng2#2正向引物:5’-CATGGATCCTCGGGCAGGAAGAGGGCCTA3’,反向引物:5’-CATCTCGAGGCGGCCGCCCGGCGTGCTTAAGATCCAGCTGAACTCGAGTTCAGCTGGATCTTAAGCACGTTTTTGGAATTCCGCGTCCTTTCCACAAG-3’。
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