CN110221070A - 组蛋白h2b单泛素化用于鉴定同源重组修复缺陷的用途 - Google Patents
组蛋白h2b单泛素化用于鉴定同源重组修复缺陷的用途 Download PDFInfo
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Abstract
本发明涉及一种基于组蛋白H2B单泛素化用于检测同源重组修复缺陷的用途,具体涉及一种基于组蛋白H2B单泛素化水平检测同源重组修复缺陷的试剂盒及其用途。所述用途是利用检测组蛋白H2B单泛素化的水平来检测具有同源重组修复缺陷,其结果有助于判别相关疾病的进展和PARP抑制剂等同源重组靶向药物治疗的响应度,包括但不限于带有乙型肝炎病毒的肝组织或肝癌,带有同源重组基因突变,表达和剪切体异常的各种类型的肿瘤。
Description
技术领域
本发明属于生物技术领域,具体涉及一种基于组蛋白H2B单泛素化用于检测同源重组修复缺陷的用途,更具体涉及一种基于组蛋白H2B单泛素化水平检测同源重组修复缺陷的试剂盒及其用途涉。具体地,本发明涉及通过检测组蛋白H2B的单泛素化(uH2B)水平确定被测样本是否具有同源重组修复功能的缺陷;所检出的同源重组修复缺陷可以由于任何调控同源重组修复机制的基因突变或功能紊乱产生(如HBx表达,BRCA1/BRCA2等同源重组修复基因的突变,剪切体变异或基因表达异常等),该检测方法是一种对同源重组修复缺陷的普适鉴定方法,而不依赖于导致修复缺陷的特定生物学原因。进一步地,通过免疫组化,免疫印迹,免疫荧光和ELISA等依赖于H2B单泛素化特异性抗体的检测方法判定H2B的单泛素化水平,可以指导该指标阴性或其水平减低的疾病(即带有同源重组修复缺陷的疾病)的靶向药物治疗和预防性早期治疗,如预防性切除手术。
背景技术
近年研究发现,同源重组缺陷是肿瘤发生发展、产生耐药性和肿瘤复发的重要生物学机制。大量临床研究研究表明多种肿瘤基因组(如乳腺癌、卵巢癌、前列腺癌、胰腺癌及胃癌)带有同源重组修复基因的突变或表达异常,导致BRCA1/BRCA2等修复蛋白的功能异常,从而促进基因组不稳定和肿瘤发生。
基于多年的研究,已有多种针对同源重组修复缺陷的靶向药物进入三期临床或获得FDA批准,例如奥拉帕尼(1-(环丙甲酰基)-4-[5-[(3,4-二氢-4-氧代-1-酞嗪基)甲基]-2-氟苯甲酰]哌嗪),Niraparib(2-[4-((3S)-3-哌啶基)苯基]-2H-吲唑-7-甲酰胺),BGB-290和Veliparib等PARP抑制剂类型的药物。这些药物在临床上的应用可以有效地治疗带有BRCA1/BRCA2基因突变的乳腺癌,卵巢癌和前列腺癌。基于最新的研究,我们还发现带有HBV病毒感染也可以导致同源重组修复缺陷,其中,病毒癌基因HBx的表达是HBV导致同源重组缺陷的标记物。因此,这些PARP类抑制剂也有希望用于治疗HBV相关/阳性的肝癌。
尽管以PARP抑制剂为代表的同源重组靶向药物具有巨大的抗肿瘤治疗潜力,但鲜有明确的诊断指标或方法能简单、准确地预测肿瘤组织中的同源重组修复缺陷,导致这类靶向药物在临床使用中的局限性。目前,只有BRCA1/BRCA2基因突变检测作为评估乳腺癌及卵巢癌同源重组缺陷的临床适用指标,用于指导PARP抑制剂靶向治疗及乳腺癌和卵巢癌的预防性切除手术。然而,大量针对乳腺癌和卵巢癌的临床研究显示,根据BRCA1/BRCA2基因突变的检测只能检出~60%带有同源重组修复缺陷的肿瘤(参见:HRDetect is apredictor of BRCA1and BRCA2deficiency based on mutational signatures.NatMed.2017.3(4):517-525.),其余40%未检出的同源重组缺陷肿瘤可能带有影响同源重组修复的其他遗传学因素,比如BRCA1/BRAC2基因启动子的甲基化异常,基因剪切突变体,其他基因突变(如ATM,RAD51,MRE11或未知变异等)。再有,由于BRCA1/BRCA2基因没有突变热点,导致临床突变检测中出现大量生物学意义不明的突变VUS(a variant of unknownsignificance)(参见:Scherr CL.etl.A preliminary investigation of geneticcounselors'information needs when receiving a variant of uncertainsignificance result:a mixed methods study.Genet Med.2015.17(9):739-46),难以判断其是否真正导致BRCA1/BRCA2的功能缺失。由于这类VUS突变的存在,导致相当大一部分肿瘤中同源重组修复缺陷的诊断困难,无法指导靶向药物在这部分肿瘤中的使用,成为乳腺癌,卵巢癌预防性手术和靶向治疗的重要障碍,进一步使同源重组缺陷的临床诊断复杂化。
由此可见,目前临床使用的通过BRCA1/BRCA2基因检测来诊断同源重组修复缺陷的肿瘤具有明显的技术缺陷和应用局限。
本发明的研究发现组蛋白H2B单泛素化是同源重组修复的重要调控因子。H2B单泛素化作为同源重组修复信号通路的末端信号,直接反映同源重组修复是否进展,而与特定修复基因的突变或者功能紊乱无关:H2B单泛素化水平减低存在于多种同源重组修复缺陷,包括BRCA1/BRAC2基因突变和功能减低、HBx表达、CRL4WDR70复合体解聚等。因此,H2B单泛素化水平可以有效揭示细胞或组织中是否具有完整的同源重组修复功能。
经研究发现,带有HBV病毒感染的肝癌也具有同源重组修复缺陷的生物学和基因组学的特征,而癌基因HBx作为分子病因,其基因表达和蛋白水平的检测可以作为具有同源重组修复缺陷的乙型肝炎病毒相关疾病和肿瘤的诊断依据。在HBx过表达的细胞中,同源重组效率以及H2B单泛素化的水平都呈减低趋势,同时大部分HBV阳性肝癌的病理组织染色呈现H2B单泛素化染色阴性或弱阳性,说明H2B单泛素化水平可以作为HBx基因检测的补充,诊断HBV相关疾病和肿瘤中的同源重组缺陷,指导同源重组靶向药物的使用。
通过病理方法研究发现,在卵巢癌及HBV病毒阳性的肝癌中,H2B单泛素化水平的评价可以预测同源重组修复的缺陷,该指标不仅与BRCA1/BRCA2基因突变有高度相关性,还可以准确检测不带有BRCA1/BRCA2的肿瘤样本中同源重组的缺陷,与HBx基因表达检测的联用,更可以预测HBV病毒阳性肝癌的同源重组修复状态,说明H2B单泛素化水平是一种可用于鉴定所有类型和所有遗传变异来源的同源重组修复缺陷,而不依赖特定基因突变或病因。
由于H2B单泛素化缺陷的细胞和肿瘤可以被PARP抑制剂靶向致死,采用该指标鉴定出的同源重组缺陷可直接用于指导同源重组靶向药物的临床使用。
由此可见,基于H2B单泛素化的检测在临床上具有取代BRCA1/BRCA2突变检测的实际意义,并且由于H2B单泛素化的检测方法为抗原-抗体的免疫识别技术,在医疗成本及可操作性等方面大大优于BRCA1/BRCA2基因突变检测,剪切体和基因表达异常,以及基因组学HRD分析等传统和高通量方法。
发明内容
本发明的目的在于提供一种基于H2B单泛素化水平的新型同源重组修复缺陷的鉴定方法,换句说是指组蛋白H2B单泛素化用于检测同源重组修复缺陷的用途,H2B单泛素化水平的同源重组修复缺陷以及通过检测H2B单泛素化水平来鉴定同源重组缺陷及其相应肿瘤和HBV感染疾病。更具体来说本发明的目的在于提供一种组蛋白H2B单泛素化生物标记物用于鉴定同源重组修复缺陷的用途,即通过检测组蛋白H2B单泛素化水平的变化来鉴定、判断同源重组修复缺陷的肿瘤和乙肝感染相关疾病及其进展,特别是与HBx基因表达检测联用,作为评估同源重组缺陷的补充手段,能有效地诊断具有同源重组修复缺陷的乙型肝炎病毒(乙肝病毒)相关疾病和肿瘤,指导同源重组靶向药物的使用和研究。
为实现本发明的目的,提供如上实施方案:
本发明提供了一种蛋白H2B单泛素化或/和检测组蛋白H2B单泛素化水平的试剂,在制造诊断、检测具有同源重组修复缺陷的肿瘤和乙肝病毒感染所致相关疾病,预示所述肿瘤和乙肝病毒感染所致相关疾病进展及其药物治疗效果的试剂盒中的用途。
上述本发明的用途,所述肿瘤包括乳腺癌、卵巢癌、宫颈癌、子宫内膜(样)癌、卵巢透明细胞癌、胃癌、胰腺癌、前列腺癌或肝癌及HBV感染引起的相关疾病。
上述本发明的用途,所述药物为PARP抑制剂,选自但不限于奥拉帕尼、rucaparib、Niraparib,BTB-290和Veliparib等。
上述本发明的用途,所述乙肝病毒(HBV)感染所致相关疾病,包括但不限于肝炎病毒感染性相关疾病,特别是乙型肝炎病毒(HBV)感染性疾病,更优选HBx表达阳性的HBV感染性疾病,更优选HBV携带者,再优选HBV感染,更优选HBV感染相关性急性肝炎、慢性肝炎、肝纤维化、肝硬化,最优选乙肝病毒阳性的肝癌和胆管细胞癌。
在另一实施方案中,本发明的一种鉴定同源重组修复缺陷的方法,包括以下过程:
1)取患者病理组织或体液;
2)检测组蛋白H2B单泛素化水平;
3)将检测结果与正常组织的单泛素化水平进行比对;
4)判断是否存在同源重组修复缺陷及其病理状态;
5)进一步的,通过比对数据结果,判断同源重组修复缺陷性肿瘤、预示所述肿瘤进展或药物治疗所述肿瘤的效果。
上述本发明的方法,所述检测组蛋白H2B单泛素化水平,所述检测包括选自免疫组化、免疫荧光、免疫印迹和ELISA的免疫检测技术;所述比对,包括判定组蛋白H2B单泛素化的阴性,或与正常组织相比较该单泛素化水平明显减少的肿瘤或相关疾病。所述组蛋白H2B单泛素化的检测,包括组蛋白H2B单泛素化的依赖H2B单泛素化特异性抗体的检测方法。对同源重组修复缺陷的鉴定,不依赖于特定的基因突变或生物学机制,包括乙型肝炎病毒HBx表达导致的同源重组修复缺陷,或BRCA1/BRCA2的功能缺陷及具有VUS特征的基因突变的同源重组缺陷肿瘤,或者,包括去甲基化酶或去乙酰化酶抑制剂可以诱导同源重组缺陷的药物处理方式及组合。
上述发明的方法中,针对肝癌及相关疾病,H2B单泛素化指标可与HBx的基因表达与蛋白水平的检测联用,作为评估同源重组缺陷的补充手段。所述检测包括但不限于免疫印迹,免疫荧光,免疫组化染色和ELISA等HBx特异性抗体相关技术,也包括原位杂交、ACD等基因表达相关技术,还包括直接检测与HBx水平直接关联的cccDNA等乙型肝炎病毒复制的指标。
在本发明中,所述同源重组修复缺陷及其相关疾病包括但不局限于BRCA1/BRCA2、RAD51、NBS1、CTIP、ATM、ATR、MRE11、RNF20/40、ARID1A/1B、SMARCD1等同源重组相关基因,组蛋白H2B和H3突变体、以及CUL4、DDB1、WDR70、19S蛋白酶体和相关去泛素化酶等基因突变和蛋白质功能障碍,HBV感染等原因导致的同源重组修复缺陷;还包括药物诱导的同源重组修复功能紊乱,包括但不限于去甲基化酶、去乙酰化酶抑制剂等可以诱导同源重组修复缺陷的药物处理方式及组合。
本发明的用途是利用H2B单泛素化水平减低来鉴定BRCA1/2(或其他相关基因突变及功能异常)突变高发型肿瘤,包括但不限于乳腺癌、卵巢癌、子宫内膜(样)癌、卵巢透明细胞癌、前列腺癌、胰腺癌及胃癌以及具有同源重组修复缺陷特征的肝癌及乙型肝炎病毒(HBV)所致的相关肝脏疾病,特别是HBV感染性相关疾病,更优选HBV携带者,再优选HBV感染,最优选HBV感染所导致的急性肝炎、慢性肝炎、肝纤维化、肝硬化、肝癌和胆管细胞癌。
本发明的方法,上述疾病组织中H2B单泛素化水平的检测,可从手术切除与活检组织中鉴定检测对象是否具有H2B单泛素化水平异常,由此判断该病理组织是否具有同源重组缺陷的特征。H2B单泛素化缺陷检测在研发过程中基于BRCA1/BRCA2突变的卵巢癌的肝癌病例及可靠临床信息,确定利用该检测指标判断同源重组修复缺陷的相关性和准确性:使用免疫组化的方法对保存良好的石蜡病理组织进行免疫组化染色,染色结果评价为阴性或弱阳性的即为H2B单泛素化缺陷型病例;这些H2B单泛素化缺陷病例对应BRCA1/BRCA2突变基因型,且包括其他方法诊断出的同源重组修复缺陷(如BRCA1/BRAC2启动子甲基化异常)。
本发明的方法,使用免疫组化的方法对HBV阴性和阳性的肝癌中H2B单泛素化水平的检测表明:带有同源重组缺陷的HBV阳性肝癌呈现H2B单泛素化染色阴性或弱阳性,而多数HBV阴性的肝癌标本则表现强阳性。说明H2B单泛素化水平可以很好地判断具有同源重组缺陷特征的肝癌。
本发明的方法,H2B单泛素化的检测可以采用临床病理组织石蜡切片的免疫组化,免疫荧光染色,或ELISA,免疫印迹等抗体相关技术,检测方法实用简单,可靠快速,便于临床操作,且相对于昂贵的高通量基因组学或突变检测具有相当好的临床经济学优势。其中,所述H2B泛素化缺陷染色检测可与其它肿瘤诊断、药物评价的生物标记物联合应用。
本发明有益效果:基于组蛋白H2B单泛素化是同源重组修复的重要调控因子,直接反映同源重组修复的进展和缺陷,因此,组蛋白H2B单泛素化可以作为生物标记物用于指示同源重组修复缺陷型疾病,通过检测组蛋白H2B单泛素化水平的变化,判断、预示源重组修复缺陷肿瘤及其相关疾病,指导PARP抑制剂等靶向药物治疗和显示药物治疗效果。
附图说明
图1 H2B单泛素化突变体和HBx表达干扰同源重组修复,利用I-SCE-I系统检测H2B单泛素化突变组(2KR)以及HBx过表达组的同源重组修复效率,qPCR结果表明H2B单泛素化突变体或HBx过表达导致同源重组效率显著降低。
图2 H2B单泛素化突变体导致染色体结构不稳定,所示细胞中期染色体的吉姆萨染色。放射线处理后,与未照射时相比,H2B单泛素化缺陷细胞(表达2KR的HL-L02细胞)中染色体畸变率显著升高,加入PARP抑制剂(奥拉帕尼)处理后则进一步加剧染色体畸变。对照HL-L02细胞中则未出现这一变化。
图3 多种同源重组修复的缺陷导致H2B单泛素化水平减低,离子射线处理细胞后,在对应的同源重组修复因子沉默细胞组以及HBx过表达组中检测H2B单泛素化水平。蛋白质免疫印迹结果显示当细胞同源重组修复缺陷时(基因沉默BRCA1,BRCA2,WDR70,CTIP,SMARCAD1,MRE11,NBS1和RAD51等或过表达HBx时),H2B单泛素化水平明显降低。
图4 PARP抑制剂对H2B泛素化水平减低的细胞和肿瘤的毒性作用,(A)克隆形成实验显示,梯度浓度PARP(奥拉帕尼)抑制剂处理同时处理H2B泛素化水平减低的细胞(HL-L02/2KR)及对照细胞(HL-L02/pLVX),二者生存率相差显著。表达2KR的细胞对低浓度的PARP抑制剂即表现出显著的敏感性,而对照组细胞在所有浓度下,均无明显的敏感性;(B)裸鼠肿瘤负荷实验显示,在PARP抑制剂处理过程中,H2B单泛素化水平减低的肿瘤HepG2/2KR始终呈现出良好的药物应答反应,肿瘤无进展。而对照细胞系HepG2/pLVX细胞肿瘤对PARP抑制剂则无明显反应,肿瘤进展情况与溶剂对照组一致。
图5 H2B单泛素化水平减低与BRCA1/BRCA2突变的相关性,免疫组化染色(IHC)检测在BRCA1/BRCA2基因野生型或突变型的高级别浆液性卵巢癌中的H2B单泛素化水平,按照IRS体系评分后利用Graphpad软件统计并作图。
图6 同源重组修复缺陷型肝脏或肝癌组织中H2B单泛素化水平减低,A-B:免疫组化染色(IHC)检测HBV血清学阴性和阳性的肝癌组织或非肿瘤肝组织切片中的H2B单泛素化水平。C:将A-B的结果按照IRS体系评分后利用Graphpad软件统计并作图。显示HBV感染组织中H2B单泛素化水平较非感染组织显著减低。
表1关键名词列表
简写 | 注释 |
uH2B | H2B单泛素化 |
HBV | 乙肝病毒 |
HBx | 乙肝病毒蛋白X |
HBV阳性肝癌 | 血清HBV感染指标阳性的肝癌 |
HBV阴性肝癌 | 血清HBV感染指标阴性的肝癌 |
表2重要试剂列表
1 | H2B单泛素化突变表达质粒(pLVX-Green-2KR) |
2 | HBx蛋白表达慢病毒质粒(pLVX-Green-HBx) |
3 | 慢病毒对照蛋白表达质粒(pLVX) |
4 | HL-7702(人源正常肝细胞系,无HBV病毒复制,无HBx表达) |
5 | HepG2(人肝癌细胞系,无HBV病毒复制,无HBx表达) |
6 | 奥拉帕尼(selleck,S1060) |
7 | H2B单泛素化抗体(CellSignalling,5546) |
8 | Tubulin抗体(Sigma,T6557) |
具体实施方式
以下非限制性实施例用来阐明本发明的精神实质,但不以此限制本发明的范围。应当理解的是,所示组分的比例变化和备选要素对本领域技术人员而言是显而易见的。
下列实施例中HL-L02细胞系(又名L-02,来源于中国科学院典型培养物保藏委员会细胞库,编号:GNHu 6)。实施例中H2B单泛素化突变体(K120R K125R)由本实验室克隆,Olaparib(KU0059436,Selleck),H2B单泛素化抗体(Millipore,05-1312)及各种抗体,质粒和化学试剂为本发明中实验用,不作为其他用途。
实施例1.H2B单泛素化突变体降低同源重组修复效率
实验方法:将HL-L02细胞接种至6cm培养皿中,37℃培养至细胞覆盖率达到50~70%,转染pLVX-Green-H2BK120RK125R(2KR)、pLVX-Green-HBx及对照质粒pLVX-Green,继续培养24h后转染I-SCE-I-HR质粒,利用I-SCE-I系统检测体内同源重组的效率:24小时后收取细胞,通过High Pure PCR Template Preparation试剂盒(Roche,11796828001)提取细胞基因组DNA,设计检测HR效率的引物(引物序列F:TGACCACCCTGACCTACG;R:CACCTTGATGCCGTTCTTCTGC),利用实时荧光定量PCR鉴定H2B单泛素突变体对体内同源重组的影响。
实验结果:本实验利用I-SceI-GFP体系(Development of Novel Visual-PlusQuantitative Analysis Systems for Studying DNA Double-Strand Break Repairs inZebrafish,Journal of Genetics and Genomics,2012,39(9):489-502),证明H2B单泛素化突变体(K120R K125R)或HBx过表达造成同源重组效率降低,显著低于对照细胞。
结论:结果表明,H2B单泛素化的缺陷导致同源重组效率降低,说明H2B单泛素化是人细胞中同源重组修复机制的重要调控点。HBx的基因表达同样导致同源重组效率降低,说明HBx的表达可以与H2B单泛素化共同指示HBV阳性肝癌及相关疾病的同源重组缺陷。
实施例2.H2B单泛素化突变体导致大量染色体断裂
实验方法:采用慢病毒感染系统构建H2B单泛素化突变细胞系HL-L02/2KR及对照细胞HL-L02/pLVX。构建好的细胞分别接种2个6cm直径培养皿,过夜培养。X射线(剂量为4Gy)处理后8小时加入秋水仙素(终浓度200ng/ml)处理1.5小时。胰酶消化,PBS清洗。去上清,加入250μl PBS重悬细胞。逐滴滴加6ml37℃预热75mM KCl。37℃温育25分钟。逐滴加入200μl固定液(甲醇:冰醋酸=3:1),轻混匀,1000rpm离心10分钟。加入5ml固定液,混匀,4℃静置20分钟。1000rpm离心10分钟,50ul固定液重悬细胞。反复固定细胞3次,最后加入500μl固定液混匀细胞,4℃保存或滴片。滴片前,载玻片-20℃预冷。将细胞混合液从大于20cm高度处滴落至载玻片上。玻片晾干。吉姆萨染色液染色10分钟,树脂封片。1000倍光学显微镜下观察拍照并进行计数。
实验结果:HL-L02细胞中过表达H2B单泛素化突变体(2KR)在X光照射后,出现异常增多的染色体断裂和星型染色体,每100次有丝分裂中的染色体畸变率高达近50%(48.8%),与对照组相比(6.7%),二者差异显著。
结论:染色体断裂和星型染色体,是典型的同源重组修复异常造成的染色体畸变。H2B单泛素化突变体在放射线处理后,其异常增高的染色体畸变说明这类突变体的基因组具有高度不稳定性,进一步说明H2B单泛素化水平减低阻碍了同源重组修复。
实施例3.同源重组修复的缺陷导致H2B单泛素化水平减低
实验方法:HL-L02细胞中用小干扰RNA(siRNA)沉默BRCA1,BRCA2,WDR70,CTIP,MRE11,NBS1和RAD51等同源重组修复基因,随机siRNA(Scramble)作为实验对照,同时转染pLVX-Green-HBx质粒。siRNA或HBx质粒转染沉默48小时后用离子射线照射(剂量为10Gy),4小时后提取细胞总蛋白,采用免疫印迹方法检测细胞中H2B单泛素化水平。
实验结果:在离子沉默射线诱导DNA断裂后,BRCA1,BRCA2,WDR70,CTIP,
SMARCAD1,MRE11,NBS1和RAD51等同源重组修复基因的沉默和癌基因HBx的表达均导致H2B单泛素化水平的减低。
结论:该结果表明,同源重组修复信号通路中关键组分的功能异常以及HBx的基因表达均会导致H2B单泛素化水平减低,而检测H2B单泛素化的水平则可以作为预测细胞和组织中同源重组修复功能的直接的和通用的指标。
实施例4 PARP抑制剂对H2B单泛素化水平减低的细胞毒性作用增强
实验方法:采用克隆形成实验验证PARP抑制剂对H2B泛素化水平减低的的细胞的特异性抑制作用。将H2B单泛素化突变细胞系HL-L02/2KR及对照细胞HL-L02/pLVX细胞分别接种于10cm直径培养皿中,设计5个药物浓度梯度,每种细胞每个梯度平行三重复。细胞密度为800个/盘。细胞完全贴壁后加入奥拉帕尼或相同体积的DMSO(对照组)。用奥拉帕尼持续处理10天,甲醇固定,吉姆萨染液染色,计算克隆数。
实验结果:克隆形成实验显示PARP抑制剂奥拉帕尼基对H2B单泛素化水平减低的的细胞具有明显毒性作用,即使最低剂量上仍然有较高的抑制效果(生存率为12%)。在高浓度(1.5uM)奥拉帕尼处理时,H2B单泛素化水平减低细胞的生存率(0.8%)远低于对照组(30%)。
结论:H2B的单泛素化水平可以评价细胞中同源重组修复能力,可以预测其对PARP抑制剂的敏感性,进而对PARP抑制剂的临床用药起指导作用。
实施例5 PARP抑制剂对H2B泛素化水平减低的肿瘤抑制作用
利用慢病毒感染体系构建肝癌细胞HepG2的H2B泛素化突变体HepG2/2KR及对照细胞HepG2/pLVX。取8周龄的无胸腺雌性裸鼠随机分为为2组,每组3只。每只小鼠于腋下及腹股沟处接种H2B单泛素化水平减低的的细胞HepG2/2KR及对照细胞系HepG2/pLVX细胞,接种量为4x106。瘤体体积约100mm3时开始注射PRPP抑制剂(奥拉帕尼)。具体用药剂量如下:奥拉帕尼164mg/kg/天,持续用药14天,取第1、3、5、7、9、10、11、12、13、14天测量肿瘤体表长短直径。根据公式:肿瘤体积=长x宽x宽/2,计算肿瘤体表体积。
实验结果:由结果可以看出与对照组相比,这类肿瘤对PRRP抑制剂有良好的应答作用。相对应的,HepG2/pLVX细胞成瘤后在整个用药阶段中呈现恶性增殖的状态。实验终点,HepG2/pLVX用药组肿瘤平均体积为HepG2/2KR用药组的4倍。结论:小鼠体内实验证明,与带有正常H2B单泛素化功能的肿瘤相比,H2B单泛素化水平减低的肿瘤对PARP抑制剂极为敏感,证明H2B单泛素化水平不仅可以作为同源重组缺陷型肿瘤预测指标,还可以作为PARP抑制剂用药效果的伴随诊断指标。结合体内或体外实验,H2B单泛素化水平减低可以直接评估同源重组缺陷和PARP敏感性的有效指标。
实施例6.BRCA1/2缺陷型高级别浆液性卵巢癌表现出H2B单泛素化缺陷
实验方法:免疫组化实验检测在BRCA1/2缺陷型高级别浆液性卵巢癌中H2B单泛素化缺陷。选取具有临床确切诊断的卵巢癌切片,脱蜡处理,切片用二甲苯浸泡两次,每次5min;随后用90%,85%,75%酒精各浸泡两次,每次5分钟,完成切片脱水。PBS洗三次,1%Triton-100处理15min,PBS清洗三次,3%H2O2(28%甲醇配制)避光处理15min;随后进行柠檬酸缓冲液抗原修复(95℃,40min,自然冷却至室温),10%羊血清封闭30min。H2B单泛素化抗体(uH2B,mouse)孵育,4℃过夜。PBS清洗3次,二抗、过氧化物酶链亲和素37℃各孵育30min,DAB显色10-15min,苏木素复染30s。显微镜下观察结果并拍照。IRS评估及统计参见(J Clin Exp Pathol.2012;5(3):187-94.Epub 2012Mar 25.Comparing of IRS andHer2as immunohistochemical scoring schemes in gastroenteropancreaticneuroendocrine tumors)。
实验结果:在正常卵巢组织组织切片中H2B单泛素化免疫组化染色呈现强阳性,一部分BRCA1/2正常基因型高级别浆液性卵巢癌中H2B单泛素化呈现强阳性(55%),其余45%表现阴性;而BRCA1/2缺陷型高级别浆液性卵巢癌中H2B单泛素化呈现90%阴性染色,H2B单泛素化染色阴性与BRCA1/BRCA2基因突变高度对应。更为重要的是,在未检出BRCA1/BRCA2基因突变的卵巢癌标本中,有19%的BRCA1基因启动子甲基化缺陷(将导致该基因不表达),说明H2B单泛素化水平的检测不仅能够准确鉴定出BRCA1/2突变的同源重组缺陷型肿瘤,而且能够鉴定非BRCA1/BRCA2突变的同源重组缺陷的肿瘤。
结论:H2B单泛素化水平的评价可应用于检测BRCA1/BRCA2或其他任何同源重组修复相关基因突变导致的同源重组缺陷,也可用于检测这些基因表达水平异常等导致的同源重组缺陷。因此,可以作为一种检测肿瘤组织同源重组修复缺陷的通用指标,而不局限于导致同源重组缺陷的病理原因。
实施例7.H2B单泛素化水平评估HBV阳性肝癌的同源重组缺陷。
实验方法:同实施例6,分别选取HBV感染阳性(同源重组缺陷)和阴性的肝癌(同源重组功能正常),以及HBV感染或非感染的非肿瘤肝脏组织,利用免疫组化做H2B单泛素化水平的检测。
实验结果:在93.75%(15/16)的HBV血清学阴性的非肿瘤肝组织中H2B单泛素化免疫组化染色呈阳性,44%(11/25)的HBV血清学阴性的肝癌组织中H2B单泛素化免疫组化染色呈阳性;而在HBV血清学阳性的非肿瘤肝组织和肝癌组织切片中H2B单泛素化染色呈强阳性的比例仅为40%(2/5)和7.4%(2/27)。将染色结果按照IRS体系评分后的统计分析显示:HBV血清学阳性非肿瘤肝组织组的H2B单泛素化水平明显低于HBV阴性非肿瘤肝组织(P=0.0002);同时,HBV血清学阳性肝癌组的H2B单泛素化水平也明显低于HBV阴性肝癌组(P=0.0191)。以上结果均表明,HBV感染组织中H2B单泛素化水平较非感染组织显著减低。
结论:H2B单泛素化水平的评价可应用于检测同源重组修复缺陷的HBV阳性的肝癌或肝脏组织,并明显区别于HBV感染阴性的组织。重要的是,现已知由于HBV感染导致肝癌的同源重组缺陷与任何修复相关基因(包括BRCA1,BRCA2等)的突变并无关联,说明通过H2B单泛素化水平的预测同源重组修复缺陷可以不依赖基因组突变病因的检测,是一种可以广泛应用的检测同源重组缺陷的方法和指标。
Claims (14)
1.一种组蛋白H2B单泛素化或/和检测组蛋白H2B单泛素化水平的试剂,在制造诊断、检测具有同源重组修复缺陷的肿瘤和乙肝病毒感染所致相关疾病,预示所述肿瘤和乙肝病毒感染所致相关疾病的进展及其药物治疗效果的试剂盒中的用途。
2.如权利要求1所述的用途,所述肿瘤包括乳腺癌、卵巢癌、宫颈癌、子宫内膜(样)癌、卵巢透明细胞癌、胃癌、胰腺癌、前列腺癌或乙肝病毒感染所致肝癌和胆管细胞癌。
3.如权利要求1所述的用途,所述乙肝病毒感染所致相关疾病,包括乙型肝炎病毒感染性疾病或乙肝病毒携带者。
4.如权利要求3所述的用途,所述乙肝病毒感染性疾病为急性肝炎、慢性肝炎、肝纤维化、肝硬化、肝癌或胆管细胞癌,优选为乙肝病毒阳性的肝癌和胆管细胞癌。
5.如权利要求1所述的用途,H2B单泛素化水平的检测,可与组织中HBx的基因表达与蛋白水平的检测联合使用,作为评估同源重组缺陷和指导PARP
抑制剂临床用药的共同指标。
6.如权利要求1所述的用途,所述药物为PARP抑制剂。
7.如权利要求6所述的用途,所述PARP抑制剂选自奥拉帕尼、rucaparib、Niraparib、BTB-290和Veliparib等。
8.一种鉴定同源重组修复缺陷的方法,包括以下过程:
1)取患者病理组织或体液;
2)检测组蛋白H2B单泛素化水平;
3)将检测结果与正常或癌旁组织的单泛素化水平进行比对;
4)判断是否存在同源重组修复缺陷及其病理状态;
5)进一步的,通过比对数据结果,判断同源重组修复缺陷性肿瘤、预示所述肿瘤进展或药物治疗所述肿瘤的效果。
9.如权利要求8所述的方法,所述检测组蛋白H2B单泛素化水平,所述检测包括选自免疫组化,免疫荧光,免疫印迹和ELISA等采用的免疫检测技术及其相关试剂。
10.如权利要求8所述的方法,所述比对,包括判定组蛋白H2B单泛素化的阴性,或与正常组织相比较该单泛素化水平明显减少的肿瘤或相关疾病。
11.如权利要求8所述的方法,所述组蛋白H2B单泛素化的检测,包括组蛋白H2B单泛素化的依赖H2B单泛素化特异性抗体的检测方法。
12.如权利要求5所述的方法,对同源重组修复缺陷的鉴定,不依赖于特定的基因突变或生物学机制,其鉴定范围包括乙型肝炎病毒感染所导致的同源重组修复缺陷,或BRCA1/BRCA2的功能缺陷及具有VUS特征的基因突变的同源重组缺陷肿瘤,或者,包括去甲基化酶或去乙酰化酶抑制剂可以诱导同源重组缺陷的药物处理方式及组合。
13.如权利要求12所述的方法,对同源重组修复缺陷的鉴定,进一步结合HBx基因表达和蛋白水平检测指标进行鉴定。
14.如权利要求12所述的方法,所述HBx基因表达和蛋白水平检测,其检测的方法,包括选自免疫印迹、免疫荧光、免疫组化染色和ELISA的HBx特异性抗体相关技术,选自原位杂交、ACD的基因表达相关技术,以及直接检测与HBx水平直接关联的cccDNA的乙型肝炎病毒复制的指标。
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