CN114369659B - 用于检测与脑动脉瘤相关的gsta4基因甲基化程度的试剂盒及其应用 - Google Patents
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Abstract
本发明公开了用于检测与脑动脉瘤相关的GSTA4基因甲基化程度的试剂盒及其应用,特点是该试剂盒内包括一对GSTA4基因DNA甲基化特异性扩增引物及一个GSTA4基因DNA甲基化特异性测序引物:其中特异性扩增上游引物具有如SEQ ID NO.1所示,特异性扩增下游引物具有如SEQ ID NO.2所示,甲基化特异性测序引物如SEQ ID NO.3所示;GSTA4基因DNA被检测序列所在区域为基因前端特定位置并通过焦磷酸测序技术进行DNA甲基化水平分析;GSTA4基因DNA甲基化水平与脑动脉瘤的患病率呈负相关;优点是该诊断试剂盒可以方便快捷地在分子水平上实现对脑动脉瘤的检测,检测效率高,针对性强。
Description
技术领域
本发明属于脑动脉瘤的辅助诊断技术领域,具体涉及一种用于检测与脑动脉瘤相关的GSTA4基因甲基化程度的试剂盒及其应用。
背景技术
脑动脉瘤(Intracranial aneurysms,IA)是脑内动脉壁的结构发育不良,或因脑外伤、动脉硬化造成动脉壁损伤或老化,使局部血管壁向外膨大形成的囊状瘤体。脑动脉瘤极易在偶发的紧张、用力、疲劳等血压升高时突然发生破裂,所引起的脑蛛网膜下腔出血及其导致的严重并发症对人生命和健康的威胁很大,死亡率和伤残率都很高。因此,脑动脉瘤被称为脑的“定时炸弹”。脑动脉瘤病因尚不甚清楚,但以先天性动脉瘤占大部分。任何年龄可发病,40-66岁常见。
流行病学调查显示,IA在普通人群中的发生率为1.0%-2.0%,在中国35~75岁成年人中为7.0%。脑动脉瘤的发病机制在很大程度上仍然不十分清楚。目前,最普遍的观念都认为脑动脉瘤的发生、形成是一个复杂的多基因、多生物因子共同作用的过程,遗传和后天不良生活因素(包括生活方式和其他社会心理因素等)共同参与这个过程。因此,越来越多的研究者们都逐渐倾向于脑动脉瘤的基因和生物因子方面的研究。近年来的研究证明,血管相关基因DNA甲基化程度的变化在脑动脉瘤的发生发展中扮演着重要角色。
谷胱甘肽S-转移酶α 4(Glutathione S-transferase alpha 4,GSTA4)基因位于6号染色体,其编码的蛋白属于α类谷胱甘肽S-转移酶,具有谷胱甘肽过氧化物酶的活性,可将脂质过氧化物转化为谷胱甘肽结合物,在机体解毒和细胞保护方面有着重要的作用。GSTA4是一种细胞溶质蛋白,广泛分布于人体各个组织中,主要在肝、肾、小肠和胃肠道等器官中有较高表达。GSTA4被称为抗氧化蛋白酶,能够通过清除活性氧积累导致的脂质过氧化,丙二醛以及4-羟基壬烯醛等副产物,进而在帕金森,阿尔兹海默症等神经功能退行性疾病中发挥脑保护作用。大鼠大脑皮层中GSTA4含量随着年龄的递增呈现出,GSTA4甚至在老年大脑皮层中增加,并且与脑部氧化应激和神经毒性的发生密切相关。在血管细胞中,GSTA4高表达能够提升保护内皮细胞和平滑肌细胞对抗内皮功能障碍和血管氧化应激的作用。小鼠动物模型研究结果显示GSTA4的表达量与血管损伤相关。由此可知,GSTA4可能在脑动脉瘤的发生发展过程中可能扮演着重要的角色。脑动脉瘤的传统诊断都是基于影像学手段进行检查,而病人往往都是出现相关症状才到医院就诊。脑动脉瘤破裂前没有任何征兆,其死亡率极高,大概1/3的脑动脉瘤患者都是发病后送往医院救治的过程中死亡。目前,国内外没有公开任何关于GSTA4基因DNA甲基化与脑动脉瘤相关的研究报道。
发明内容
本发明所要解决的技术问题是提供一种检测方便、针对性强、检测准确率高的用于检测与脑动脉瘤相关的GSTA4基因甲基化程度的试剂盒及其应用,其中GSTA4基因DNA甲基化水平与脑动脉瘤的患病率呈负相关,可通过检测GSTA4基因DNA甲基化程度辅助诊断脑动脉瘤。
本发明解决上述技术问题所采用的技术方案为:一种用于检测与脑动脉瘤相关的GSTA4基因甲基化程度的试剂盒,包括一对GSTA4基因甲基化特异性扩增引物及一个GSTA4基因甲基化特异性测序引物,其中所述的GSTA4基因甲基化特异性扩增上游引物的核苷酸序列如SEQ ID NO.1所示:5'-AGGGAAAAAAAAGAAGAGAGAAAGG-3’;
所述的GSTA4基因甲基化特异性扩增下游引物的核苷酸序列如SEQ ID NO.2所示:5’-Biotin-TTTAACACTATCCAAAATACCTTACAAA-3’;
所述的GSTA4基因甲基化特异性测序引物的核苷酸序列如SEQ ID NO.3所示:5’-GGAGATAGATTTGGAGTTTA-3’。
进一步,所述的GSTA4基因DNA被检测序列所在区域为基因前端区域Chr6: 52,995,261-52,995,336内。
进一步,所述的Chr6: 52,995,261-52,995,336区域内9个CpG位点的具体位置为:
。
进一步,所述的GSTA4基因DNA被检测序列采用焦磷酸测序技术对GSTA4基因内的9个CpG位点进行DNA甲基化水平分析。
上述用于检测与脑动脉瘤相关的GSTA4基因启动子区甲基化程度的试剂盒在制备检测脑动脉瘤试剂或者辅助诊断脑动脉瘤试剂中的应用。
与现有技术相比,本发明的优点在于:本发明首次公开了用于检测与脑动脉瘤相关的GSTA4基因DNA甲基化程度的试剂盒及其应用,GSTA4基因域的低甲基化水平导致GSTA4基因的高表达,从而引起脑部动脉血管的紊乱引发疾病。因此,GSTA4基因DNA甲基化水平与脑动脉瘤的患病率呈负相关。因此,以检测GSTA4基因DNA甲基化水平为基础的检测试剂盒可以方便快捷地在分子水平上实现对脑动脉瘤的检测,检测效率高,针对性强,同时,以GSTA4基因甲基化为靶点的药物为脑动脉瘤辅助诊断、检测和筛选的一种创新用途。
综上所述,本发明以检测GSTA4基因DNA甲基化水平为基础的检测试剂盒可以方便快捷地在分子水平上实现对脑动脉瘤的检测,检测效率高,针对性强,有利于脑动脉瘤的早期发现和及时治疗。
附图说明
图1为GSTA4基因被检测序列所在区域具体位置为hg38,Chr6: 52,995,261-52,995,336,该区域所检测的9个CpG甲基化位点分布位置;
图2为GSTA4基因甲基化水平检测结果示例,图中所示百分数为对应CpG位点的甲基化程度,如图示有CpG1到CpG9的甲基化程度分别为15%,10%,7%,6%,11%,5%,7%,7%,7%;
图3为脑动脉瘤与对照组间GSTA4基因DNA甲基化差异(*p<0.05, **p<0.01);
图4为不同性别脑动脉瘤与对照组之间GSTA4基因平均DNA甲基化差异(脑动脉瘤患者GSTA4基因DNA甲基化程度显著低于对照组,**p<0.01,ns: p>0.05);
图5为GSTA4基因DNA甲基化与脑动脉瘤的敏感性和特异性(女性AUC = 0.79,最佳临界点时的敏感度为62.5%,特异性为79.2%)。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
具体实施例
1、研究对象的收集
募集愿意参加研究的志愿者,根据脑动脉瘤的国际诊断标准,将志愿者分为病例组和对照组,并采用调查表的形式调查志愿者的一般情况,同时采取静脉血,进行一般血生化检测,具体如下:从某医院神经外科收集经血管造影术证实的脑动脉瘤患者48例(24男;24女,平均年龄46.63±6.04),同时收集性别匹配、年龄严格配对的经CT证实没有心脑血管病历史的48名正常人作为对照组(平均年龄48.08±5.69)。记录所有研究对象空腹抽血检测血脂、血糖等一般生化指标,同时抽取静脉血3ml入EDTA抗凝管中,于4℃离心机,3000rpm离心20分钟,分别将上清血浆和中间层白细胞保存-80℃低温冰箱,以备用于标本统一提取基因组DNA。
2、基因组DNA的提取
应用Lab-Aid 820全自动核酸提取仪提取上述步骤得到的样本的全血基因组DNA,再通过核酸蛋白测定仪检测所得DNA的浓度,以供GSTA4基因DNA甲基化水平的检测。
3、DNA甲基化水平测定
本研究采用焦磷酸测序技术对GSTA4基因前端区域Chr6: 52,995,261-52,995,336内的9个CpG位点(如图1所示)进行了DNA甲基化水平分析,图1所示GSTA4基因被检测序列所在区域为基因前端区域hg38,Chr6: 52,995,261-52,995,336内的9个CpG位点,其具体位置为:
。
此技术的基本原理:用重亚硫酸盐处理DNA样品后,再应用聚合酶链反应(PCR)扩增,可使发生甲基化的胞嘧啶(C)碱基保持不变,而使未发生甲基化的C转变成了尿嘧啶(U),然后通过测序引物进行PCR测序,从而得到哪个位点发生了甲基化。本次研究采用PyroMark Assay Design软件进行引物设计,用于实验的PCR扩增引物和测序引物如下:
GSTA4基因DNA甲基化特异性上游引物的核苷酸序列(Forward primer) SEQ IDNO.1如下:5'-AGGGAAAAAAAAGAAGAGAGAAAGG-3’;
GSTA4基因DNA甲基化特异性下游引物的核苷酸序列(Reverse primer) SEQ IDNO.2如下:5’-Biotin-TTTAACACTATCCAAAATACCTTACAAA-3’;
GSTA4基因DNA甲基化特异性测序引物的核苷酸序列(Sequencing primer) SEQID NO.3如下:5’-GGAGATAGATTTGGAGTTTA-3’。
DNA甲基化水平测定的具体步骤:
第一步:亚硫酸盐转化。采用QIAGEN EpiTect® 亚硫酸氢盐处理试剂盒(EpiTechBisulfite Kits;Qiagen; #59104)对样本DNA进行亚硫酸氢盐转化,按照说明书操作,经上样,洗涤,洗脱等步骤得到转化后的DNA。
第二步:PCR反应。取第一步中转化过的DNA样本20ng加入到Pyromark PCR试剂盒(Pyromark PCR Kit; Qiagen; #978703),并加入上述一对GSTA4基因甲基化特异性扩增引物,进行PCR扩增,扩增条件:首先95℃ 3 min的变性;接着94℃ 30s,56℃ 30s,72℃ 1min,共40个循环的退火反应;然后延伸反应72℃ 5min。
第三步:焦磷酸测序的前期准备:在96孔板中预先加入45μl含有0.3μM上述甲基化特异性测序引物的退火缓冲液(PyroMark Annealing Buffer; Qiagen; #979009);将需要使用的混匀的琼脂糖珠总量(每样本3μl)转移到一个Eppendorf管中;在琼脂糖珠中加入结合缓冲液(PyroMark Binding Buffer; Qiagen; #979006),使得平均每个样品约有50μl的体积,将混合物混匀;将以上混合物加入PCR产物(50μl反应体积)中,每样本50μl;将PCR产物在常温下混匀10分钟,使得磁珠与生物素结合;在真空预备工作站中,四个样品板中依次加入180ml的高纯水、70%乙醇、洗涤缓冲液(PyroMark Wash Buffer; Qiagen; #979008)和120ml的变性缓冲液(PyroMark Denaturation Solution; Qiagen; #979007);打开真空预备工作站的泵,将真空准备工具在高纯水中清洗30秒;然后将真空准备工具(PyroMarkVacuum Prep Filter Probes; Qiagen; #979010)移到PCR板中,抓取琼脂糖珠(在磁珠与PCR产物结合后三分钟内完成此操作);拿起PCR板,检查是否大部分磁珠都被吸附在了真空准备工具上;将真空准备工具放入70%乙醇中5秒;然后移到变性缓冲液中5秒;再移到洗涤缓冲液中清洗5-10秒;关掉泵;将真空准备工具放入含有测序引物的板中,摇动,释放琼脂糖珠(测序引物也可最后加入);使用高纯水清洗真空准备工具;将放有样品的PSQ96板放在加热板上加热到80℃ 2分钟,再冷却到室温,即可进行焦磷酸测序反应。
第四步:焦磷酸测序:在PyroMark Q24焦磷酸测序仪上,采用Pyromark Gold Q24试剂盒(Pyromark Gold Q24 Reagents; Qiagen; #978802)对第三步中的PSQ96板中的样本进行测序,然后应用PyroMark CpG软件对结果进行甲基化分析(甲基化水平检测结果见图2)。图2中所示为GSTA4基因甲基化水平检测结果示例。图中所示百分数为对应CpG位点的甲基化程度,如图示有CpG1到CpG9的甲基化程度分别为15%,10%,7%,6%,11%,5%,7%,7%,7%。
4、数据分析
本次研究采用GraphPad Prism 8对数据进行整理分析。本项研究设计的引物能够检测出GSTA4基因内Chr6: 52,995,261-52,995,336区域内的9个CpG位点的甲基化情况。通过病例对照组比较,我们发现脑动脉瘤患者中GSTA4基因CpG1(p<0.01), CpG2(p<0.01),CpG3(p<0.01), CpG4(p<0.01), CpG5(p<0.05), CpG6(p<0.01), CpG7(p<0.01), CpG8(p<0.01), CpG9(p<0.05)以及平均甲基化程度(p<0.01)均低于对照组(图3)。性别分组分析的结果显示,女性脑动脉瘤患者中GSTA4基因平均甲基化水平显著低于对照组(p < 0.001,图4),而男性患者与对照组间的甲基化没有差异(p>0.05)。ROC曲线分析结果显示GSTA4基因甲基化水平在所有研究对象中线下面积AUC = 0.60,男性人群中AUC面积为0.55,而在女性人群中AUC面积为0.79(p<0.01,图5),最佳临界点时的敏感度62.5%,特异性为79.2%。
上述研究结果显示,脑动脉瘤患者血液中GSTA4基因DNA甲基化显著低于健康对照组,并且这种差异仅仅出现在女性人群中。因此,GSTA4基因DNA甲基化程度的检测有望作为脑动脉瘤早期诊断的一个预测因子。
综上所述,本发明一种检测GSTA4基因DNA甲基化程度的试剂盒及其应用,在辅助诊断脑动脉瘤的检测试剂盒具有准确可靠、灵活快速和经济节约的优点。本发明采用上述试剂盒对GSTA4基因DNA甲基化水平进行检测,能够快速、可靠地为脑动脉瘤的辅助诊断、检测或筛查药物提供借鉴。
上述说明并非对本发明的限制,本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内,做出的变化、改型、添加或替换,也应属于本发明的保护范围。
序 列 表
<110> 宁波市第一医院
<120> 用于检测与脑动脉瘤相关的GSTA4基因甲基化程度的试剂盒及其应用
<160> 3
<170> PatentIn version 3.1
<210> 1
<211> 25
<212> DNA
<213> GSTA4基因甲基化特异性扩增上游引物(5'-AGGGAAAAAAAAGAAGAGAGAAAGG-3’ )
<400> 1
<210> 2
<211> 28
<212> DNA
<213>GSTA4基因甲基化特异性扩增下游引物(5’-Biotin-TTTAACACTATCCAAAATACCTTACAAA-3’ )
<400> 2
<210> 3
<211> 20
<212> DNA
<213>GSTA4基因甲基化特异性测序引物(5’-GGAGATAGATTTGGAGTTTA-3’)
<400> 3
Claims (5)
1.一种用于检测与脑动脉瘤相关的GSTA4基因甲基化程度的试剂盒,其特征在于包括一对GSTA4基因甲基化特异性扩增引物及一个GSTA4基因甲基化特异性测序引物,其中所述的GSTA4基因甲基化特异性扩增上游引物的核苷酸序列如SEQ ID NO.1所示:5'-AGGGAAAAAAAAGAAGAGAGAAAGG-3’;
所述的GSTA4基因甲基化特异性扩增下游引物的核苷酸序列如SEQ ID NO.2所示:5’-Biotin-TTTAACACTATCCAAAATACCTTACAAA-3’;
所述的GSTA4基因甲基化特异性测序引物的核苷酸序列如SEQ ID NO.3所示:5’-GGAGATAGATTTGGAGTTTA-3’。
2.根据权利要求1所述的用于检测与脑动脉瘤相关的GSTA4基因甲基化程度的试剂盒,其特征在于:所述的GSTA4基因DNA被检测序列所在区域为基因前端区域Chr6: 52,995,261-52,995,336内。
3.根据权利要求2所述的用于检测与脑动脉瘤相关的GSTA4基因甲基化程度的试剂盒,其特征在于:所述的Chr6: 52,995,261-52,995,336区域内9个CpG位点的具体位置为:
。
4.根据权利要求3所述的用于检测与脑动脉瘤相关的GSTA4基因甲基化程度的试剂盒,其特征在于:所述的GSTA4基因DNA被检测序列采用焦磷酸测序技术对GSTA4基因内的9个CpG位点进行DNA甲基化水平分析。
5.一种用于检测与脑动脉瘤相关的GSTA4基因启动子区甲基化程度的试剂盒在制备检测脑动脉瘤试剂或者辅助诊断脑动脉瘤试剂中的应用。
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