CN104946785B - 一种基于wdr5基因用于结直肠癌辅助诊断的试剂盒 - Google Patents
一种基于wdr5基因用于结直肠癌辅助诊断的试剂盒 Download PDFInfo
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Abstract
本发明公开了一种基于WDR5基因用于结直肠癌辅助诊断的试剂盒，包括试剂A和/或试剂B；试剂A是用于检测WDR5基因表达水平的引物对；试剂B是用于检测WDR5蛋白含量的试剂，优选抗WDR5蛋白的抗体。本发明通过对结直肠正常组织和癌组织中的WDR5基因mRNA及编码蛋白的表达水平进行检测，发现WDR5在癌组织中显著高表达。在肠癌细胞中过表达WDR5能检测到，癌细胞表现出更强的迁移能力。基于以上发现开发得到一种用于结直肠癌辅助诊断的试剂盒，其对于更好的了解结直肠癌的发生发展机理，制定结直肠癌风险评估和早期检测的有效措施具有重要意义。
Description
技术领域
本发明属于分子诊断领域，具体涉及基于与结直肠癌相关的基因WDR5而开发得到的一种用于结直肠癌辅助诊断的试剂盒。
背景技术
大肠癌(CRC)是胃肠道常见的恶性肿瘤。在西方发达国家，CRC的发病率处于第2位。据国际癌症研究组织(IARC)提供的数据显示，亚洲地区，尤其是经济发达区，CRC的发生率也迅速增长。美国国家癌症数据库(US hospital-based oncology database)的研究结果表明，50岁以下成年人的CRC发病率在过去10年内增加了2.1％。美国安德森癌症研究中心(M.D.Anderson Cancer Center)的Yi Qian Nancy You等人2011年12月12日在《内科学文献》(Archives of Internal Medicine)上发表《Young-Onset Colorectal Cancer:IsIt Time to Pay Attention》称，年轻型的CRC的中位发病年龄为44岁，其中72.5％在40岁至49岁发病。
种种现实表明CRC如今已成为常见病。随着经济发展，发病率越来越高，且趋于年轻化，对人类的健康有着极大的影响。而影响结直肠癌死亡率最重要的因素是发生转移，且针对转移性结直肠癌的治疗效率很有限。
肿瘤分子标志物是肿瘤细胞在癌变过程中由于基因的表达水平的变化而增加或减少的抗原和其他生物活性物质，可用于肿瘤的诊断、分期、检测肿瘤进程，和评定药物的治疗效果；它对肿瘤的临床治疗带来了巨大的影响，尤其当它能够在临床病症出现之前被检测到，或者用于治疗效果实时监测时，具有更大的意义。同时，肿瘤分子标志物具有敏感，方便，经济、无创的特征，成为大规模筛查的重要手段。
目前已发现的结直肠癌分子标志物有CEA、CA-199等，但是尚无理想的分子标志物能够预测肠癌的转移。因此，寻找找到能够对结直肠癌特异的，具有提示作用的肿瘤标志物具有重要的意义。
WDR5基因(WD repeat domain 5，Gene ID：11091)能编码含7个WD模体的蛋白质，文献报道其编码蛋白能够参与多种细胞功能的调节，包括细胞凋亡和转录抑制等。WDR5蛋白能够促进异源三聚体的形成，对脊椎动物的生长，hox基因的激活等具有十分重要的意义。在成骨细胞、软骨细胞、骨髓基质细胞中表达并加速骨细胞分化，同时它能够参与细胞自我更新和重编程的过程。因此，它的异常表达可能引起肿瘤的发生发展，在临床上，也许能够应用WDR5的表达特征进行诊断预测。
发明内容
本发明的目的在于提供一种用于结直肠癌辅助诊断的试剂盒，该试剂盒能特异性地检测WDR5基因的表达水平有无上调，作为结直肠癌诊断的依据。
本发明的目的通过下述技术方案实现：
一种用于结直肠癌辅助诊断的试剂盒，包括试剂A和/或试剂B；
试剂A是用于检测WDR5基因表达水平的引物对，也即用于在QRT-PCR中定量扩增WDR5特异片段的QRT-PCR引物对，所述引物对为以下引物中的任意一对：
引物对1：其序列如SEQ ID NO:3和SEQ ID NO:4所示；
引物对2：其序列如SEQ ID NO:7和SEQ ID NO:8所示；
引物对3：其序列如SEQ ID NO:9和SEQ ID NO:10所示；
引物对4：其序列如SEQ ID NO:11和SEQ ID NO:12所示；
引物对5：其序列如SEQ ID NO:13和SEQ ID NO:14所示；
引物对6：其序列如SEQ ID NO:15和SEQ ID NO:16所示；
引物对7：其序列如SEQ ID NO:17和SEQ ID NO:18所示；
引物对8：其序列如SEQ ID NO:19和SEQ ID NO:20所示；
引物对9：其序列如SEQ ID NO:21和SEQ ID NO:22所示；
引物对10：其序列如SEQ ID NO:23和SEQ ID NO:24所示；
引物对11：其序列如SEQ ID NO:25和SEQ ID NO:26所示；
引物对12：其序列如SEQ ID NO:27和SEQ ID NO:28所示；
引物对13：其序列如SEQ ID NO:29和SEQ ID NO:30所示；
引物对14：其序列如SEQ ID NO:31和SEQ ID NO:32所示；
引物对15：其序列如SEQ ID NO:33和SEQ ID NO:34所示；
引物对16：其序列如SEQ ID NO:35和SEQ ID NO:36所示；
引物对17：其序列如SEQ ID NO:37和SEQ ID NO:38所示；
引物对18：其序列如SEQ ID NO:39和SEQ ID NO:40所示；
引物对19：其序列如SEQ ID NO:41和SEQ ID NO:42所示；
引物对20：其序列如SEQ ID NO:43和SEQ ID NO:44所示；
试剂B是用于检测WDR5蛋白含量的试剂，优选抗WDR5蛋白的抗体。
通过实验发现：与正常肠上皮组织相比，WDR5(WD Repeat Domain 5，Gene ID：11091)蛋白在肿瘤组织中呈高水平表达；因此，对肠上皮组织中WDR5表达水平的检测可作为结直肠癌诊断的依据；
所述的高水平表达是指肿瘤组织内WDR5mRNA及蛋白表达水平高于正常对照；所述的肿瘤细胞为TNM I-IV期结直肠癌病人肿瘤组织细胞。
本发明相对于现有技术具有如下的优点及效果：
本发明通过对结直肠正常组织和癌组织中的WDR5基因mRNA及编码蛋白的表达水平进行检测，发现WDR5在癌组织中显著高表达。在肠癌细胞中过表达WDR5能检测到癌细胞表现出更强的迁移能力。基于以上发现开发得到一种用于结直肠癌辅助诊断的试剂盒，其对于更好的了解结直肠癌的发生发展机理，制定结直肠癌风险评估和早期检测的有效措施具有重要意义。
附图说明
图1是定量PCR检测WDR5在结直肠癌组织中mRNA水平。
图2是免疫组织化学染色检测WDR5在结直肠癌组织中蛋白水平。
图3是免疫组织化学染色示例WDR5肿瘤组织(箭头所示)和癌旁组织(箭头所示)的表达量。
图4是在HCT116细胞中过表达WDR5进行细胞划痕实验。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
下述实施例中的实施方法，如无特别说明，均为常规方法。
以下是实施例所用到的部分实验材料来源：
结直肠癌细胞株HT-29、HCT116购自美国菌种保藏委员会(American TypeCulture Collection，ATCC)。
细胞培养基DMEM，McCoy5A、胎牛血清购自Gibco公司。
实时荧光定量PCR试剂盒SYBR Green PCR Kit购自Invitrogen公司。抗体anti-WDR5(AF5810)购自R&D System公司。
逆转录用dNTP混合物(10mM)、RNA酶抑制剂(40u/ul)、MMLV逆转录酶购买自Invitrogen公司。
克隆用PremixPrime STAR HS酶购买自TAKARA公司。
实施例中所用到的其它原始试剂和材料均可商购得到。
实施例1、临床样本收集
对于mRNA含量测定标本：选取中山大学肿瘤防治中心2010年2月至2012年8月结直肠癌各期标本共16例，于术后取新鲜组织保存于液氮中；对于蛋白含量测定样本：选取中山大学肿瘤防治中心2000年9月至2006年1月结直肠癌各期标本共100例，石蜡标本室温保存，切取的白片保存于4℃冰箱。
对于每个入选病例，入选标准为初诊、经病理学诊断为结直肠单发腺癌、无术前放化疗，TNM I、II、III期病例要求行根治术。每例病人包含肿瘤和正常对照(距离切缘5cm以上的正常粘膜)。
对于所有纳入研究的病例，其临床病理资料均来自中山大学肿瘤防治中心病案报告，相应的随访资料来自随访科。病人的肿瘤分级均按照UICC/AJCC(第七版)的标准分级。手术病人在术后两年内每三个月随访一次，术后三到四年内每半年随访一次，术后五年及以后每一年随访一次，随访时记录病人复发/转移/死亡情况并记录日期，末次随访日期是2014年12月31日。
实施例2、定量PCR检测WDR5在结直肠癌组织中mRNA水平
1)使用TRIZOL法提取16例结直肠癌组织及其正常对照，分别取2μg总RNA逆转录为cDNA。
2)查询NCBI的Gene数据库，得到WDR5和GAPDH(内参)的cDNA序列，根据序列设计PCR引物如下：
3)建立以下反应体系，每个样本设置三个反应复孔：
混匀以上组分，加入8联管各孔，盖紧盖子，排除气泡，离心使液体聚集到管底。热循环参数如下：95℃ 10min；95℃ 30s，60℃ 30s，72℃ 40s，40个循环；95℃ 1min，60℃30s，95℃ 30s。
结果如图1。
图1所示，所有肿瘤样本(16/16)比其正常对照有显著的WDR5mRNA水平上调(P<0.05)。
图1证实WDR5在结直肠癌的肿瘤组织的含量显著高于正常对照。
实施例3、WDR5蛋白含量在结直肠癌组织标本中的测定
1)实验时取出100例结直肠癌病例石蜡切片白片，经二甲苯脱蜡、梯度酒精水化、0.3％H2O2去组织过氧化物酶、枸橼酸盐溶液微波修复、R&D System公司anti-WDR5(AF5810)抗体(1:100)和相应种属二抗孵育、二氨基联苯胺(3,3’-diaminobenzidine)和苏木素显色、盐酸酒精分化、梯度酒精脱水，二甲苯透明步骤后，中性树胶封片。
2)组织标本的染色评估由200×倒置显微镜拍照，癌旁、癌组织各取5个视野/病例，然后用inForm1.4.0软件对图片进行分析。细胞中WDR5的染色强度分为：阴性(0)，弱阳性(1)，中等阳性(2)和强阳性(3)四个等级。判断每个标本染色的最终指标为强度×阳性率(H-score)。
结果如图2-3所示。
图2证明WDR5蛋白在肿瘤组织中的含量(H-score)显著高于癌旁组织：WDR5在肿瘤组织中表达阳性率为43.8％，H-score评分为52.99；而WDR5在癌旁组织中表达阳性率为19.5％，H-score评分为22.43(P<0.001，P<0.001)。
图3证明WDR5蛋白在肿瘤组织中的含量显著高于癌旁组织。
实施例4、WDR5基因的克隆
1.培养HT-29细胞
用含10％胎牛血清McCoy 5A培养基培养HT-29细胞，融合度达到80％时，用TRIZOL收集细胞。
2.RNA抽提
(1)利用TRIZOL法从HT-29细胞中抽提总RNA，用紫外分光光度计测定RNA的浓度和纯度。
(2)逆转录反应
①在PCR管中加入以下试剂：
总RNA 2ug
随机引物(0.5ug/ul) 1ul
无RNA酶双蒸灭菌水 补至10ul
②置PCR仪中70℃、5min，立即放入冰中静置至少2min；
③再在上述PCR管中依次加入以下试剂：
④置PCR仪中42℃、1h；
(3)以NCBI数据库上公布的WDR5基因序列为对象设计克隆引物
WDR5：F：5’-CCCAAGCTTCATGGCGACGGAGGAGAAG-3’(SEQ ID NO:5)
R：5’-CCGGAATTCTTAGCAGTCACTCTTCCACAG-3’(SEQ ID NO:6)
(4)扩增WDR5的CDS区序列
用逆转录得到的cDNA模板进行扩增，具体体系如下：
反应条件：
(5)酶切和连接
用HindIII和EcoRI内切酶对步骤(4)得到的片段在37℃进行酶切，并在16℃中过夜连接至表达载体pcDNA3.1(+)上。鉴定合格即得所需带WDR5基因的表达质粒。
(6)同样的方法构建带EGFR的表达质粒。
实施例5、伤口愈合/细胞划痕实验
(1)采用Wound healing方法。培养板用记号笔进行标记，方便后续拍照定位。
(2)在HCT116细胞中用lipo2000法转染实施例4中获得的WDR5表达质粒及对照EGFP表达质粒，得到HCT116-WDR5和HCT116-EGFP细胞。
(3)HCT116-WDR5和HCT116-EGFP细胞消化下来后，接入6孔板，约5×105个。
(4)培养24小时待细胞贴壁后，用0.1mL枪头垂直于孔板制造划痕，尽量保证各划痕宽度一直。
(5)吸去细胞培养液，用PBS冲洗孔板3次，洗干净划痕产生的细胞碎片。
(6)加入无血清培养基培养。
(7)每隔6小时在标记的位置拍照，观察细胞愈合情况。
(8)收集图片数据分析结果。
结果见图4，可以看出稳定过表达WDR5的HCT116细胞的迁移能力比对照株的显著增强，说明WDR5有促进直肠癌细胞转移的功能。
以上实验表明，WDR5是一个与结直肠癌密切相关的基因。WDR5的mRNA和蛋白表达水平在结直肠癌病人的肿瘤组织中显著上调。同时，WDR5上调的细胞的迁移能力显著强于对照组细胞。基于上述发现，我们开发了一种用于结直肠癌辅助诊断的试剂盒，所述试剂盒可以包括以下试剂中的至少一种：用于检测WDR5基因表达水平的引物、用于检测WDR5蛋白含量的试剂。同时，本发明提供了一个潜在的治疗靶点。本发明对于更好地理解结直肠癌的发生机理，制定结直肠癌风险评估和早期检测的有效措施具有极其重要的意义。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (1)
1.试剂A在制备用于结直肠癌辅助诊断的试剂盒中的应用，其特征在于：所述的试剂A是用于检测WDR5基因表达水平的引物对，其序列如SEQ ID NO:3和SEQ ID NO:4所示。
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