CN116990517A - RNA结合蛋白Musashi在神经母细胞瘤诊断和预后中的应用 - Google Patents
RNA结合蛋白Musashi在神经母细胞瘤诊断和预后中的应用 Download PDFInfo
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Abstract
本发明公开了RNA结合蛋白Musashi在神经母细胞瘤诊断和预后中的应用,涉及分子生物医学技术领域。本发明提供了检测样本中MSI2蛋白表达水平的试剂在制备恶性神经母细胞瘤诊断或预后产品中的应用,本发明为神经母细胞瘤的诊断或药物疗效评估的提供了一种准确度更高的新型标志物,克服目前传统肿瘤标志物n‑Myc在神经母细胞瘤诊断中存在的潜在漏诊风险,同时也发掘出了MSI2蛋白新的医药价值。
Description
技术领域
本发明涉及分子生物医学技术领域,具体涉及RNA结合蛋白Musashi在神经母细胞瘤诊断和预后中的应用。
背景技术
神经母细胞瘤(Neuroblastoma,NB)是最常见的儿童颅外恶性实体肿瘤之一,其发病率占儿童恶性肿瘤发生率的10%,死亡率占儿童肿瘤死亡人数的15%,严重威胁着儿童的健康和生命。目前,神经母细胞瘤的治疗仍以手术治疗为主,辅以放化疗,干细胞移植以及免疫治疗。肿瘤细胞恶性增殖及转移仍是神经母细胞瘤治疗和预后不佳的主要原因,65%的患儿初诊时已有骨髓、骨骼或远处淋巴结转移。NB是一种好发于儿童交感神经脊部位以肾上腺最常见的颅外实体瘤。根据INRG(International Risk Neuroblastoma Group)分类系统,NB可分为极低危、低危、中危及高危组。在组织细胞学分类上,可将神经母细胞瘤分为ganglioneuroma(GN),ganglioneuroblastoma intermixed,ganglioneuroblastomanodular(GNB)和neuroblastoma(NB)等四类。其中极低危组,如部分M-S(INRG分期)/IV-S期(INSS分期)患者,无需治疗肿瘤可自我消退。但是,对于高危组NB,纵使给予高强的综合治疗,包括化疗、手术、放疗,其2年生存率仅为19%。
近年来,随着各种临床治疗手段如免疫治疗,干细胞自体移植等的不断改进与完善,放化疗的联合应用,NB患者的5年生存率已经超过90%,但是对于高危NB患者,肿瘤细胞的恶性增殖和转移成为治疗失败的主要原因。传统肿瘤标准物n-Myc在患者组织中表达灵敏度不高,存在漏诊风险。因此,开发新型的NB组织学肿瘤标志物,既能针对早期NB患者也有较好的诊断效能,又能对恶性NB患者临床诊断治疗与预后转归有充分预警作用。
发明内容
本发明的目的在于克服现有技术中肿瘤标志物n-Myc在神经母细胞瘤诊断中存在潜在漏诊风险,提供RNA结合蛋白Musashi在神经母细胞瘤诊断和预后中的应用,为神经母细胞瘤的诊断或药物疗效评估的提供了一种准确度更高的新型标志物。
为实现上述目的,本发明采取的技术方案为:检测样本中MSI2蛋白表达水平的试剂在制备恶性神经母细胞瘤诊断或预后产品中的应用。RNA结合蛋白Musashi(MSI)家族在哺乳动物中包含两个成员,即MSI1和MSI2。本申请发明人通过前期的实验筛选和验证,发现恶性神经母细胞瘤患者的病理组织中的MSI2蛋白水平相较于分化程度较好的神经母细胞瘤显著上调。MSI2蛋白表达水平可作为新型肿瘤标志物应用于神经母细胞瘤的诊断与预后中,临床价值较高,为神经母细胞瘤患者的诊治与预后转归提供新的思路。
作为本发明所述应用的优选实施方式,所述样本包括临床病理组织。
作为本发明所述应用的优选实施方式,所述MSI2蛋白表达水平在恶性神经母细胞瘤患者中上调。
作为本发明所述应用的优选实施方式,所述预后包括预后受试者的临床结局、预后受试者的治疗效果或预后受试者的生存。
作为本发明所述应用的优选实施方式,所述MSI2蛋白表达水平上调的恶性神经母细胞瘤患者预后差。
作为本发明所述应用的优选实施方式,所述产品包括诊断剂、试剂盒、芯片、试纸或孔板。
本发明还提供检测样本中MSI2蛋白表达水平的试剂在制备用于神经母细胞瘤病程进展监控的产品中的应用。
作为本发明所述应用的优选实施方式,所述MSI2蛋白表达水平随着神经母细胞瘤分期进展呈上升趋势。
本发明还提供一种用于诊断恶性神经母细胞瘤、评估恶性神经母细胞瘤患者预后效果或评估神经母细胞瘤病程进展监控的试剂盒,所述试剂盒包括用于检测样本中MSI2蛋白表达水平的试剂。
本发明的有益效果:本发明提供了检测样本中MSI2蛋白表达水平的试剂在制备恶性神经母细胞瘤诊断或预后产品中的应用,本发明为神经母细胞瘤的诊断或药物疗效评估的提供了一种准确度更高的新型标志物,克服目前传统肿瘤标志物n-Myc在神经母细胞瘤诊断中存在的潜在漏诊风险,同时也发掘出了MSI2蛋白新的医药价值。
附图说明
图1为增殖程度不同的NB患者临床病理组织中MSI2蛋白水平检测图,其中,NB为肿瘤细胞增殖快速的神经母细胞瘤组;GNB为肿瘤细胞增殖较快的神经节母细胞瘤组;GN为肿瘤细胞增殖较缓慢的神经节瘤组;A-B为NB组、GNB组、GN组的MSI2蛋白水平;C为MSI2蛋白水平与NB患者预后生存的关系图;D-E为WB法检测结果;F为ki67阳性组、ki67阴性组的MSI2蛋白水平。
图2为转移性NB患者病理新鲜冰冻组织MSI2蛋白水平检测图,其中,A为生物大数据预测MSI2在转移性NB患者中表达升高;B为检测转移性和非转移性NB患者中表达水平;C为NB患者外周病理组织MSI2蛋白水平的诊断效能评估。其中NB的临床分期:1-2期以及4s期为非转移性组;3-4期为转移性组。
图3为NB患者临床病理组织中MSI2蛋白水平与常见诊断性标志物n-Myc的诊断效能;其中A-B为临床病理组织中MSI2和n-Myc蛋白表达水平;C为NB患者中MSI2低水平n-Myc低水平、MSI2低水平n-Myc高水平、MSI2高水平n-Myc低水平以及MSI2高水平n-Myc高水平四种类型所占的比例;D为四种类型的H-Score分数与对应的生存预后时间统计分析图;E为MSI2水平、肿瘤标志物n-Myc的诊断效能。
具体实施方式
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1
本实施例通过实验检测增殖程度不同的NB患者临床病理组织中MSI2蛋白水平,具体实验方法如下:
(1)收集2010-2015年期间经确诊的123例神经母细胞瘤患者的石蜡组织样本(其中NB 65例,GNB 31例,GN 27例),检测NB组织中MSI2蛋白的表达水平以及定位情况,具体操作如下:制作的石蜡切片经过白片脱蜡水化(60℃烘箱烘烤50min),于二甲苯中脱蜡3次,每次20min;转入无水乙醇中浸泡2次,每次10min,洗净残余二甲苯后,乙醇溶液:95%、90%、80%、70%,依次浸泡水化,每级浸泡5min,后转移至蒸馏水中。切片利用10mmol/L柠檬酸盐缓冲液(pH=6.0)的抗原修复液高压锅加热后自然冷却进行抗原修复;PBST洗3次,每次3min;然后利用3%的过氧化氢进行阻断,正常非免疫山羊血清(试剂B)封闭30min;去除血清,MSI2单克隆抗体(抗体稀释度均为1:300),4℃孵育过夜。PBST洗3次滴加50μl生物素标记的鼠兔通用二抗(试剂C)室温孵育20-30min,PBST洗3次分别滴加50μl链霉素抗生物素-过氧化物酶溶液(试剂D)室温孵15-20min,PBST洗3次。DAB显色反应,每切片滴加100μl新鲜配制的DAB工作液,光镜下观察显色结果,当阳性显色明显时,适时自来水冲洗终止反应,用苏木素复染1min,自来水冲洗返蓝。而后梯度酒精(依次为70%,80%,90%,95%,100%,各3min)脱水干燥,二甲苯透明(新鲜二甲苯2次,每次10min),中性树胶封片,光镜下观察分析结果,并拍照记录。
组织化学评分(H-SCORE):将染色好的组织芯片放在研究级正置显微镜(BX51,OLYMPUS)100X镜头下进行观察,根据细胞内MSI2染色强度和MSI2阳性细胞百分比计算MSI2得分多少。染色强度评分标准:不着色0分,黄色1分,棕黄色2分,黄褐色3分;阳性细胞所占比例评分标准:阳性细胞数<10%者为0分,10%-40%者为1分,40%-70%者2分,大于70%者3分。按照公式H-SCORE=∑(i×Pi),i代表染色强度,Pi代表染色细胞的百分数,计算组织芯片中MSI2得分。
(2)通过蛋白印迹技术(Western blottting)检测NB新鲜临床病理组织中MSI2蛋白水平,具体步骤为:将新鲜神经母细胞瘤冰冻组织标本经PBS洗净再吸干,称取50mg,加含蛋白酶及磷酸酶抑制剂的1ml 1x SDS缓冲液进行充分裂解,然后在匀浆机上匀浆1-2min,冰上超声震碎,接着煮沸蛋白,使其充分裂解变性,上离心机进行离心30min(12000rpm,4℃),弃去沉渣,收集上清,即为组织蛋白。接着使用Bio-rad的DC试剂盒,具体步骤详见试剂说明书。根据蛋白定量结果,计算每个样品的上样体积,配好上样缓冲液(按1:9比例与10X变性缓冲液充分混匀),100℃煮沸,待其离心后,上样于预先制好的胶孔中。进行SDS-PAGE电泳-转膜-封闭-一抗孵育-二抗孵育-ECL显影,以β-actin为内参,检测肿瘤组织和细胞株中MSI2的含量。
(3)分别对组织芯片进行MSI2免疫组化染色,运用H-Score系统,根据MSI2得分多少分为高表达组(H-Score≥6分)和低表达组(H-Score<6分),查阅123例患者的临床病理资料(ki67阳性比例),分别统计MSI2高表达组和低表达组的ki67阳性比例,分析比较MSI2表达水平是否与NB患者ki67阳性比例存在正相关性。
(4)对2010-2015年期间符合入组标准的123例患者(其中NB 67例,GNB 31例,GN25例)做了生存预后随访调查。期间通过随访跟踪调查,电话,门诊等形式咨询患者情况,出现联系不上患者或是患者死亡现象,则视为生存预后随访调查终点事件。在本研究中,我们把已收集的研究对象的生存死亡比例同组织芯片的MSI2的表达情况进行统计学分析,明确MSI2的表达水平与患者生存预后是否存在负相关。
结果如图1所示。图1A-B显示:NB组(5.80±0.98)相较于GNB组(3.94±1.05)以及GN组(2.04±0.81),临床病理组织中MSI2蛋白水平显著增多;图1C显示:MSI2蛋白水平与NB患者预后生存明显存在负相关关系,说明MSI2表达高水平的NB患者生存预后较差,MSI2蛋白水平表达低的NB患者生存预后较好;图1D-E显示:WB法检测结果显示,与GNB组相比,NB组临床病理组织中MSI2蛋白水平同样升高;图1F显示:ki67阳性组MSI2蛋白水平较高,ki67阴性组MSI2蛋白水平较低,说明MSI2在增殖较快的NB患者中表达升高。
实施例2
本实施例通过实验检测转移性NB患者病理组织标本中MSI2蛋白水平并探讨MSI2在转移性NB患者中的诊断效能,具体方法为:基于转移性和非转移性NB患者病理组织中的MSI2蛋白水平绘制ROC曲线,根据AUC大小确定最佳cutoff值,此时特异性和灵敏性最高。
实验结果如图2所示。图2A显示:GEO公共数据中NB中MSI2表达水平升高,并于患者预后成负相关关系;图2B显示:与非转移性NB组中MSI蛋白水平(3.27±1.28)相比,转移性NB组中MSI2蛋白水平(8.35±1.16)表达增多;图2C显示:NB临床病理组织中MSI2蛋白水平的AUC高达0.9442,说明NB临床病理组织中MSI2水平对诊断NB具有较好的诊断功能。
实施例3
本实施例比较NB患者临床病理组织中MSI2蛋白水平与常见诊断性标志物n-Myc的诊断效能,具体方法为:分别根据123例NB患者病理组织中的MSI2和n-Myc蛋白表达水平绘制相应的ROC曲线,然后比较MSI2和n-Myc的ROC曲线下面积(AUC)以确定两者在诊断NB的效能高低。
实验结果如图3所示。图3A-B显示:临床病理组织中MSI2和n-Myc蛋白表达水平;图3C显示:在65例NB患者中,分别统计MSI2低水平n-Myc低水平、MSI2低水平n-Myc高水平、MSI2高水平n-Myc低水平以及MSI2高水平n-Myc高水平四种类型所占的比例,结果显示在NB患者中,MSI2高水平比例高于n-Myc高水平组,说明与传统肿瘤标志物n-Myc相比,新型标志物MSI2阳性率较高,存在较高灵敏度,避免漏诊风险;图3D显示:将这四种类型的H-Score分数与对应的生存预后时间进行统计分析,结果表明,MSI2高表达的患者死亡率高于n-Myc高表达的患者死亡率,这提示与传统肿瘤标志物n-Myc相比,MSI2可以较好地预测患者NB的生存预后程度;图3E显示:临床病理组织中MSI2水平的AUC高达0.9442,而传统的肿瘤标志物n-Myc水平的AUC仅为0.8094,这提示在NB临床组织中,MSI2水平比传统肿瘤标志物n-Myc具有更好的诊断效能。
最后应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (9)
1.检测样本中MSI2蛋白表达水平的试剂在制备恶性神经母细胞瘤诊断或预后产品中的应用。
2.根据权利要求1所述的应用,其特征在于,所述样本包括临床病理组织。
3.根据权利要求1所述的应用,其特征在于,所述MSI2蛋白表达水平在恶性神经母细胞瘤患者中上调。
4.根据权利要求1所述的应用,其特征在于,所述预后包括预后受试者的临床结局、预后受试者的治疗效果或预后受试者的生存。
5.根据权利要求1所述的应用,其特征在于,所述MSI2蛋白表达水平上调的恶性神经母细胞瘤患者预后差。
6.根据权利要求1所述的应用,其特征在于,所述产品包括诊断剂、试剂盒、芯片、试纸或孔板。
7.检测样本中MSI2蛋白表达水平的试剂在制备用于神经母细胞瘤病程进展监控的产品中的应用。
8.根据权利要求7所述的应用,其特征在于,所述MSI2蛋白表达水平随着神经母细胞瘤分期进展呈上升趋势。
9.一种用于诊断恶性神经母细胞瘤、评估恶性神经母细胞瘤患者预后效果或评估神经母细胞瘤病程进展监控的试剂盒,其特征在于,所述试剂盒包括用于检测样本中MSI2蛋白表达水平的试剂。
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