CN116256520A - 脑脊液penk试剂盒及其在髓母细胞瘤转移检测预判中的应用 - Google Patents
脑脊液penk试剂盒及其在髓母细胞瘤转移检测预判中的应用 Download PDFInfo
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Abstract
本发明属生物技术领域，涉及肿瘤诊断试剂盒，具体涉及脑脊液PENK蛋白试剂盒及其在髓母细胞瘤转移检测预判中的应用。本发明中，设计了包括鼠抗人PENK生物素标记单抗、链霉素抗生素蛋白‑过氧化酶工作液等组分的试剂盒，然后通过ELISA的一般步骤，对采集的脑脊液样本进行检测。根据脑脊液中PENK蛋白浓度做出相应的预判，当PENK蛋白浓度上升至10倍以上时，可初步判断为髓母细胞瘤脑脊液转移。本发明方法用于髓母细胞瘤转移预测，不仅可以对高危人群进行筛查，而且能够指导肿瘤分期，为寻找灵敏、特异的髓母细胞瘤脑脊液标志物提供基础。
Description
技术领域
本发明属生物技术领域，涉及肿瘤诊断试剂盒，具体涉及脑脊液PENK试剂盒及其在髓母细胞瘤转移检测预判中的应用。所述试剂盒通过检测脑脊液中的PENK蛋白，为寻找灵敏、特异的髓母细胞瘤转移脑脊液标志物提供基础。
背景技术
现有技术公开了髓母细胞瘤是危害人们身体健康的重大脑疾病，尤其是髓母细胞瘤是最常见的儿童恶性脑肿瘤，约占儿童肿瘤的20％，临床实践显示成人发病后预后也很差。2016WHO将髓母细胞瘤按照不同分子亚型：SHH，WNT，GROUP3，GROUP4。目前临床实践中髓母细胞瘤的标准治疗方案为：手术切除+术后放疗+术后化疗，患者5年生存率为52％，而出现远处转移病人5年生存率仅为26％，肿瘤转移是髓母细胞瘤常见的致死原因之一；对髓母细胞瘤病人术后出现转移进行预测，以便及时调整术后放化疗计量、延长患者生存期，成为越来越多的科学家和医生关注课题。
目前临床对于髓母细胞瘤转移的分级仍然采用1969年的chang分级，该分级根据患者的影像学及脑脊液脱落细胞学为依据对髓母细胞瘤进行分级，由于相关技术及研究的缺乏，尚不能对髓母细胞瘤病人脑脊液成分及生物信息充分认知，该chang分级尚不能对髓母细胞瘤患者的转移起到预测的作用。目前国际上对于髓母细胞瘤转移患者脑脊液成分研究少之又少，转移病人的预测研究迫在眉睫。
研究公开了蛋白质作为动物生命活动的基础，在人类各个生物过程起到至关重要的作用；肿瘤的转移同样需要蛋白质和相关肽段的参与，研究显示，髓母细胞瘤转移通常是通过肿瘤细胞混杂在脑脊液中，延蛛网膜下腔播散，最后定植在脊髓或者中枢神经系统以外的各个系统，因此，检测髓母细胞瘤脑脊液蛋白的变化预判髓母细胞瘤转移的将是可行的。
基因测序技术的变革使针对患者的个体化分析以及相应的个体化治疗成为可能，精准医疗随之而产生。然而，业内公知，疾病的发生来自于遗传与后天环境两方面，建立在基因测序基础上的、仅考虑遗传因素的精准医疗注定不能到达“精准”，何况遗传性致病因素要由基因转化为蛋白缺陷方能得以致病；因此，业内研究者认为，无论从验证基因缺陷角度还是从环境致病因素角度，蛋白质分析将是精准医疗的下一个里程碑。蛋白质谱技术，即通常被成为液相－质谱联用(液质联用，LC/MS/MS)，是独立于抗体技术之外的两大蛋白质分析技术之一，其原理是以肽段在色谱柱中的保留时间(Retention time，RT)和肽段及其在高能碎裂后的离子特征(质量/电荷比例，即质荷比m/z)推导被检肽段的氨基酸序列，由于被检肽段是来自特定酶解后某蛋白质，因而可以根据多个含有特征氨基酸序列的肽段，推导该蛋白质的存在(即鉴定，identification)；继而可以根据肽段离子(又称为母离子，precursor ion)及其碎裂离子(又称为子离子，fraction ion)在质谱中的响应程度对肽段及其对应的蛋白质进行定量。2014年末，《Nature Methods》杂志介绍了了2015年值得关注的技术，其中就包括DIA质谱分析技术。传统从液态中识别蛋白质采用的是：数据依赖液态活检方法(DDA)，它将蛋白质样品消化成肽段，离子化并通过质谱进行分析。在全扫描质谱图中，高于噪音的肽段信号被选择性裂解，产生随机(MS/MS)质谱，能够与数据库中的图谱相匹配。数据依赖液态活检方法(DIA)：在DDA的基础上扫描范围内对母离子进行无缝碎裂，相比DIA能够采集到更多更全的信息。这实现了准确的肽段定量，而不限于分析预先定义的肽段。上述新型的DIA方法将有助于本领域更全面的分析脑脊液中肽段及蛋白。
前已述及，疾病的发生、进程、转归是一个复杂的生物过程，绝不是单一蛋白标志物可以全面指示的。与抗体技术相比，质谱的强项是建立在氨基酸序列基础上的、定量地瞬时监测大量的蛋白变化。因而，质谱的临床应用必将是从整体上监测一个与疾病特点相关的panel才能发挥质谱的特点。从质谱原理角度讲，质谱对于某蛋白的定量是基于对该蛋白酶解后各个肽段的综合质谱丰度计算得出，而肽段的质谱丰度是由该肽段在质谱仪中被高能击碎的多个碎片离子的质谱丰度综合计算得出，因而肽段的碎片离子丰度是质谱的最小定量单元。事实证明，不同肽段离子化程度、不同离子在质谱仪中的响应不尽相同，因此传统质谱分析中使用各个肽段信息推导相应蛋白的含量可能导致定量分析的稳定性和敏感性下降，其后果是某些差异蛋白不能被发现。基于此，本申请的研究团队在肽段水平挑选稳定、敏感的标志物并组成panel族群进行综合评判的临床诊断，其不但使分子数据对疾病的反映更加敏感，同时也通过扩大监测panel的内容，提高了诊断的自身纠错能力；后者，尤其是在监测肽段数目受限的绝对定量质谱分析中，较以蛋白为基础诊断单位的常规质谱分析显示出巨大优势。更具体的，本申请的发明人拟提供脑脊液PENK试剂盒及其在髓母细胞瘤转移检测预判中的应用。
发明内容
本发明的主要目的是基于现有技术的基础，提供脑脊液PENK试剂盒及其在髓母细胞瘤转移检测预判中的应用，本发明的脑脊液PENK试剂盒将有助于预测髓母细胞瘤远处转移的发生。
本发明中，通过DIA方法检测髓母细胞瘤术后病人脑脊液中蛋白质及肽段的变化；获得转移病人脑脊液中肽段的变化，建立一个多肽段的髓母细胞瘤预测模型，补充评价髓母细胞瘤转移分级过于依赖影像学的问题，并应用于相关治疗方案和预后评估。
本发明中，基于前期的基础研究中：采用大样本髓母细胞瘤病人数据，分析其不同分子亚型后，探索不同分子亚型髓母细胞瘤对于术后放化疗的敏感性，发现SHH,WNT亚型对于术后化疗较为敏感，结果发表于《Plos one》上等有关的基础，本发明通过检测髓母细胞瘤术后为转移患者、转移患者脑脊液中肽段及离子含量的变化，利用统计学分析揭示转移患者脑脊液中PENK蛋白的明显下调，该结果将能够最大程度上预测髓母细胞瘤转移的发生，为髓母细胞瘤转移靶向治疗提供理论依据。
具体的，
本发明提供了脑脊液PENK蛋白在用于制备髓母细胞瘤转移诊断试剂盒中的用途，所述的试剂盒针对待测脑脊液样品，用ELISA试剂盒检测该脑脊液中PENK蛋白浓度，根据结果可用于预评断髓母细胞瘤转移的发生概率。
本发明提供了一种脑脊液PENK试剂盒，其包括组分：固相96孔酶标板；鼠抗人PENK纯化单抗(eBioscience)；鼠抗人PENK生物素标记单抗(eBioscience)；链霉素抗生素蛋白-过氧化酶工作液(Abcam)；PENK蛋白标准品(R&D Systems)；ABTS底物；0.05％Tween20-PBS洗涤液；0.1％BSA-PBS稀释液；终止液。
进一步，本发明提供了所述的脑脊液PENK试剂盒检测脑脊液中PENK蛋白浓度的方法，其包括步骤：蛋白标准品配制，包被封闭鼠抗人PENK，标准品及待测样本加样，一抗孵育，酶标抗体孵育，底物显色，酶标仪检测，绘制标准曲线，计算样本浓度。
进一步，本发明提供了所述的脑脊液PENK试剂盒在用于脑胶质瘤转移早期诊断预测中的应用，其包括：取待测样本，测脑脊液PENK浓度上升至10倍以上时，可初步预测髓母细胞瘤转移。
本发明公开的检测脑脊液PENK蛋白的试剂盒可以准确、快速地检测脑脊液中的相关肽段含量，进而能够对髓母细胞瘤术后病人是否转移进行预测，具有较高的临床应用价值。
本发明的PENK蛋白试剂盒能根据患者脑脊液中PENK蛋白变化从而预测髓母细胞瘤的转移风险，筛查高危人群，早期预测髓母细胞瘤转移。
附图说明
图1显示了配有Ultimate 3000系统的Dionex仪器；其中，该数据非依赖液态活检设备：Dionex，配有捕获柱(Acclaim PepMap 100C18 75um×2cm)和分析柱(AcclaimPepMap RSLC C18 75um×25cm)的Ultimate 3000系统上分析样品。
图2显示了Skyline软件处理脑脊液中的肽段和离子数据。
具体实施方式
本发明结合实施例和相应附图做进一步阐释说明，以下实施例仅用于说明目的，不用于限制本发明范围。
实施例1
DIA方法分析脑脊液中肽段的含量：
脑脊液样本准备：将细胞以20000g在4℃下在冷冻离心机中离心10分钟以除去不溶物和细胞，在每例脑脊液中加入1M DTT至终浓度5mM，在56℃下还原1小时，冷却至室温后，加入0.5M IAM至10mM的终浓度，并在室温下进行烷基化45分钟，L-半胱氨酸在室温下终浓度为20mM，终止烷基化反应20分钟；加入1M TEAB终浓度为0.1M，胰蛋白酶(胰蛋白酶：样品，1：50，m/m)酶水解过夜，第二天再次加胰蛋白酶4小时；加入甲酸至终浓度1％，终止酶水解；用C18萃取柱提取产物，脱盐后用真空浓缩器干燥提取；使用Spikemix complex溶解样品(Spikemix用于监测仪器的平行度)，转化为质谱进行检测；在机器上的每个样品丰度，通过浓度转换为等体积进行分析；
DIA方法采用质谱分析仪：Dionex，配有捕获柱(Acclaim PepMap 100C18 75um×2cm)和分析柱(Acclaim PepMap RSLC C18 75um×25cm)的Ultimate 3000系统上分析样品；
质谱仪设置参数：通过从缓冲液A(2％ACN，0.1％FA)/缓冲液B(80％ACN，0.1％FA)的线性梯度分离肽段(2ug消化物)，上样和清洗步骤的总运行时间是120分钟，梯度为2-35％缓冲液B(105min)和35-80％缓冲液B(15min)，流速为300nl/min；
对于DDA，质谱仪在数据相关模式下进行以下设置：质谱仪以数据相关的TOP15模式运行，设置如下：MS1质量范围350-1400m/z，MS1和MS2的分辨率分别为70000和17500，碎裂能量分别为24％，27％，30％NCE，MS1和MS2的AGC分别为3E6和1E4，动态排除设置为10秒；
对于DIA，质谱仪以独立于数据的模式运行，具有以下设置：全扫描，分辨率为35000；AGC为3E6；质量范围390-1210m/z；其次是二级扫描分辨率为17500；AGC3E6；碎裂能量是30％NCE；得到的数据采用Skyline软件进行分析：
实施例2、DIA方法检测脑脊液PENK浓度
收集标本：在征得2006-2014年就诊于华山医院神经外科的29例髓母细胞瘤病人(包括14例未转移病人、15例出现远处转移病人)的同意后，对其进行腰穿，每人留取5ml脑脊液，储存于-80℃冰箱；
检测目标浓度：脑脊液样品在冰浴中解冻30-60min，离心1500rpm×5min，取上清，按照上述的方法，采用DIA、ELISA方法检测PENK含量；
结果显示，DIA方法检测到的PENK相关肽段，经过处理换算为蛋白质，转移-非转移髓母细胞瘤患者脑脊液差别高达10倍以上；
对比术后初始PENK含量，预测标准为：如果PENK含量增高至10倍以上，可考虑该患者出现转移概率较大，
实验结果表明，DIA方法检测脑脊液PENK浓度，可用于评判诊断胶质瘤的灵敏度、特异度。
Claims (4)
1.脑脊液PENK蛋白在用于制备髓母细胞瘤转移诊断试剂盒中的用途，所述的试剂盒针对待测脑脊液样品，用ELISA试剂盒检测该脑脊液中PENK蛋白浓度，根据结果用于预评断髓母细胞瘤转移的发生概率。
2.一种脑脊液PENK蛋白试剂盒，其特征在于，包括组分：固相96孔酶标板；鼠抗人PENK纯化单抗(eBioscience)；鼠抗人PENK生物素标记单抗(eBioscience)；链霉素抗生素蛋白-过氧化酶工作液(Abcam)；PENK蛋白标准品(R&D Systems)；ABTS底物；0.05％Tween20-PBS洗涤液；0.1％BSA-PBS稀释液；终止液。
3.根据权利要求1所述的用途，其特征在于，所述的髓母细胞瘤转移诊断试剂盒通过下述方法检测脑脊液中的PENK蛋白浓度，包括步骤：蛋白标准品配制，包被封闭鼠抗人PENK，标准品及待测样本加样，一抗孵育，酶标抗体孵育，底物显色，酶标仪检测，绘制标准曲线，计算样本浓度。
4.根据权利要求1或3所述的用途，其特征在于，测待测样本中脑脊液PENK浓度上升至10倍以上时，可初步预测为：髓母细胞瘤转移。
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