CN112266957A - 预测cis临床孤立综合征转归的组合物 - Google Patents
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Abstract
本发明涉及一种预测CIS临床孤立综合征转归的组合物，所述组合物包括如下的一个或多个检测试剂，检测CO4A，AP0B，ITIH4和/或KNTC1的试剂。
Description
技术领域
本发明属于CIS临床孤立综合征转归领域。具体的，本发明公开了一种组合物，其包含蛋白CO4A，AP0B，ITIH4和KNTC1。
背景技术
多发硬化(Multiple Sclerosis，MS)是一种中枢神经系统的慢性自身免疫性炎性疾病，急性期以白质多发性脱髓鞘病变为主要特征，慢性期由于胶质纤维增生而形成钙化斑。由于本病发病迅猛，主要侵犯中枢神经系统，目前的治疗策略主要针对免疫系统的调节以减少炎症及脱髓鞘的发生，而胶质增生、神经变性等病变依然存在，患者病程缓慢进行性加重，最终导致终生残疾，给家庭及社会带来严重的负担。
根据2010年最新的McDonald标准，MS的诊断主要基于磁共振成像(MRI)及时间空间的播散证据，即不同时间发生的，中枢神经系统白质至少两个不同区域的病变证据。所谓“临床确诊型MS”，即患者表现为复发缓解的病程，而且有CNS白质不同区域至少两处病变的征象。然而，约85％的病人在首次就诊时并不完全具备上述特征，而仅仅在临床上表现为脱髓鞘发作的症状，如急性视神经炎、局部髓鞘炎或脑干综合征等，这种首次的临床脱髓鞘发作被称为临床孤立综合征(Clinically Isolated Syndrome，CIS)；同时，在MS的诊断上还存在“拟诊型MS”，即患者有多灶性白质病变的证据，但只有一次临床发作，或至少有两次临床发作的病史，而只有单一病灶的体征；此外，用于MS诊断或辅助诊断的技术，如MRI、脑脊液检测、自身免疫抗体筛查等，其检测结果并不是MS所特有的，这些都大大增加了MS疾病早期诊断的不确定性。研究表明CIS患者的疾病转归不尽相同，部分患者多年后也并不出现进一步的MS疾病症状，因此预测CIS患者的疾病转归并给于相应患者影响疾病进程药物的治疗显得尤为重要。
目前已有多项报道对CIS患者的临床转归进行了研究，并试图找出可能的预测标志物。目前已有多项报道对CIS患者的临床转归进行了研究，并试图找出可能的预测标志物，其中MRI初始病灶数目、抗MOG及MBP抗体、脑脊液寡聚IgG条带等被认为是“热点候选标志物”，然而结果并不尽人意。以MRI初始病灶数为例，CHAMPS实验表明，38％的CIS患者在3年的随访中进展为临床确诊型MS，但其脑部应有两处或更多3mm以上的病变，其中一处病变必须在脑室周围；ETOMS实验发现，在至少有4处T2病变或三处T2病变及一处钆增强病变的CIS患者中，有45％在2年内进展为MS；而在PreCISe实验中，T2图像上有两处或更多大于6mm病变的患者在2年内有42.9％成为MS。虽然这些实验都在一定程度上预测了CIS患者的疾病转归，但各个标准之间的差异缺乏在大样本中的验证阻碍了其临床应用的可行性。此外，有研究显示，“未经治疗的疑似MS患者长期预后较差”可能是人为放大所致。例如Rodriguez经过长达40年的随访发现在特发性视神经炎患者中只有40％发展为临床确诊型MS。因此，对于CIS患者的诸如视神经炎之类的症状，并非如我们所认为的那样，而可能只是脱髓鞘疾病的良性表现形式，并且是一种十分常见的表现形式。因此，迫切需要找到一种能特异、敏感地预测患者是否由CIS发展为“临床确诊型MS”的指标，从而指导临床的早期干预，降低疾病的致残率及死亡率。
不幸的是，虽然大量研究报道了海量的生物标志物，提供了可能的候选，帮助我们理解疾病的病理机制，但是到目前为止依然没有任何可靠的蛋白质生物标志物可以应用到临床，用以预测疾病的发生及转归。如EP0071654通过血液中的抗体检测多发硬化。CN1307237A是通过人髓质素的免疫测定方法诊断多发性硬化。WO2007/137410则是通过检测代谢物的特异性小分子用以诊断。WO2011/10166利用了人类4号染色体上肌醇聚磷酸盐-4-磷酸酶-类型2基因(INPP4B)在RS13102150位点上的SNP来诊断或预诊多发硬化症，或检测患者多发硬化症的风险。WO2017/103633则是从外周血或脑脊液中的IL-33的表达水平来诊断多发硬化。
发明内容
本发明的目的在于提供一种预测CIS临床孤立综合征转归的方法及其组合物。
本申请采用“入组—随访—再分类”的研究策略，以CIS组(即随访结束仍未进展为MS)，MS组(包括由CIS在随访中进展为MS的患者)，正常对照组及脑血管炎组(OIND，以排除炎症导致的误差)为研究对象，通过在不同人群血液样本中的蛋白质组芯片的筛查及后续的验证，寻找可以特异预测CIS转归的差异表达蛋白质组。
本发明第一方面提供了一种预测CIS临床孤立综合征转归的组合物，其特征在于所述组合物包括如下的一个或多个检测试剂，检测CO4A，AP0B，ITIH4和/或KNTC1的试剂。
在一种实施方式中，所述检测试剂为蛋白或核酸。
进一步的，所述检测试剂为特异性抗体或特异性引物或探针。
本发明另一方面提供了一种标记物用于制备预测CIS临床孤立综合征转归试剂的用途，其特征在于所述标记物包含CO4A，AP0B，ITIH4和/或KNTC1中的一个或多个。
在一种实施方式中，所述标记物在不同CIS转归中差异表达。
进一步的，所述预测方法为，检测样本中的标记物水平，分析差异表达的标记物。
进一步的，所述差异表达为高表达或低表达。
进一步的，所述样本来源于体液，具体的可以是血液。
进一步的，所述CIS临床孤立综合征发展为多发硬化。
本发明另一方面提供了预测CIS临床孤立综合征的方法。
在一种实施方式中，所述方法还可以联合UTE序列成像，评价CIS患者脑内脱髓鞘的程度一并判断。
在一种实施方式中，所述标记物包含CO4A，AP0B，ITIH4和/或KNTC1中的一个或多个。
与现有技术相比，本发明共发现4个在CIS临床孤立综合征不同转归的差异表达蛋白，分别来自于胶原蛋白4α亚基(CO4A)、载脂蛋白B(APOB)中间α球蛋白抑制因子H4蛋白(ITIH4)、着丝点关联蛋白1(KNTC1)。根据蛋白的表达水平差异，来预测CIS临床孤立综合征。提高了CIS临床孤立综合征预测的准确性。并建立神经影像学层面疾病的预测标准，对于研判疾病转归及疾病的早期诊断和治疗奠定了基础。
附图说明
图1：iTRAQ技术流程示意图。
图2：差异表达蛋白质的生物信息学分析流程。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
实施例一不同人群样本收集及临床资料采集
首先，采用“入组—随访—再分类”的研究策略，所有患者均经过两步筛查，并最终确定患者的入组情况。以CIS组(即随访结束仍未进展为MS)，MS组(包括由CIS在随访中进展为MS的患者)，正常对照组及脑血管炎组(OIND，以排除炎症导致的误差)为研究对象，采集不同人群血液样本蛋白质组芯片的筛查及后续的验证，寻找可以特异预测CIS转归的差异表达蛋白质组，并以此为基础进行UTE序列成像研究，评价CIS患者脑内脱髓鞘的程度，从而建立神经影像学层面的预测标准。
CIS患者的纳入标准如下：(1)中国汉族人群；首次发作，未经任何药物治疗；(2)无IgG寡克隆带；(3)入组MRI表现不符合Barkhof标准49；(4)在3年的随访中依然没有向MS转化。
MS患者的纳入标准：(1)中国汉族人群；符合2010版McDonald诊断标准，临床确诊为RRMS患者；(2)排除其他神经系统疾病；(3)无其他严重系统性疾病，如高血压、糖尿病等；(4)入组时诊断为CIS患者，而在随后的3年随访中转变为RRMS的患者；(5)入组时至少有一项MRI表现满足Barkhof标准。
OIND患者的纳入标准：(1)中国汉族人群；(2)排除CIS及MS；(3)临床诊断为脑血管炎的患者。
患者的排除标准：(1)合并有类风湿性关节炎、风湿热等其他风湿免疫疾病的患者；(2)合并有恶性肿瘤的患者；(3)合并其他严重系统性疾病的患者；(4)不符合CIS/MS/OIND诊断标准的患者；(5)母亲孕期有明确用药史、毒物接触史、高温暴露史者；(6)流行病学资料、临床资料和影像资料不完整者；(7)未获得知情同意者。
正常对照的纳入排除标准：(1)对照的采集来自健康献血者，年龄、性别、籍贯和受教育程度与患者组匹配；(2)所有对照的资料经一位高年资神经科专科医生、一位影像科医生盲法评估为非CIS/MS/OIND；(3)排除明确的CIS/MS/OIND疾病家族史；(4)无脑外伤史；(5)无严重的器质性病变，目前未服用任何药物；(6)采血前1周内未吸烟、饮酒、熬夜、服用刺激性食物等。
严格按照纳入排除标准采集样本资料，所有患者或患者一级亲属签署知情同意书。最终拟收集散发CIS/MS/ONID患者及正常对照样本100例(共400例)，所有被试抽取外周血8-10ml，提取血浆蛋白质，制备工作液备用。
通过蛋白质组芯片的筛查及后续的验证，寻找可以特异预测CIS转归的差异表达蛋白质组，从而建立CIS转归的预测标准。
实施例二蛋白质表达量分析
基于iTRAQ技术(图1)寻找四组被试差异表达蛋白质，在大样本中对所得数据进行Elisa检验，并通过体外功能试验检测目的蛋白导致疾病的可能机制。同时，根据不同蛋白质含量或作用模式对被试进行分组，探索蛋白质含量或作用模式对脑内髓鞘质UTE成像的影响。
不同人群蛋白质组学研究及数据分析，在4组人群中每组选择5例表型均一的被试进行iTRAQ检测。采用iTRAQ技术进行蛋白质表达量分析：
(1)蛋白质提取：选择相应的蛋白质提取试剂提取蛋白质；
(2)蛋白质定量：取各组相同蛋白质SDS-APGE电泳，银染定量，根据每个涌道染色情况调整，使每组样本量相同；
(3)酶切，标记：按照酶：蛋白质＝1：20的比例加入trypsin酶，37℃酶解过夜，加入113,114,115,116,117,118,119,121标记试剂标记；
(4)等量混合各管标记好的样品，真空冷冻干燥；
(5)第一维强阳离子柱(SCX)分离：根据峰型和时间共收取20个梯度，真空离心浓缩后，用50μL RPLC A相[5％ACN，0.1％甲酸(TEDIA,Fairfield,USA)]溶解，进行第二维分析；
(6)第二维反相液质联用RPLC-MS：流动相A液为5％(体积分数)乙腈和0.1％甲酸溶液，流动相B液为95％乙腈和0.1％甲酸溶液；(7)质谱鉴定：MS扫描范围400-1800，MS/MS扫描范围100-2000。
实施例三差异表达蛋白质数据筛选及生物信息学分析
将上述所得数据通过ProteinPilot 3.0软件检索SwissProt数据，提取检索蛋白质的相关信息，并进行如下生物信息学分析(图2)；在数据中根据已知相关基因及其信号通路进行更有效的筛选；按照“患者共享，非患者不携带”的原则，进行筛选，尽量在所有患病个体的集合中取交集。最后进行突变危害程度的预测，以找到候选的差异表达蛋白质。
通过以上生物信息学分析，共发现4个差异表达蛋白。分别来自于胶原蛋白4α亚基(CO4A)、载脂蛋白B(APOB)中间α球蛋白抑制因子H4蛋白(ITIH4)、着丝点关联蛋白1(KNTC1)。
Claims (6)
1.一种预测CIS临床孤立综合征转归的组合物，其特征在于所述组合物包括如下的一个或多个检测试剂，检测CO4A，AP0B，ITIH4和/或KNTC1的试剂。
2.如权利要求1所述的组合物，其特征在于所述检测试剂为蛋白或核酸。
3.如权利要求1所述的组合物，其特征在于所述检测试剂为特异性抗体或特异性引物或探针。
4.一种标记物用于制备预测CIS临床孤立综合征转归试剂的用途，其特征在于所述标记物包含CO4A，AP0B，ITIH4和/或KNTC1中的一个或多个。
5.如权利要求4所述的用途，其特征在于所述标记物在不同CIS转归中差异表达。
6.如权利要求4所述的用途，其特征在于所述差异表达为高表达或低表达。
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