CN113436675B - 基于血液滤过方式清除体外循环突触核蛋白的方法 - Google Patents
基于血液滤过方式清除体外循环突触核蛋白的方法 Download PDFInfo
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Abstract
本发明公开了基于血液滤过方式清除体外循环突触核蛋白的方法，属于医学领域。具体公开：获取血液中突触核蛋白二聚体的比例S；根据大量的临床试验数据或者文献历史数据，获取血液滤过的血流速度和交换膜面积；根据上述确定的血流速度和交换膜面积进行血液滤过，采用线性回归法构建不同突触核蛋白二聚体的比例S与血液滤过透析膜孔径数值D的计算模型；利用上述计算模型设置血液滤过参数，即能预期体外循环突触核蛋白清除率。本发明首次发现了血液滤过有助于降低体外循环α‑突触核蛋白水平，因此开发一种基于α‑突触核蛋白二聚体比例构建设置血液滤过参数的计算模型，以为临床医学上缓解α‑突触核蛋白相关毒性症状提供了科学数据和新方案。
Description
技术领域
本发明涉及医学领域，特别是涉及一种基于血液滤过方式清除体外循环突触核蛋白的方法。
背景技术
α-突触核蛋白清除障碍可导致帕金森病(PD)及多系统萎缩(MSA)等α-突触核蛋白谱氏病发生发展，导致机体免疫失调和神经炎症反应。血液净化术可有效清除血液中毒性物质，如炎症因子、自身抗体和毒性蛋白分子等；例如已报道血浆或腹膜透析术可有效清除机体的Aβ毒性蛋白和炎症因子。PD及MSA等α-突触核蛋白谱氏病与AD存在相似的发病机制，缘于类似Aβ样扩散性质相似的α-突触核致病蛋白清除障碍，导致神经免疫失调和炎症反应。目前尚无有效清除α-突触核蛋白抑制免疫炎症的方法。
血液滤过技术是指在血液净化过程中不使用透析液，而是在血管通路中持续补充一定量的置换液，与血液充分混合，再以相同的速度进行超滤，以达到清除体内过多的水和毒素的目的。与血液透析相比，血液滤过具有对血流动力学影响小、中分子物质清除率高等优点。α-突触核蛋白在不同的影响因素下会表现出许多种形态，包括舒展态、溶解前球型态、α-螺旋态、β-片层态、二聚体态、寡聚体态、以及不可溶的无定型态和纤维态等。不同形态的α-突触核蛋白具有不同分子量，而二聚体、寡聚体态下α-突触核蛋白的毒性作用尤为显著。上述毒性α-突触核蛋白的分子量刚好在血液滤过适应范围内，根据个体情况调整血液滤过方式可有助于清除外周循环毒性α-突触核蛋白，起到改善α-突触核蛋白相关毒性症状的作用。目前血液滤过多根据需滤过物质的分子量设定滤过参数，而α-突触核蛋白具有二聚体的结构，使得每个患者的α-突触核蛋白谱系的分子量分布不同，目前尚无针对蛋白二聚体或多聚体的血液滤过参数设置参考研究。
发明内容
本发明的目的是提供一种基于血液滤过方式清除体外循环突触核蛋白的方法，以解决上述现有技术存在的问题，通过设计一种清除体外循环突触核蛋白的计算模型，可以根据突触核蛋白二聚体含量的不同，设计不同的血液滤过参数，进而能预期清除突触核蛋白的效率，这为临床医学上缓解α-突触核蛋白相关毒性症状提供了科学数据和新方案。
为实现上述目的，本发明提供了如下方案：
本发明提供一种基于血液滤过方式构建体外循环突触核蛋白清除率计算模型的方法，包括以下步骤：
获取血液中突触核蛋白二聚体的比例S；
根据大量的临床试验数据或者文献历史数据，获取血液滤过的血流速度和交换膜面积；
根据上述确定的血流速度和交换膜面积进行血液滤过，采用线性回归法分析不同突触核蛋白二聚体的比例S与血液滤过交换膜孔径数值D的相关性，构建S与D两者间的计算模型；
采用所述突触核蛋白二聚体的比例S和所述计算模型确定的交换膜孔径进行血液滤过，即能预期体外循环突触核蛋白清除率。
优选的是，所述计算模型的公式为：
D＝5.44S+1.7376，R2＝0.7671；
其中，D为交换膜孔径，S为突触核蛋白二聚体比例，R2为相关系数。
优选的是，通过酶联免疫吸附剂测定方法、生物学活性测定方法、酶联免疫斑点检测以及其他分子生物学测定方法获取血液中突触核蛋白二聚体的比例S。
本发明还提供所述的方法在清除体外循环突触核蛋白中的应用。
优选的是，应用于α-突触核蛋白谱氏病中突触核蛋白的清除。
本发明还提供一种基于血液滤过方式清除体外循环突触核蛋白的方法，包括以下步骤：
获取血液中突触核蛋白二聚体的比例S；
通过所述的计算模型，获取血液滤过所需的交换膜孔径D；
根据所述交换膜孔径选择透析膜进行血液滤过，之后再检测血液中突触核蛋白水平，以评估体外循环突触核蛋白的清除情况。
进一步地，所述血液滤过方式包括交换膜孔径、血流速度和交换膜的面积等参数，参数设定根据个体α-突触核蛋白二聚体的比例。
进一步地，个体携带α-突触核蛋白二聚体的比例的测定方法包括使用ELISA、生物学活性测定方法、酶联免疫斑点试验及其他分子生物学测定方法。
进一步地，所述α-突触核蛋白的特异性抗体包括单克隆抗体、多克隆抗体。所述α-突触核蛋白的特异性抗体包括完整的抗体分子、抗体的任何片段或修饰片段等。只要所述片段能够保留与α-突触核蛋白的结合能力即可。用于蛋白质水平的抗体的制备时本领域技术人员公知的，并且本发明可以使用任何方法来制备所述抗体。
在本发明的上下文中，α-突触核蛋白基因表达产物包括人α-突触核蛋白以及人α-突触核蛋白的部分肽。所述α-突触核蛋白的部分肽含有与α-突触核蛋白相关疾病的功能域。
“α-突触核蛋白”包括人α-突触核蛋白以及人α-突触核蛋白的任何功能等同物。所述功能等同物包括人α-突触核蛋白保守性变异蛋白质、或其活性片段，或其活性衍生物，等位变异体、天然突变体、诱导突变体、在高或低的严格条件下能与人α-突触核蛋白的DNA杂交的DNA所编码的蛋白质。
α-突触核蛋白是具有下列氨基酸序列的蛋白质：
(1)由如下SEQ ID NO.1所示的氨基酸序列组成的蛋白质；
MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTKEQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSEEGYQDYEPEA
(2)将SEQ ID NO.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与SEQ ID NO.1所示的氨基酸序列具有相同功能的由SEQ ID NO.1所示的氨基酸序列衍生的蛋白质。取代、缺失或者添加的氨基酸的个数通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个。
(3)与SEQ ID NO.1所示的氨基酸序列具有至少80％同源性(又称为序列同一性)，更优选地，与SEQ ID NO.1所示的氨基酸序列至少约90％至95％的同源性，常为96％、97％、98％、99％同源性的氨基酸序列构成的多肽。
在本发明的具体实施方案中，所述α-突触核蛋白是具有SEQ ID NO.1所示的氨基酸序列的蛋白质。
通常，已知的是，一个蛋白质中一个或多个氨基酸的修饰不会影响蛋白质的功能。本领域技术人员会认可改变单个氨基酸或小百分比的氨基酸或对氨基酸序列的个别添加、缺失、插入、替换是保守修饰，其中蛋白质的改变产生具有相似功能的蛋白质。提供功能相似的氨基酸的保守替换表是本领域公知的。
通过添加一个氨基酸或多个氨基酸残基修饰的蛋白质的例子是α-突触核蛋白的融合蛋白。对于与α-突触核蛋白融合的肽或者蛋白质没有限制，只要所得的融合蛋白保留α-突触核蛋白的生物学活性即可。
本发明的α-突触核蛋白也包括对SEQ ID NO.1所示的氨基酸序列的非保守修饰，只要经过修饰的蛋白质仍然能够保留α-突触核蛋白的生物学活性即可。在此类修饰蛋白质中突变的氨基酸数目通常是10个或者更少，例如6个或者更少，例如3个或者更少。
在本发明的上下文中，“α-突触核蛋白相关毒性症状”既包括判断受试者是否已经患有突触核蛋白相关毒性症状、也包括判断受试者是否存在患有突触核蛋白相关毒性症状的风险。
在本发明的上下文中，“缓解α-突触核蛋白相关毒性症状”从症状的状态变化来分，可以包括症状的缓解、疾病的完全治愈。
由于α-突触核蛋白被认为是PD的致病物质。α-突触核蛋白自身聚合能力强，形成比α-突触核蛋白单体毒性更强的寡聚体和纤维。因此，判断一种方法是否具有改善α-突触核蛋白毒性的功能就可以通过检测其是否具有抑制和清除体外α-突触核蛋白的水平以及具有阻断α-突触核蛋白的毒性的作用来判断。
在本发明的具体实施方式中，提取的是外周血中的总蛋白来进行α-突触核蛋白水平检测的研究。本领域技术人员可知，脑组织中的细胞表达α-突触核蛋白会脱落到外周血中进行循环，即可溶性α-突触核蛋白，因此血液中α-突触核蛋白水平可间接反应脑组织中α-突触核蛋白基因的表达情况。根据本发明的研究成果可知，通过提取体液尤其是外周血可检测α-突触核蛋白水的水平和构成均可用于判断受试者是否出现α-突触核蛋白相关毒性症状。
本发明公开了以下技术效果：
本发明首次发现了血液滤过有助于降低体外循环α-突触核蛋白水平，因此，基于临床经验确定血流速度和交换膜面积的前提下，研究了α-突触核蛋白与交换膜孔径的相关性，并构建α-突触核蛋白与交换膜孔径的计算模型，基于此，即能实现体外循环α-突触核蛋白清除率20±2％的目的。可见，本发明开发一种经校正的血液滤过方法清除外周血中α-突触核蛋白水平的方法，这对于缓解α-突触核蛋白相关的毒性症状、或者减少罹患α-突触核蛋白相关疾病的风险具有重要意义，因此，本发明弥补既往的毒性α-突触核蛋白清除方法的空白，该技术根据个体情况调整血液滤过方式，具有及时性、特异性和有效性清除外周循环毒性α-突触核蛋白，起到改善α-突触核蛋白相关毒性症状的作用，可为临床应用提供了科学依据和新方案。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为透析前后帕金森病受试者的UPDRS和HY分级评分结果；
图2为透析前后帕金森病受试者的HAMA、HAMD、MMSE、MoCA、非运动量表以及日常生活能力问卷评分结果；
图3为交换膜孔径与突触核蛋白比例分布图。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
实施例1
1、体外验证血液滤过可以降低体外循环α-突触核蛋白水平
采用费森尤斯医疗器械有限公司生产的HF-80S高通量透析器构建体外血液滤过模型，根据实验要求设置不同的滤过速度、滤过面积和交换膜孔径。采用新鲜牛全血3L，将红细胞压积调整为(31±0.5)％，于(36.7±0.5)℃水浴恒温存储；根据实验要求加入不同的α-突触核蛋白二聚体组合，模拟患者人体循环血液。将模拟患者血液经过体外血液滤过模型，收集和测试滤过前后突触核蛋白水平，计算清除效率。
2、通过上述试验测定了不同比例α-突触核蛋白二聚体存在情况，透析速度、透析膜面积以及α-突触核蛋白二聚体清除效率之间存在如下的关系，见表1。
表1
由表1可以看出，不同比例α-突触核蛋白二聚体，设置不同的透析速度和透析膜面积，其清除效率差别不大，都在20％左右，也就是说，透析速度和透析膜面积并非是影响α-突触核蛋白二聚体清除效率的关键因素。
因此，为达到α-突触核蛋白清除效率为20±2％的目的，在透析液流速设置为200ml/min和滤过面积为1.35m2下，研究不同α-突触核蛋白二聚体比例，采用相应交换膜的孔径数值分布情况，如图3所示。
由图3可以易得，α-突触核蛋白比例与交换膜的孔径相关性的计算模型公式为：D＝5.44S+1.7376，R2＝0.7671；其中，D为交换膜孔径，S为突触核蛋白二聚体比例，R2为相关系数。
实施例2
临床上测定α-突触核蛋白的方法
1、外周血和样本的收集
PD患者来自广州医科大学附属第一医院，共50例，年龄57-66岁，所有病例被诊断为PD，其诊断标准参照帕金森病的UK脑库临床诊断标准。对照人群共40例，选自广州医科大学附属第一医院常规体检人群，所有入试者均排除血脂代谢、神经系统慢性退行性疾病等疾病，年龄55-68岁。所有研究对象均签署了对该检测项目的知情同意书，进行UPDRS评分，并且提供了外周血用于α-突触核蛋白的检测。
2、构建测定α-突触核蛋白的检测试剂盒
(1)为了定量测定α-突触核蛋白的含量，用两种不同对人突触核蛋白蛋白的单克隆抗α-突触核蛋白抗体，包括使用抗α-突触核蛋白单克隆抗体作为捕获抗体，和生物素化小鼠抗人α-突触核蛋白单克隆抗体作为检测抗体。
(2)在96孔检测板上捕获抗体以4.0μg/ml的最终浓度在PBS缓冲液中室温包埋过夜。
(3)用Wash Buffer(含0.05％Tween20的PBS缓冲溶液,PH＝7.2)清洗3遍后，在25℃条件下，用Reagent Diluents(1％BSA in PBS)将检测板封闭1h。
3、血清和可溶性α-突触核蛋白的测定
(1)取全血250μl(或0.25g)至RNase-Free过滤柱中，13000rpm离心2分钟，收集上清液。
(2)所有试验中均使用可溶性人TRME2-FC和人a-突触核蛋白-FC作为标准品。
(3)在包被待测板中加入100μl新鲜复温好的血清和标准品室温在孵育2h，用WashBuffer清洗后加入至100μl终浓度为可溶性α-突触核蛋白的检测抗体(终浓度：35.0ng/ml)和a-突触核蛋白检测抗体(终浓度：25.0ng/ml)室温孵育2h。
(4)用Wash Buffer清洗后，加入100μl辣根过氧化物酶标记的链酶亲和素在室温下避光孵育20min。
(5)用Wash Buffer清洗后，加入100μl显色剂(R&D Catalog#DY999)在室温下避光孵育20min。
(6)通过添加50μl 2N硫酸(R&D Catalog#DY994)来中止颜色的发展。
(7)在450纳米处测定测定每孔的OD值,同时采用540纳米处测定的OD值来校正。
4、统计学方法
实验都是按照重复3次来完成的，结果数据都是以平均值±标准差的方式来表示，采用SPSS21.0统计软件来进行统计分析的，两者之间的差异采用t检验，认为当P<0.05时具有统计学意义。
实施例3
基于血液滤过方式构建体外循环突触核蛋白清除率计算模型的方法，其具体应用如下：
1、具体病例
患者女，81岁，已婚，因震颤13年入院，13年前患者发现左上肢震颤，震颤多在休息时出现，运动、紧张时可消失，无明显加重或缓解因素，中山大学附属第一医院就诊，予以“美多芭0.125g BID+辅酶Q10 10mg QD”，震颤无进一步加重。3年前，症状加重，夜间难以入睡，需口服“氯硝安定2mg”方可入睡。2年前右上肢亦出现震颤，震颤为静止性，频率同左侧，震颤幅度较左侧稍轻，并出现便秘，大便3-4日/次，干结难解，有时需使用石蜡油或开塞露方可解出，遂调整口服药为“美多芭0.25g TID+森福罗0.25mg TID+珂丹0.2g TID”，1年前出现行走不稳，表现为起步困难，行走过程中步幅越来越小，转弯时易跌倒，并出现下颌震颤，震颤在静息时明显，出现颈部活动费力、食欲减退。扣纽扣、穿拖鞋等动作较不灵活，有忧郁情绪，加用“倍爱欣1粒QD”，症状无明显改善，1周前感觉乏力加重，并自觉出现呼吸困难，吸氧后无好转，多在下午3时出现，到夜间8-9时可自行消失。起病以来精神可，睡眠、大便差(同上述)，小便可。入院查体示：体温36.5℃，脉搏78次/分，呼吸20次/分，血压117/60mmHg。心胸腹无特殊。神志清晰，言语较含糊，对答切题，查体合作，面具脸，慌张步态。短时记忆减退，定向力、视空间功能大致正常，执行力下降。嗅觉减退，粗侧视野无缺损，眼球运动灵活，双侧瞳孔等大等圆，d＝3mm，对光反射、辐辏反射灵敏。粗侧双耳听力下降，weber试验居中，AC＞BC。下颌震颤。余颅神经查体未见异常。颈部肌张力3级，呈齿轮样增高，颏胸距2横指，左侧轮替试验较不灵活。行走时左手摆幅较右侧小。后拉试验(+)。双上肢肌张力呈齿轮样增高，肌力5级，腱反射+++。双下肢肌张力正常，肌力5-级，腱反射+。左下肢直腿抬高试验(+)。浅感觉、深感觉、皮层复合觉未见明显异常。病理征未引出。脑膜刺激征阴性。入院诊断：帕金森病；病态窦性综合征，植入心脏起搏器；冠状动脉粥样硬化性心脏病。
完善检查和签署知情同意：患者为帕金森中晚期，服药后病情无明显好转。近期症状加重，行走不稳、下颌震颤，并出现下午至晚间的呼吸困难、胸闷感。考虑帕金森病为α-突触核蛋白谱氏病，存在机体免疫失调和神经炎症反应血液净化术可有效清除血浆中的毒性物质，如炎性因子、自身抗体及毒性蛋白分子；例如已报道血浆或腹膜透析术可有效清除Aβ毒性蛋白和炎症因子。帕金森病和阿尔兹海默病存在相似的发病机制，缘于类似Aβ样扩散性质相似的α-突触核致病蛋白的清除障碍，导致神经免疫失调和炎症反应。目前在帕金森病临床治疗中尚无有效清除免疫炎症的方法，血液净化技术可能有助于清除外周神经免疫炎症分子，并在帕金森病临床防治中起到重要作用。尽管患者年龄偏大，但能够耐受α-突触核蛋白谱氏病外周血毒性蛋白及免疫炎症因子血液净化术临床试验，向患者及家属(儿媳)告知血液净化治疗中存在的风险及治疗转归不一定能达到患者及家属的预期，患者及家属经过慎重考虑后，表示知情理解并签字为证。拟下周行血液净化术。
2、实施方案
利用实施例2的方法获取受试者血液中α-突触核蛋白的比例，结果显示受试者脑脊液突触核蛋白二聚体占比在0.035左右，并根据本发明的计算模型公式，得到交换膜孔径为1.928nm。之后制定治疗方案。
现行血液净化治疗：选择血液灌流参数如下，交换膜孔径为2nm，血流速度200ml/min，交换膜面积为1.35m2；行血液灌流治疗2小时每次，共3次。
治疗前后患者症状改变：患者震颤、行动迟缓较入院时好转。查体:神志清晰，言语较含糊，对答切题，查体合作，面具脸，慌张步态。短时记忆减退，定向力、视空间功能大致正常，执行力下降。嗅觉减退，粗侧视野无缺损，眼球运动灵活，瞳孔等大等圆，d＝3mm，对光反射、辐辏反射灵敏。粗侧双耳听力下降，weber试验居中，AC＞BC。下颌震颤。余颅神经查体未见异常。颈部肌张力2级，呈齿轮样增高，颏胸距2横指，左侧轮替试验较不灵活。行走时左手摆幅较右侧小。后拉试验(-)。双上肢肌张力齿轮样增高，肌力5级，腱反射+++。双下肢肌张力正常，肌力5级，腱反射+。左下肢直腿抬高试验(+)。浅感觉、深感觉、皮层复合觉未见明显异常。病理征未引出。脑膜刺激征阴性。
治疗后复测脑脊液突触核蛋白浓度，清除效率约为0.232。同时，还对比了透析前后帕金森病患者症状的的多项评分进行的比对，发现透析后帕金森病受试者症状得到明显的改善，见图1和图2。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
序列表
<110> 广州医科大学附属第一医院（广州呼吸中心） 江西省乐云健康科技有限公司
<120> 基于血液滤过方式清除体外循环突触核蛋白的方法
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Claims (5)
1.一种基于血液滤过方式构建体外循环突触核蛋白清除率计算模型的方法，其特征在于，包括以下步骤：
获取血液中突触核蛋白二聚体的比例S；
根据大量的临床试验数据或者文献历史数据，获取血液滤过的血流速度和交换膜面积；
根据上述确定的血流速度和交换膜面积进行血液滤过，采用线性回归法分析不同突触核蛋白二聚体的比例S与血液滤过透析膜孔径数值D的相关性，构建S与D两者间的计算模型；
采用所述突触核蛋白二聚体的比例S和所述计算模型确定的透析膜孔径进行血液滤过，即能预期体外循环突触核蛋白清除率；
所述计算模型的公式为：
D＝5.44S+1.7376，R2＝0.7671；
其中，D为透析膜孔径，S为突触核蛋白二聚体比例，R2为相关系数。
2.如权利要求1所述的方法，其特征在于，通过酶联免疫吸附剂测定方法、生物学活性测定方法或酶联免疫斑点检测方法获取血液中突触核蛋白二聚体的比例S。
3.如权利要求1所述的方法在清除体外循环突触核蛋白中的应用。
4.如权利要求3所述的应用，其特征在于，应用于α-突触核蛋白谱氏病中突触核蛋白的清除。
5.一种基于血液滤过方式清除体外循环突触核蛋白的方法，其特征在于，包括以下步骤：
获取血液中突触核蛋白二聚体的比例S；
通过权利要求1中所述的计算模型，获取血液滤过所需的交换膜孔径D；
根据所述交换膜孔径选择透析膜进行血液滤过，之后再检测血液中突触核蛋白水平，以评估体外循环突触核蛋白的清除情况。
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| CN102099809A (zh) * | 2008-06-20 | 2011-06-15 | 诺华公司 | 识别蛋白质中大分子结合区域和易聚集区域的方法及其用途 |
| DE102010032081A1 (de) * | 2010-07-23 | 2012-01-26 | Siemens Aktiengesellschaft | Nachweis lebender, zirkulierender oder disseminierter Zellen bzw. Zellbestandteile in Blut oder Knochenmark nach Filtration von Blut |
| CN103983778A (zh) * | 2014-05-08 | 2014-08-13 | 首都医科大学宣武医院 | 一种检测受实验者体液体外促进疾病相关蛋白聚合能力的方法 |
| CN109273050A (zh) * | 2018-08-03 | 2019-01-25 | 五邑大学 | 一种用于解析复杂多聚体蛋白受体的脉冲电子顺磁共振数据的系统及方法与应用 |
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| CN102099809A (zh) * | 2008-06-20 | 2011-06-15 | 诺华公司 | 识别蛋白质中大分子结合区域和易聚集区域的方法及其用途 |
| DE102010032081A1 (de) * | 2010-07-23 | 2012-01-26 | Siemens Aktiengesellschaft | Nachweis lebender, zirkulierender oder disseminierter Zellen bzw. Zellbestandteile in Blut oder Knochenmark nach Filtration von Blut |
| CN103983778A (zh) * | 2014-05-08 | 2014-08-13 | 首都医科大学宣武医院 | 一种检测受实验者体液体外促进疾病相关蛋白聚合能力的方法 |
| CN109273050A (zh) * | 2018-08-03 | 2019-01-25 | 五邑大学 | 一种用于解析复杂多聚体蛋白受体的脉冲电子顺磁共振数据的系统及方法与应用 |
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