WO2020125530A1 - D-丝氨酸作为标志物在制备抑郁症诊断试剂盒中的应用及抑郁症诊断试剂盒 - Google Patents

D-丝氨酸作为标志物在制备抑郁症诊断试剂盒中的应用及抑郁症诊断试剂盒 Download PDF

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WO2020125530A1
WO2020125530A1 PCT/CN2019/124721 CN2019124721W WO2020125530A1 WO 2020125530 A1 WO2020125530 A1 WO 2020125530A1 CN 2019124721 W CN2019124721 W CN 2019124721W WO 2020125530 A1 WO2020125530 A1 WO 2020125530A1
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depression
serine
diagnosis
diagnostic kit
subject
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屠洁
周辛夷
肖倩
谢丽
鲁艺
杨帆
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Shenzhen Institute of Advanced Technology of CAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/304—Mood disorders, e.g. bipolar, depression

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  • the invention relates to the technical field of diagnosis of depression, in particular to the application of D-serine as a marker in the preparation of a diagnostic kit for depression and a diagnostic kit for depression.
  • depression is a negative emotion. If the duration is too long, it will form a pathological mental illness, such as depression.
  • Depression is a mental illness caused by a variety of reasons and the main symptom is persistent depression. Its clinical manifestations are diverse, often accompanied by anxiety, lack of pleasure and social disorders. Depression is a common disease in psychiatry, and the World Health Organization predicts that by 2020, depression will become the second most common disease.
  • the first object of the present invention is to provide D-serine as a marker in the preparation of a diagnostic kit for depression, so as to alleviate the technical problems in the prior art.
  • the second object of the present invention is to provide a diagnostic kit for depression, which detects whether or not depression is detected by detecting the content of D-serine, and provides a new strategy for the diagnosis of depression.
  • the third object of the present invention is to provide the use of D-serine in the preparation of a medicament for treating depression.
  • the invention provides the application of D-serine as a marker in the preparation of a diagnostic kit for depression.
  • the detection sample of the depression diagnosis kit is cerebrospinal fluid.
  • the depression diagnosis kit monitors the concentration of D-serine in the cerebrospinal fluid of the subject to determine whether the subject has depression.
  • the concentration of D-serine in the cerebrospinal fluid of the subject is lower than the normal value of 51%, the subject is judged to have depression.
  • the depression diagnosis kit monitors the concentration of D-serine in the cerebrospinal fluid of the subject to determine the severity of depression in the subject.
  • the depression is moderate depression and above.
  • the present invention also provides a diagnostic kit for depression, which monitors the concentration of D-serine in the cerebrospinal fluid of the subject to determine whether the subject has depression.
  • the kit includes a D-serine assay buffer, a probe, a D-serine enzyme mixture, a developer enzyme mixture, and a D-serine standard.
  • the concentration of D-serine in the cerebrospinal fluid of the subject is detected by detecting the fluorophore formed by the reaction of the oxidized intermediate product of D-serine metabolism with the probe.
  • the invention also provides the application of D-serine in the preparation of a medicament for treating depression.
  • the present invention finds that D-serine is closely related to depression by studying metabolic substances in abnormal lesions in the brain tissue of patients with depression. Therefore, it is proposed for the first time to apply D-serine as a marker to the field related to the preparation of a diagnostic kit for depression To overcome the shortcomings of the unobjective diagnosis of the indicators used in the diagnosis of depression, and thus provide a new and more accurate objective diagnosis standard for the diagnosis of later depression, and further provide a diagnosis of depression A new way of thinking.
  • the depression diagnosis kit provided by the invention can quickly and accurately detect the concentration of D-serine in the cerebrospinal fluid of the subject, thereby effectively and accurately determining whether the subject has depression and the severity of depression.
  • D-serine provided in the present invention in the preparation of a medicament for treating depression can increase the excitability of neurons in related brain regions through the release of D-serine, thereby improving depression-like behavior.
  • FIG. 1A is a graph showing the results of successful immunohistochemistry of the transgenic animal provided in Example 1 of the present invention.
  • 1B is a graph showing the results of the time spent by mice in each group provided in Example 1 of the present invention.
  • 1C is a graph showing the interaction time between each group of mice and strange mice provided in Example 1 of the present invention.
  • 1D is a graph showing the results of the immobility incubation period of the mice in each group provided in Example 1 of the present invention.
  • Example 2 is a graph showing the ability of mouse glial cells to secrete D-serine in each group provided in Example 2 of the present invention.
  • the numerical range “a to b” represents an abbreviated representation of any combination of real numbers between a and b, where a and b are both real numbers.
  • the numerical range “6-22” means that all real numbers between “6-22” have been listed in this article, and "6-22" is just an abbreviated representation of these numerical combinations.
  • the form of the lower limit and the upper limit of the "range" disclosed in the present invention may be one or more lower limits and one or more upper limits, respectively.
  • each reaction or operation step may be performed sequentially or in order.
  • the reaction methods herein are performed sequentially.
  • Depression is also known as depressive disorder, with a significant and persistent mood depression as its main clinical feature, and it is the main type of mood disorder. Clinically, it can be seen that the depressed mood is not commensurate with their situation.
  • the depression of emotions can range from depression to grief, inferiority and depression, even pessimism, and suicide attempts or behaviors; even stiffness; some cases have obvious anxiety and motor agitation; In severe cases, mental symptoms such as hallucinations and delusions may occur. Therefore, objective and accurate diagnosis of depression is the first condition for clinical and psychological treatment.
  • D-serine is an endogenous brain information co-transmission substance. Its molecular formula is C3H7NO3, molecular weight is 105.09, and its chemical structural formula is:
  • the present invention takes depression model mice as an object to study the relationship between the D-serine content in cerebrospinal fluid of normal mice and model mice and depression, and the results show that the level of D-serine in the depression model mouse group is relatively high. The normal group declined.
  • the present invention provides the use of D-serine as a marker in the preparation of a diagnostic kit for depression.
  • the present invention finds that D-serine is closely related to depression by studying metabolic substances in abnormal lesions in the brain tissue of patients with depression. Therefore, it is proposed for the first time to apply D-serine as a marker to the field related to the preparation of a diagnostic kit for depression To overcome the shortcomings of the unobjective diagnosis of the indicators used in the diagnosis of depression, and thus provide a new and more accurate objective diagnosis standard for the diagnosis of later depression, and further provide a diagnosis of depression A new way of thinking.
  • test sample of the depression diagnosis kit is cerebrospinal fluid.
  • Cerebrospinal fluid is a colorless and transparent liquid produced by the choroid plexus in the ventricle. It circulates on the surface of the brain and spinal cord, and is related to the systemic circulation through the internal cerebral venous system.
  • the main functions are: 1 to protect the brain and spinal cord from external shock damage; 2 to regulate changes in intracranial pressure; 3 to supply nutrients to the brain and spinal cord and to transport away metabolites; 4 to regulate the alkali reserve of the nervous system and maintain normal PH value.
  • cerebrospinal fluid will also undergo various changes.
  • the present invention is based on the current cerebrospinal fluid is generally used for the examination of nervous system diseases, creatively using cerebrospinal fluid as a detection sample of mental illness depression, can accurately and objectively diagnose depression.
  • the depression diagnosis kit determines whether the subject has depression by monitoring the concentration of D-serine in the cerebrospinal fluid of the subject.
  • the subject when the concentration of D-serine in the cerebrospinal fluid of the subject is lower than 51% of the normal value, the subject is judged to have depression.
  • the concentration of D-serine markers in the cerebrospinal fluid detected by the diagnostic kit for depression is lower than the normal value of 51%, and it is reasonable to make a more objective diagnosis of depression.
  • the normal concentration of D-serine in a typical cerebrospinal fluid is the concentration of D-serine in the cerebrospinal fluid of a non-depressed population measured by conventional measurement methods.
  • the depression diagnosis kit monitors the concentration of D-serine in the cerebrospinal fluid of the subject to determine the severity of depression in the subject.
  • the depression is moderate depression and above.
  • Taking the value of the concentration of D-serine in the cerebrospinal fluid of the subject as an indicator of the severity of depression can better reflect the objectivity of the diagnosis or help to improve the accuracy of the diagnosis.
  • D-serine as a marker can not only be applied in the preparation of a diagnostic kit for depression. It can also be used in the field of evaluating the efficacy of depression, for evaluating the therapeutic effect of related depression drugs. Therefore, the application of D-serine as a marker also belongs to the protection scope of the present invention.
  • the present invention also provides a diagnostic kit for depression, which monitors the concentration of D-serine in the cerebrospinal fluid of the subject to determine whether the subject has depression.
  • the depression diagnosis kit provided by the invention can quickly and accurately detect the concentration of D-serine in the cerebrospinal fluid of the subject, thereby effectively and accurately determining whether the subject has depression and the severity of depression.
  • the kit includes D-serine assay buffer, probe, D-serine enzyme mixture, developer enzyme mixture, and D-serine standard.
  • the concentration of D-serine in the cerebrospinal fluid of the subject is detected by detecting the fluorophore formed by the reaction of the oxidized intermediate product of D-serine metabolism with the probe.
  • the present invention proposes the use of D-serine as a marker in the diagnosis or evaluation of therapeutic effects of depression, for example, the use of D-serine as a marker in the preparation of a kit for diagnosis or evaluation of depression, which is easy to understand and corresponding Relevant reagents or antibodies that can detect the concentration or level of D-serine can be used to prepare a diagnostic kit for depression diagnosis or curative effect evaluation, which also belongs to the protection scope of the present invention.
  • the present invention first proposes the application of D-serine as a marker in the preparation of a kit for diagnosis or evaluation of depression. Therefore, as long as it is based on this application, all kits used for the diagnosis or evaluation of the efficacy of depression, as long as it uses D-serine as a marker to detect the level of D-serine in the sample, is within the protection scope of the present invention.
  • the invention also provides the application of D-serine in the preparation of a medicament for treating depression.
  • the release of D-serine can increase the excitability of neurons in related brain regions, thereby improving depression-like behavior.
  • D-serine is closely related to depression by studying metabolic substances of abnormal lesions in the brain tissue of patients with depression. Therefore, it is proposed for the first time to apply D-serine as a marker to the preparation of a diagnostic kit for depression In the field of depression, overcoming the shortcomings of the unobjective diagnosis of the indicators used in the diagnosis process of depression, and thus providing a new and more accurate objective diagnosis standard for the diagnosis of later depression, and further diagnosis of depression Provides a new idea.
  • the experimental animals, drugs and reagents used in the embodiments of the present invention are all from regular and easy-to-purchase channels:
  • D-serine assay buffer, probe, D-serine enzyme mixture, developer enzyme mixture and D-serine standard products were purchased from Kasma Mall; tamoxifen was purchased from sigma; mice were purchased from Beijing Vital Lihua Laboratory Animal Technology Limited company; microplate reader was purchased from biotek.
  • this embodiment establishes a rodent depression animal model that can be induced in adulthood.
  • tamoxifen-induced transgenic animals start to move earlier than other mice, indicating their depression-like behavior.
  • the D-serine level was detected by the following method:

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Abstract

一种D-丝氨酸作为标志物在制备抑郁症诊断试剂盒中的应用及抑郁症诊断试剂盒，涉及抑郁症诊断技术领域，通过研究抑郁症患者大脑组织中异常病变的代谢物质，研究结果发现D-丝氨酸与抑郁症密切相关，因此首次提出将D-丝氨酸作为标志物应用到制备抑郁症诊断试剂盒相关的领域中，克服目前在抑郁症诊断过程中的所用指标存在的诊断不客观的缺陷，进而为后期抑郁症的诊断提供了一种全新的更加准确的客观诊断的标准，进一步为抑郁症的诊断提供了一种新的思路。抑郁症诊断试剂盒，能够快速准确的检测出受试者脑脊液中D-丝氨酸的浓度，从而有效准确地判断受试者是否患有抑郁症及抑郁症的严重程度。

Description

D-丝氨酸作为标志物在制备抑郁症诊断试剂盒中的应用及抑郁症诊断试剂盒 技术领域
本发明涉及抑郁症诊断技术领域，尤其是涉及一种D-丝氨酸作为标志物在制备抑郁症诊断试剂盒中的应用及抑郁症诊断试剂盒。
背景技术
人的抑郁情绪属于负性情绪，持续时间过长会形成病理性精神疾病，如抑郁症。抑郁症是一种由多种原因导致的以持续情绪低落为主要症状的精神疾病，其临床表现为多样，往往伴随焦虑、快感缺失和社交障碍等。抑郁症是精神科常见疾病，世界卫生组织预计，到2020年，抑郁症将会成为第二大常见疾病。
抑制症发病机制复杂，涉及多系统、多环节的功能失调，可表现为多种生化物质的动态平衡异常。但令人遗憾的是，抑郁症的病因至今尚不完全清楚，并且，目前临床上对抑郁症的诊断多数依赖于各种量表进行的评分法，如比较常用的焦虑自评量表(Self-rating anxiety scale,SAS)或抑郁自评量表(Self-rating depression scale,SDS)。而不管哪种形式的量表，都属于主观诊断范畴，缺乏客观的依据和标准。目前，尚缺乏与抑郁症诊断相关的生物标志物等客观指标。
有鉴于此，特提出本发明。
发明内容
本发明的第一个目的在于提供D-丝氨酸作为标志物在制备抑郁症诊断试剂盒中的应用，以缓解现有技术中存在的技术问题。
本发明的第二个目的在于提供一种抑郁症诊断试剂盒，该试剂盒通过检测D-丝氨酸的含量来诊断是否患有抑郁症，为抑郁症的诊断提供了一个新的策略。
本发明的第三个目的在于提供D-丝氨酸在制备治疗抑郁症的药物中的应 用。
本发明提供了D-丝氨酸作为标志物在制备抑郁症诊断试剂盒中的应用。
进一步地，所述抑郁症诊断试剂盒的检测样本为脑脊液。
进一步地，所述抑郁症诊断试剂盒通过监测受试者脑脊液中D-丝氨酸的浓度来判断受试者是否患有抑郁症。
进一步地，当受试者脑脊液中D-丝氨酸的浓度低于正常值51％时，判断所述受试者患有抑郁症。
进一步地，所述抑郁症诊断试剂盒通过监测受试者脑脊液中D-丝氨酸的浓度来判断受试者患有抑郁症的严重程度。
进一步地，所述抑郁症为中度抑郁症及中度以上的抑郁症。
另外，本发明还提供了一种抑郁症诊断试剂盒，所述抑郁症诊断试剂盒通过监测受试者脑脊液中D-丝氨酸的浓度来判断受试者是否患有抑郁症。
进一步地，所述试剂盒包括D-丝氨酸测定缓冲液、探针、D-丝氨酸酶混合物、显影剂酶混合物和D-丝氨酸标准品。
进一步地，通过检测被氧化后的D-丝氨酸代谢中间产物与探针反应形成的荧光团来检测受试者脑脊液中D-丝氨酸的浓度。
另外，本发明还提供了D-丝氨酸在制备治疗抑郁症的药物中的应用。
本发明通过研究抑郁症患者大脑组织中异常病变的代谢物质，研究结果发现D-丝氨酸与抑郁症密切相关，因此首次提出将D-丝氨酸作为标志物应用到制备抑郁症诊断试剂盒相关的领域中，克服目前在抑郁症诊断过程中的所用指标存在的诊断不客观的缺陷，进而为后期抑郁症的诊断提供了一种全新的更加准确的客观诊断的标准，进一步为抑郁症的诊断提供了一种新的思路。
本发明提供的抑郁症诊断试剂盒，能够快速准确的检测出受试者脑脊液中D-丝氨酸的浓度，从而有效准确地判断受试者是否患有抑郁症及抑郁症的严重程度。
本发明提供的D-丝氨酸在制备治疗抑郁症的药物中的应用，通过D-丝氨酸的释放能够增加相关脑区的神经元兴奋性，进而改善抑郁样行为。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，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1A为本发明实施例1提供的转基因动物构建成功的免疫组化结果图；
图1B为本发明实施例1提供的各组小鼠在鼠室中度过的时间的结果图；
图1C为本发明实施例1提供的各组小鼠与陌生小鼠的交互时间的结果图；
图1D为本发明实施例1提供的各组小鼠不动潜伏期的时间的结果图；
图2为为本发明实施例2提供的各组小鼠胶质细胞分泌D-丝氨酸能力的结果图。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是：
本发明中，如果没有特别的说明，本文所提到的所有实施方式以及优选实施方法可以相互组合形成新的技术方案。
本发明中，如果没有特别的说明，本文所提到的所有技术特征以及优选特征可以相互组合形成新的技术方案。
本发明中，除非有其他说明，数值范围“a～b”表示a到b之间的任意实数 组合的缩略表示，其中a和b都是实数。例如数值范围“6～22”表示本文中已经全部列出了“6～22”之间的全部实数，“6～22”只是这些数值组合的缩略表示。
本发明所公开的“范围”以下限和上限的形式，可以分别为一个或多个下限，和一个或多个上限。
本发明中，除非另有说明，各个反应或操作步骤可以顺序进行，也可以按照顺序进行。优选地，本文中的反应方法是顺序进行的。
除非另有说明，本文中所用的专业与科学术语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法或材料也可应用于本发明中。
下面对本发明提供的D-丝氨酸作为标志物在制备抑郁症诊断试剂盒中的应用及抑郁症诊断试剂盒进行具体说明。
抑郁症又称抑郁障碍，以显著而持久的心境低落为主要临床特征，是心境障碍的主要类型。临床可见心境低落与其处境不相称，情绪的消沉可以从闷闷不乐到悲痛欲绝，自卑抑郁，甚至悲观厌世，可有自杀企图或行为；甚至发生木僵；部分病例有明显的焦虑和运动性激越；严重者可出现幻觉、妄想等精神病性症状。因此，对抑郁症进行客观、准确的诊断是临床和心理治疗的首要条件。
D-丝氨酸(D-serine)是一种内源性脑信息协传物质，它的分子式为C3H7NO3，分子量为105.09，化学结构式为：
Figure PCTCN2019124721-appb-000001
现有的研究显示，D-丝氨酸水平和抑郁症的相关性并不明确。
而本发明以抑郁症模型小鼠为对象，研究正常小鼠与模型小鼠脑脊液中D-丝氨酸含量与抑郁症之间的关系，而结果显示，抑郁症模型小鼠组的D-丝氨酸水平较正常组有所下降。
基于此，一方面，本发明提供了D-丝氨酸作为标志物在制备抑郁症诊断试剂盒中的应用。
本发明通过研究抑郁症患者大脑组织中异常病变的代谢物质，研究结果发现D-丝氨酸与抑郁症密切相关，因此首次提出将D-丝氨酸作为标志物应用到制备抑郁症诊断试剂盒相关的领域中，克服目前在抑郁症诊断过程中的所用指标存在的诊断不客观的缺陷，进而为后期抑郁症的诊断提供了一种全新的更加准确的客观诊断的标准，进一步为抑郁症的诊断提供了一种新的思路。
在一些优选的实施方式中，所述抑郁症诊断试剂盒的检测样本为脑脊液。
脑脊液是由脑室内脉络丛产生的一种无色透明的液体，它循环流动于脑和脊髓表面，经脑内静脉系统与体循环相关联。主要功能是①保护大脑和脊髓免受外界震荡损伤；②调节颅内压力变化；③供给大脑、脊髓营养物质并运走代谢产物；④调节神经系统碱储量，维持正常PH值等。当脑组织或脊髓发生病变和外伤时，脑脊液也会发生各种改变。
本发明在目前脑脊液一般用于神经系统疾病检查的基础上，创造性地将脑脊液作为精神疾病抑郁症的检测样本，能够准确、客观地对抑郁症进行诊断。
在一些优选的实施方式中，所述抑郁症诊断试剂盒通过监测受试者脑脊液中D-丝氨酸的浓度来判断受试者是否患有抑郁症。
在一些优选的实施方式中，当受试者脑脊液中D-丝氨酸的浓度低于正常值51％时，判断所述受试者患有抑郁症。
本发明的研究结果显示，抑郁症模型组的D-丝氨酸的水平均低于正常值51％，明显低于对照组。因此，采用抑郁症诊断试剂盒所检测脑脊液中的D-丝氨酸标志物的浓度低于正常值51％，有理由作出相应的抑郁症的更加客观的诊断结果。其中，典型的脑脊液中的D-丝氨酸的正常浓度为通过常规测量方法测量得到的非抑郁人群脑脊液中的D-丝氨酸的浓度。在一些优选的实施方式中，所述抑郁症诊断试剂盒通过监测受试者脑脊液中D-丝氨酸的浓度来判断受试者患有抑郁症的严重程度。
在一些优选的实施方式中，所述抑郁症为中度抑郁症及中度以上的抑郁症。
以受试者脑脊液中D-丝氨酸浓度的数值为划分抑郁症严重程度的指标，更能体现出诊断的客观性或有助于提高诊断结果的准确性。
容易理解，以D-丝氨酸作为标志物不仅可以在制备抑郁症诊断试剂盒中的进行应用。还可以用于抑郁症的疗效评估等领域中，用于评价相关抑郁症药物的治疗效果，因此，将在D-丝氨酸作为标志物这方面的应用也属于本发明的保护范围。
在另一方面，本发明还提供了一种抑郁症诊断试剂盒，所述抑郁症诊断试剂盒通过监测受试者脑脊液中D-丝氨酸的浓度来判断受试者是否患有抑郁症。
本发明提供的抑郁症诊断试剂盒，能够快速准确的检测出受试者脑脊液中D-丝氨酸的浓度，从而有效准确地判断受试者是否患有抑郁症及抑郁症的严重程度。
在一些优选的实施方式中，所述试剂盒包括D-丝氨酸测定缓冲液、探针、D-丝氨酸酶混合物、显影剂酶混合物和D-丝氨酸标准品。
在一些优选的实施方式中，通过检测被氧化后的D-丝氨酸代谢中间产物与探针反应形成的荧光团来检测受试者脑脊液中D-丝氨酸的浓度。
其中，典型的Ex/Em＝535/587nm。
本发明在提出了将D-丝氨酸作为标志物在抑郁症诊断或疗效评估中的应用，例如将D-丝氨酸作为标志物制备抑郁症诊断或疗效评估的试剂盒中的应用，容易理解，相应的可检测D-丝氨酸浓度或水平的相关试剂或抗体均可以用于制备抑郁症诊断或疗效评估试剂盒，其也属于本发明的保护范围。
容易理解，本发明首次提出了D-丝氨酸作为标志物在制备抑郁症诊断或疗效评估试剂盒中的应用。因此，只要是基于该应用，所有的用于抑郁症诊断或疗效评估的试剂盒，只要其以D-丝氨酸为标志物，检测样本中的D-丝氨酸的水平，即属于本发明的保护范围。
另外，本发明还提供了D-丝氨酸在制备治疗抑郁症的药物中的应用。通过D-丝氨酸的释放能够增加相关脑区的神经元兴奋性，进而改善抑郁样行为。
综上，本发明通过研究抑郁症患者大脑组织中异常病变的代谢物质，研究结果发现D-丝氨酸与抑郁症密切相关，因此首次提出将D-丝氨酸作为标志物应用到制备抑郁症诊断试剂盒相关的领域中，克服目前在抑郁症诊断过程中的所用指标存在的诊断不客观的缺陷，进而为后期抑郁症的诊断提供了一种全新的更加准确的客观诊断的标准，进一步为抑郁症的诊断提供了一种新的思路。
为了有助于更清楚的理解本发明的内容，现结合具体的实施例详细介绍如下。
如无特别说明，本发明实施例中使用的实验动物、药品及试剂均来源为正规而易购渠道：
D-丝氨酸测定缓冲液、探针、D-丝氨酸酶混合物、显影剂酶混合物和D-丝氨酸标准品购自喀斯玛商城；tamoxifen购自sigma；小鼠购自北京维通利华实验动物技术有限公司；酶标仪购自biotek。
实施例1 小鼠抑郁症模型的建立
抑郁症的发病不同于其他遗传性的精神疾病，往往在成年之后才发病，为此，本实施例建立了一种可在成年期诱导的啮齿类抑郁动物模型。
转基因动物的构建：突变DISC1蛋白融合带HA-标记的雌激素受体的配体，在小鼠成年时给予它莫西芬药物能诱导该蛋白表达，可诱发抑郁样行为。结果如图1A所示，从图中可以看出HA在大脑皮层、海马体和伏隔核均有表达，说明转基因动物构建成功。
本实施例建立的啮齿类抑郁动物模型的确立是通过社交障碍和强迫游泳争扎时间减少来体现的：
社交障碍：三箱社交实验
1.实验鼠放入箱体中适应10min；
2.一侧箱体金属笼中放入5周龄的陌生小鼠，另一侧只放入金属笼，实验鼠自由探索10min；统计与金属笼小鼠和金属笼的交互时间。
3.将另一侧的金属笼放入另一只5周龄的陌生小鼠，实验鼠自由探索10min。分别统计与旧小鼠和新小鼠的交互时间。
结果如图1B和图1C所示，从图1B中可以看出，它莫西芬诱导该转基因动物后，在有陌生小鼠的箱体中时间显著降低，说明其出现了社交障碍；从图1C中可以看出，它莫西芬诱导该转基因动物后，与陌生小鼠交互的时间和金属笼交互的时间没有差异，没有表现出野生型小鼠对新奇老鼠的偏好性，说明该转基因鼠出现了社交障碍。
强迫游泳：准备一个水缸，装上25°温水，水深约10cm，将实验鼠放进水中10min，统计其开始不动的时间。
结果如图1D所示，从图中可以看出，它莫西芬诱导的转基因动物开始不 动的时间比其它老鼠早，说明了其抑郁样行为。
上述结果说明，本实施例的模型建立了可诱导成年发病的抑郁症动物模型显示，对比野生型小鼠或者未诱导转基因表型的动物，其社交行为出现障碍，而且在强迫游泳中其挣扎时间也显著减少。
实施例2 D-丝氨酸水平检测
本实施例通过如下方法对D-丝氨酸水平进行检测：
1.在体微透析取得小鼠样品；
2.对样品进行过柱纯化；
3.用本发明提供的抑郁症诊断试剂盒制作D-丝氨酸的标准曲线；
4.同样的方法，将标准品替换为脑脊液样品；
5.孵育60min，37℃；
6.用酶标仪进行检测Ex/Em＝539/587nm，终点模式。
7.根据标准曲线所得的公式进行计算。
抑郁症大脑中的特定神经环路出现了功能异常，而这种异常往往是由于具部神经细胞功能异常造成的。研究显示，在抑郁症模型中，神经胶质细胞的功能出现了异常，小鼠出现抑郁样行为。本发明实施例1中建立的抑郁症小鼠模型在体微透析实验结果也证明，在此模型小鼠中，其胶质细胞的代谢能力显著低于野生型小鼠，脑脊液中的D-serine水平下降(结果如图2所示)。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (10)

  1. D-丝氨酸作为标志物在制备抑郁症诊断试剂盒中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述抑郁症诊断试剂盒的检测样本为脑脊液。
  3. 根据权利要求2所述的应用，其特征在于，所述抑郁症诊断试剂盒通过监测受试者脑脊液中D-丝氨酸的浓度来判断受试者是否患有抑郁症。
  4. 根据权利要求3所述的应用，其特征在于，当受试者脑脊液中D-丝氨酸的浓度低于正常值51％时，判断所述受试者患有抑郁症。
  5. 根据权利要求3所述的应用，其特征在于，所述抑郁症诊断试剂盒通过监测受试者脑脊液中D-丝氨酸的浓度来判断受试者患有抑郁症的严重程度。
  6. 根据权利要求1-5任一项所述的应用，其特征在于，所述抑郁症为中度抑郁症及中度以上的抑郁症。
  7. 一种抑郁症诊断试剂盒，其特征在于，所述抑郁症诊断试剂盒通过监测受试者脑脊液中D-丝氨酸的浓度来判断受试者是否患有抑郁症。
  8. 根据权利要求7所述的抑郁症诊断试剂盒，其特征在于，所述试剂盒包括D-丝氨酸测定缓冲液、探针、D-丝氨酸酶混合物、显影剂酶混合物和D-丝氨酸标准。
  9. 根据权利要求8所述的抑郁症诊断试剂盒，其特征在于，通过检测被氧化后的D-丝氨酸代谢中间产物与探针反应形成的荧光团来检测受试者脑脊液中D-丝氨酸的浓度。
  10. D-丝氨酸在制备治疗抑郁症的药物中的应用。
PCT/CN2019/124721 2018-12-18 2019-12-12 D-丝氨酸作为标志物在制备抑郁症诊断试剂盒中的应用及抑郁症诊断试剂盒 Ceased WO2020125530A1 (zh)

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