CN111549117B - Biomarker and application thereof in Parkinson - Google Patents

Abstract

Translated fromChinese

本发明公开了一种生物标志物及其在帕金森中的应用，所述生物标志物为FAM189A2，经过研究，首次发现了FAM189A2在帕金森中表达上调，且FAM189A2可以影响神经细胞的增殖，提示可以将FAM189A2应用于帕金森的诊断和个性化治疗。

The invention discloses a biomarker and its application in Parkinson's disease. The biomarker is FAM189A2. After research, it was found for the first time that the expression of FAM189A2 is up-regulated in Parkinson's disease, and FAM189A2 can affect the proliferation of nerve cells, suggesting that FAM189A2 can be applied to the diagnosis and personalized treatment of Parkinson's.

Description

Translated fromChinese

一种生物标志物及其在帕金森中应用A biomarker and its application in Parkinson's

技术领域technical field

本发明涉及生物技术领域，涉及一种生物标志物及其在帕金森中应用。The invention relates to the field of biotechnology, and relates to a biomarker and its application in Parkinson's disease.

背景技术Background technique

帕金森病(Parkinson's disease,PD)是第二大最常见的神经系统退行性疾病，发病率仅次于阿尔茨海默病(Alzheimer's disease,AD)。其典型的特征是：黑质致密部多巴胺能神经元进行性死亡，由此导致的基底神经节多巴胺缺乏以及病变区残存神经元内形成的异常蛋白包涵体，包括路易小体(Lewy Bodies,LBs)和路易突起(Lewy Neurites,LNs)。其中异常折叠和过表达的a-突触核蛋白(a-synuclein,a-syn)已被证实为LBs和LNs的核心成分(Dehay,B.,Bourdenx,M.，et al.Targeting alpha-synuclein for treatment ofParkinson's disease：mechanistic and therapeutic considerations.Lancet Neurol,2015,14(8),855-866.)。选择性的多巴胺能神经元丢失导致主要的运动症状:运动徐缓，静止性震颤和肌强直(Gibb WR and Lees AJ.The relevance of the Lewy body to thepathogenesis ofidiopathic Parkinson's disease.J Neurol Neurosurg Psychiatry,1988,51(6):745-52.)。此外PD患者也有一些“非运动”症状，如嗅觉减弱、睡眠障碍、疲劳、情绪改变、认知障碍、植物神经功能紊乱和抑郁等(Titova N,Qamar MA,and ChaudhuriKR.The Nonmotor Features of Parkinson's Disease.Int Rev Neurobiol,2017,132:33-54.)。Parkinson's disease (PD) is the second most common neurodegenerative disease, second only to Alzheimer's disease (Alzheimer's disease, AD). Its typical features are: progressive death of dopaminergic neurons in the substantia nigra compacta, resulting in a lack of dopamine in the basal ganglia and abnormal protein inclusions, including Lewy bodies (LBs), formed in the remaining neurons in the lesion area. ) and Lewy Neurites (LNs). The abnormally folded and overexpressed a-synuclein (a-synuclein, a-syn) has been confirmed as the core component of LBs and LNs (Dehay, B., Bourdenx, M., et al.Targeting alpha-synuclein for treatment of Parkinson's disease: mechanical and therapeutic considerations. Lancet Neurol, 2015, 14(8), 855-866.). Selective loss of dopaminergic neurons results in major motor symptoms: bradykinesia, resting tremor and rigidity (Gibb WR and Lees AJ. The relevance of the Lewy body to thepathogenesis of idiopathic Parkinson's disease. J Neurol Neurosurg Psychiatry, 1988, 51 (6):745-52.). In addition, PD patients also have some "non-motor" symptoms, such as decreased sense of smell, sleep disturbance, fatigue, mood changes, cognitive impairment, autonomic dysfunction and depression (Titova N, Qamar MA, and ChaudhuriKR. The Nonmotor Features of Parkinson's Disease . Int Rev Neurobiol, 2017, 132:33-54.).

目前科学家们认为PD是遗传和环境因素共同作用导致的，主要包括:a-Syn聚积及其在细胞间的传递、线粒体功能障碍、氧化应激和神经炎症。但是其精确的分子机制还没有完全研究清楚。充分认识PD病理进程中生物指标的改变以及阐明PD病理学机制，提高预防和治疗PD的水平、延缓PD病理进程，是PD研究者们亟待解决的关键问题。At present, scientists believe that PD is caused by the joint action of genetic and environmental factors, mainly including: a-Syn accumulation and its transmission between cells, mitochondrial dysfunction, oxidative stress and neuroinflammation. However, its precise molecular mechanism has not been fully studied. Fully understanding the changes of biological indicators in the pathological process of PD, clarifying the pathological mechanism of PD, improving the level of prevention and treatment of PD, and delaying the pathological process of PD are the key issues that PD researchers need to solve urgently.

发明内容Contents of the invention

为了弥补现有技术的不足，本发明的目的在于提供一种可用于帕金森病诊断和治疗的生物标志物，从而为患者制定靶向性调控策略来治疗PD。In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a biomarker that can be used for the diagnosis and treatment of Parkinson's disease, so as to formulate targeted regulation strategies for patients to treat PD.

为了实现上述目的，本发明采用如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:

本发明提供了检测FAM189A2水平的试剂在制备诊断帕金森病的产品中的应用。The invention provides the application of the reagent for detecting the level of FAM189A2 in the preparation of products for diagnosing Parkinson's disease.

进一步，所述试剂包括通过RT-PCR、实时定量PCR、免疫检测、原位杂交或芯片检测FAM189A2表达水平的试剂。Further, the reagents include reagents for detecting the expression level of FAM189A2 by RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization or chips.

进一步，所述用RT-PCR检测FAM189A2表达水平的试剂至少包括一对特异扩增FAM189A2基因的引物；所述用实时定量PCR检测FAM189A2表达水平的试剂至少包括一对特异扩增FAM189A2基因的引物；所述用免疫检测检测FAM189A2表达水平的试剂：与FAM189A2蛋白特异性结合的抗体；所述用原位杂交检测FAM189A2表达水平的试剂包括：与FAM189A2基因的核酸序列杂交的探针；所述用芯片检测FAM189A2表达水平的试剂包括：蛋白质芯片和基因芯片；其中，蛋白质芯片包括与FAM189A2蛋白特异性结合的抗体，基因芯片包括与FAM189A2基因的核酸序列杂交的探针。Further, the reagent for detecting the expression level of FAM189A2 by RT-PCR includes at least a pair of primers for specifically amplifying the FAM189A2 gene; the reagent for detecting the expression level of FAM189A2 by real-time quantitative PCR includes at least a pair of primers for specifically amplifying the FAM189A2 gene; The reagent for detecting the expression level of FAM189A2 by immunoassay: an antibody specifically binding to the FAM189A2 protein; the reagent for detecting the expression level of FAM189A2 by in situ hybridization includes: a probe hybridized with the nucleic acid sequence of the FAM189A2 gene; The reagents for detecting the expression level of FAM189A2 include: a protein chip and a gene chip; wherein, the protein chip includes an antibody specifically binding to the FAM189A2 protein, and the gene chip includes a probe hybridized with the nucleic acid sequence of the FAM189A2 gene.

本发明还提供了一种诊断帕金森病的产品，所述产品包括检测FAM189A2水平的试剂，所述产品包括但不限于芯片、试剂盒。The present invention also provides a product for diagnosing Parkinson's disease, the product includes a reagent for detecting the level of FAM189A2, and the product includes but not limited to a chip and a kit.

进一步，所述试剂包括所述试剂包括特异性针对FAM189A2探针、引物和/或抗体。Further, the reagents include probes, primers and/or antibodies specific for FAM189A2.

进一步，特异性针对FAM189A2的引物序列如SEQ ID NO.1～2所示。Further, the primer sequences specific for FAM189A2 are shown in SEQ ID NO.1-2.

本发明提供了FAM189A2在筛选治疗帕金森的候选药物中的应用。The invention provides the application of FAM189A2 in screening candidate drugs for treating Parkinson's disease.

本发明提供了FAM189A2的抑制剂在制备治疗帕金森病药物中的应用。The invention provides the application of the inhibitor of FAM189A2 in the preparation of the medicine for treating Parkinson's disease.

进一步，所述抑制剂为降低FAM189A2的表达水平的试剂。Further, the inhibitor is an agent that reduces the expression level of FAM189A2.

进一步，所述试剂选自核酸分子、脂类、小分子化学药、核酸构建体。Further, the reagent is selected from nucleic acid molecules, lipids, small molecule chemicals, and nucleic acid constructs.

进一步，所述核酸分子为干扰RNA。Further, the nucleic acid molecule is interfering RNA.

进一步，干扰RNA的序列如SEQ ID NO.5～6所示。Further, the sequence of the interfering RNA is shown in SEQ ID NO.5-6.

本发明提供了一种治疗帕金森的药物组合物，所述药物组合物包括前述的FAM189A2的抑制剂。The present invention provides a pharmaceutical composition for treating Parkinson's disease, which comprises the aforementioned inhibitor of FAM189A2.

进一步，所述药物组合物还包括药学上可接受的载体。Further, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

本发明的优点和有益效果：Advantages and beneficial effects of the present invention:

本发明首次发现了FAM189A2基因与帕金森的发生发展相关，对于帕金森的发病机制的研究具有重要的科研价值以及临床意义，为帕金森的分子诊断和治疗提供了新的手段。The invention discovers for the first time that the FAM189A2 gene is related to the occurrence and development of Parkinson's, which has important scientific research value and clinical significance for the research on the pathogenesis of Parkinson's, and provides a new means for the molecular diagnosis and treatment of Parkinson's.

附图说明Description of drawings

图1是FAM189A2基因的表达情况图。Fig. 1 is a graph showing the expression of the FAM189A2 gene.

具体的实施方式specific implementation

本发明首次发现了FAM189A2在帕金森患者中呈现高表达，FAM189A2可作为独立的预测因子应用于帕金森的诊断，同时本发明通过实验证明了FAM189A2的表达水平影响神经细胞的增殖，从而为帕金森的治疗提供了一种新的手段。The present invention found for the first time that FAM189A2 is highly expressed in Parkinson's patients, and FAM189A2 can be used as an independent predictor in the diagnosis of Parkinson's. At the same time, the present invention has proved through experiments that the expression level of FAM189A2 affects the proliferation of nerve cells, thereby providing a basis for Parkinson's disease. The treatment provides a new means.

术语“生物标志物”是其在组织或细胞中的表达水平与正常或健康细胞或组织的表达水平相比发生改变的任何基因或蛋白。The term "biomarker" is any gene or protein whose expression level in a tissue or cell is altered compared to that of a normal or healthy cell or tissue.

本领域技术人员将认识到，本发明的实用性并不局限于对本发明的标志物基因的任何特定变体的基因表达进行定量。作为非限制性的实例，标志物基因具有基因ID：9413所示的编码序列或氨基酸序列。一种代表性的核苷酸序列如NM_001127608.2所示，相对应的蛋白序列如NP_001121080.1所示。Those skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the marker genes of the present invention. As a non-limiting example, the marker gene has the coding sequence or amino acid sequence shown in Gene ID: 9413. A representative nucleotide sequence is shown in NM_001127608.2, and the corresponding protein sequence is shown in NP_001121080.1.

发明中的“多核苷酸”包括RNA、cDNA、基因组DNA、合成形式及混合的聚合物，有义链和反义链，及可以是化学或生物化学修饰的，或者可以含有非天然或衍生的核苷酸碱基，这易为本领域技术人员所意识到。这种修饰包括例如标记、甲基化、用类似物取代一或多个天然发生的核苷酸、核苷酸间修饰如无电荷的键连(例如甲基磷酸酯，磷酸三酯，磷酸酰胺，氨基甲酸酯等)、带电荷的键连(例如硫代磷酸酯，二硫代磷酸酯等)、悬垂部分(pendentmoieties)(例如多肽)、嵌入物(例如吖啶、补骨脂素等)、螯合剂、烷化剂，及修饰的键连(例如α端基异构核酸等)。也包括合成分子，其具有模拟多核苷酸通过氢键及其它化学相互作用结合指定序列的能力。这种分子为本领域已知，包括例如其中在分子主链中肽键取代磷酸键。"Polynucleotide" in the context of the invention includes RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, sense and antisense strands, and may be chemically or biochemically modified, or may contain non-natural or derived Nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g. methylphosphonate, phosphotriester, phosphoamide , carbamate, etc.), charged linkages (such as phosphorothioate, phosphorodithioate, etc.), pendent moieties (such as polypeptides), intercalators (such as acridine, psoralen, etc. ), chelating agents, alkylating agents, and modified linkages (eg, alpha-anomeric nucleic acids, etc.). Also included are synthetic molecules that have the ability to mimic the binding of polynucleotides to a given sequence through hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, wherein peptide bonds replace phosphate bonds in the backbone of the molecule.

基于FAM189A2在帕金森中表达上调的发现，可以利用本领域内已知的任何方法测定基因表达。本领域技术人员应当理解，测定基因表达的手段不是本发明的重要方面。可以在转录或翻译(即蛋白)水平上检测生物标志物的表达水平。Based on the finding that FAM189A2 is upregulated in Parkinson's, gene expression can be measured using any method known in the art. Those skilled in the art will appreciate that the means by which gene expression is measured is not an essential aspect of the invention. Expression levels of biomarkers can be detected at the transcriptional or translational (ie, protein) level.

作为一种可选择的实施方案，在转录水平上检测生物标志物的表达水平。利用核酸杂交技术进行具体DNA和RNA测量的多种方法是本领域技术人员已知的。一些方法涉及电泳分离(例如，用于检测DNA的Southern印迹和用于检测RNA的Northern印迹)，但是也可以在不利用电泳分离的情况下进行DNA和RNA的测量(例如，通过斑点印迹)。基因组DNA(例如，来自人)的Southern印迹可用于筛选限制性片段长度多态性(RFLP)，以检测影响本发明多肽的遗传病症的存在。可以检测所有形式的RNA，包括但不限于信使RNA(mRNA)、微RNA(miRNA)、核糖体RNA(rRNA)和转运RNA(tRNA)。核酸杂交形式的选择不是关键的。多种核酸杂交形式包括但不限于夹心测定和竞争或替代测定。As an alternative embodiment, the expression level of the biomarker is detected at the transcriptional level. Various methods for making specific DNA and RNA measurements using nucleic acid hybridization techniques are known to those skilled in the art. Some methods involve electrophoretic separation (eg, Southern blots for DNA and Northern blots for RNA), but measurements of DNA and RNA can also be performed without electrophoretic separation (eg, by dot blot). Southern blots of genomic DNA (eg, from humans) can be used to screen for restriction fragment length polymorphisms (RFLPs) to detect the presence of genetic disorders affecting the polypeptides of the invention. All forms of RNA can be detected, including but not limited to messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA). The choice of nucleic acid hybridization format is not critical. Various nucleic acid hybridization formats include, but are not limited to, sandwich assays and competition or displacement assays.

作为另一种可选择的实施方案，在蛋白水平上检测生物标志物的表达水平。使用基于抗体的方法进行具体蛋白测量的多种方法是本领域技术人员已知的。可使用的示例性方法包括但不限于免疫印迹法(蛋白质印迹(western blot))、酶联免疫吸附测定(ELISA)、免疫组织化学、流式细胞术、流式细胞术微珠阵列和质谱法。ELISA的一些类型是常用的，包括直接ELISA、间接ELISA和夹心ELISA。As another alternative embodiment, the expression level of the biomarker is detected at the protein level. Various methods for making specific protein measurements using antibody-based methods are known to those skilled in the art. Exemplary methods that can be used include, but are not limited to, immunoblotting (western blot), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flow cytometry, flow cytometry bead arrays, and mass spectrometry . Several types of ELISA are commonly used, including direct ELISA, indirect ELISA, and sandwich ELISA.

本发明提供了FAM189A2在筛选治疗帕金森的候选药物中的应用。The invention provides the application of FAM189A2 in screening candidate drugs for treating Parkinson's disease.

使用FAM189A2筛选治疗帕金森的药物的步骤如下：The steps to use FAM189A2 to screen drugs for Parkinson's disease are as follows:

在实验组中，向培养体系中加入待测化合物，并测定FAM189A2的表达水平；在对照组中，向同样的培养体系中不加入待测化合物，并测定FAM189A2的表达水平；其中，如果实验组中FAM189A2的表达水平小于对照组，则说明该待筛选的物质为治疗帕金森的候选药物。In the experimental group, the compound to be tested was added to the culture system, and the expression level of FAM189A2 was determined; in the control group, the compound to be tested was not added to the same culture system, and the expression level of FAM189A2 was determined; wherein, if the experimental group The expression level of FAM189A2 in the drug is lower than that of the control group, indicating that the substance to be screened is a candidate drug for treating Parkinson's disease.

在本发明中，所述的方法还包括：对上面步骤获得的药物进一步测试其抑制帕金森的效果，若测试化合物对帕金森有显著的抑制效果，则说明该化合物为预防或治疗帕金森的药物。In the present invention, the method also includes: further testing the effect of inhibiting Parkinson's on the drug obtained in the above steps, if the test compound has a significant inhibitory effect on Parkinson's, it means that the compound is effective for preventing or treating Parkinson's drug.

所述培养体系包括(但不限于)细胞体系、亚细胞体系、溶液体系、组织体系、器官体系或动物体系(如动物模型，优选非人哺乳动物的动物模型，如鼠、兔、羊、猴等)等。The culture system includes (but not limited to) a cell system, a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably an animal model of a non-human mammal, such as a mouse, a rabbit, a sheep, a monkey etc.

当将通过本发明的筛选方法分离的化合物作为药物施用于人或其它哺乳动物，其包括但不限于小鼠、大鼠、豚鼠、兔、猫、犬、羊、猪、牛、猴子、狒狒、黑猩猩时，分离的化合物可以直接施用，或者可以利用已知的药物制备方法配制成各种剂型。例如，根据需要，所述药物可以作为糖衣片剂、胶囊剂、酏剂和微胶囊口服施用；或者用水或任何其它药物可接受的液体配制成无菌溶液或悬浮液，以注射剂的形式非口服施用。例如，可以将化合物以一般接受的药物施用方式所需的单位剂型(unit dose)、与药学可接受的载体或介质混合在一起，所述载体或介质包括但不限于无菌水、生理盐水、植物油、乳化剂、悬浮剂、表面活性剂、稳定剂、调味剂、赋形剂(excipient)、媒介物(vehicle)、防腐剂、粘合剂等。根据这些制剂中有效成分的含量，可以获得在指定范围内的合适的给药量。When the compound isolated by the screening method of the present invention is administered to humans or other mammals, including but not limited to mice, rats, guinea pigs, rabbits, cats, dogs, sheep, pigs, cows, monkeys, baboons, In the case of chimpanzees, the isolated compound can be administered directly, or can be formulated into various dosage forms using known pharmaceutical preparation methods. For example, the drug can be administered orally as sugar-coated tablets, capsules, elixirs, and microcapsules; or formulated into sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid, and parenterally in the form of injections, as needed. apply. For example, the compound can be mixed together in unit dosage form (unit dose) required by generally accepted pharmaceutical administration methods, together with a pharmaceutically acceptable carrier or medium, which includes but not limited to sterile water, physiological saline, Vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc. Depending on the content of the active ingredient in these preparations, an appropriate dosage within the specified range can be obtained.

本发明提供了FAM189A2的抑制剂在制备治疗帕金森病药物中的应用。The invention provides the application of the inhibitor of FAM189A2 in the preparation of the medicine for treating Parkinson's disease.

作为可选择的实施方式，所述抑制剂包括：通过干扰RNA抑制FAM189A2基因表达的双链核糖核酸，或基于FAM189A2抗原蛋白的肿瘤疫苗、或用于抑制FAM189A2蛋白活性的蛋白质。As an alternative embodiment, the inhibitor includes: double-stranded ribonucleic acid inhibiting the expression of FAM189A2 gene by interfering RNA, or a tumor vaccine based on FAM189A2 antigen protein, or a protein used to inhibit the activity of FAM189A2 protein.

作为本发明的一种优选的实施方式，所述抑制剂是针对FAM189A2的干扰RNA。As a preferred embodiment of the present invention, the inhibitor is an interfering RNA against FAM189A2.

在本发明中，所述RNA干扰(RNA interference，RNAi)是指内源性或外源性双链RNA(double-stranded RNA，dsRNA)介导的细胞内mRNA发生特异性降解，从而导致靶基因的表达沉默，产生相应的功能表型缺失的现象。RNAi技术是一种典型的负调控机制，使用该技术可以特异性剔除或关闭特定基因的表达，该技术已被广泛用于探索基因功能、基因治疗及新药开发领域。以细胞为基础的RNAi筛选在功能基因学研究方面具有许多优势，主要表现在大多数细胞类型都能使用RNAi方法，并且相对较容易下调或沉默目的基因的表达。为了进一步提高siRNA片段的有效性，通过同源性比对(NCBI BLAST)来过滤siRNA序列，以提高siRNA片段的特异性并减少RNAi干扰的脱靶效应，最后，通过实验筛选干扰效果最佳的siRNA应用于本发明中。In the present invention, the RNA interference (RNA interference, RNAi) refers to the specific degradation of intracellular mRNA mediated by endogenous or exogenous double-stranded RNA (double-stranded RNA, dsRNA), thereby causing target gene Silencing of expression results in a corresponding loss of functional phenotype. RNAi technology is a typical negative regulatory mechanism, which can specifically knock out or turn off the expression of specific genes. This technology has been widely used in the fields of exploring gene functions, gene therapy and new drug development. Cell-based RNAi screening has many advantages in functional genetics research, mainly in that most cell types can use RNAi methods, and it is relatively easy to down-regulate or silence the expression of target genes. In order to further improve the effectiveness of siRNA fragments, siRNA sequences were filtered by homology comparison (NCBI BLAST) to improve the specificity of siRNA fragments and reduce the off-target effect of RNAi interference. Finally, the siRNA with the best interference effect was screened through experiments applied in the present invention.

本发明提供了一种治疗帕金森的药物组合物，所述药物组合物包括针对FAM189A2的抑制剂以及药学上可接受的载体。The invention provides a pharmaceutical composition for treating Parkinson's disease, which comprises an inhibitor against FAM189A2 and a pharmaceutically acceptable carrier.

在本发明中药学上可接受的载体包括但并不限于：稀释剂、赋形剂如水等、填充剂如淀粉、蔗糖等；粘合剂如纤维素衍生物、藻酸盐、明胶和聚乙烯吡咯烷酮；湿润剂如甘油；崩解剂如琼脂、碳酸钙和碳酸氢钠；吸收促进剂季铵化合物；表面活性剂如十六烷醇；吸附载体如高岭土和皂粘土；润滑剂如滑石粉、硬脂酸钙和镁、聚乙二醇等。In the present invention, pharmaceutically acceptable carriers include, but are not limited to: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginate, gelatin and polyethylene Pyrrolidone; wetting agents such as glycerin; disintegrants such as agar-agar, calcium carbonate and sodium bicarbonate; absorption enhancers quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; Calcium and magnesium stearate, polyethylene glycol, etc.

本发明的药物还可与其他治疗帕金森的药物联用，多种药物联合使用可以大大提到治疗的成功率。The medicine of the present invention can also be used in combination with other medicines for treating Parkinson's disease, and the combined use of multiple medicines can greatly improve the success rate of treatment.

在本发明的上下文中，“诊断帕金森病”既包括判断受试者是否已经患有帕金森病、也包括判断受试者是否存在患有帕金森病的风险。In the context of the present invention, "diagnosing Parkinson's disease" includes not only judging whether the subject has Parkinson's disease, but also judging whether the subject is at risk of suffering from Parkinson's disease.

在本发明的上下文中，“治疗帕金森病”从疾病的状态变化来分，可以包括疾病的缓解、疾病的完全治愈。In the context of the present invention, "treating Parkinson's disease" is divided from the state change of the disease, and may include the remission of the disease and the complete cure of the disease.

统计学分析Statistical analysis

在本发明的具体实施例中，实验都是按照至少重复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，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。In the specific embodiment of the present invention, the experiment is all completed according to repeating at least 3 times, and the result data is all expressed in the mode of mean ± standard deviation, and the difference between the two adopts t test, thinks when P< 0.05 is statistically significant.

下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

实施例1检测FAM189A2的表达水平Example 1 Detection of the expression level of FAM189A2

1、样品收集1. Sample collection

各收集80例正常人血液和帕金森病患者血液样本，写明样本名称、编号、取样日期、样本处理过程等情况。Collect 80 normal blood samples and blood samples from patients with Parkinson's disease, and write down the sample name, serial number, sampling date, sample processing process, etc.

正常对照组排除标准：(1)帕金森病及帕金森病家族史；(2)阿尔茨海默病，癫痫、脑血管疾病等其他神经系统疾病史；(3)有既往精神病史，如精神分裂症、重度抑郁者、精神发育迟滞及躯体疾病所致精神障碍者；(4)文盲、色盲、语言障碍或其它原因无法沟通者。Exclusion criteria for the normal control group: (1) Parkinson's disease and family history of Parkinson's disease; (2) Alzheimer's disease, epilepsy, cerebrovascular disease and other nervous system disease history; (3) previous history of mental illness, such as psychosis Schizophrenia, severe depression, mental retardation and mental disorders caused by physical diseases; (4) Illiteracy, color blindness, language barriers or other reasons unable to communicate.

帕金森病组排除标准：(1)文盲、色盲、语言障碍或其它原因无法沟通者；(2)存在药物中毒、内分泌代谢性疾病、重度抑郁等其它原因导致认知功能障碍或痴呆者；(3)继发性帕金森综合征、脑血管疾病、脑炎、颅脑外伤或手术者；(4)恶性肿瘤及其他严重疾病者。Exclusion criteria for Parkinson's disease group: (1) People who are illiterate, color blind, language barrier or unable to communicate due to other reasons; (2) People with cognitive impairment or dementia caused by drug poisoning, endocrine and metabolic diseases, severe depression and other reasons; ( 3) Secondary Parkinson's syndrome, cerebrovascular disease, encephalitis, craniocerebral trauma or surgery; (4) Malignant tumors and other serious diseases.

2、RNA样品的制备2. Preparation of RNA samples

使用天根公司的TRNzol Universal总RNA提取试剂(DP424)提取RNA，具体步骤详见说明书。RNA was extracted using TRNzol Universal Total RNA Extraction Reagent (DP424) from Tiangen Company, and the specific steps were detailed in the instruction manual.

3、逆转录：3. Reverse transcription:

使用TIANGEN公司的反转录试剂盒(KR106)进行操作，具体步骤如下：Use the reverse transcription kit (KR106) of TIANGEN Company to operate, and the specific steps are as follows:

去除基因组DNA反应，在试管中加入5×DNA Buffer 2.0μl，RNA 1μg，加入去RNA酶ddH₂O使总体积至10μl，水浴锅中42℃加热3min，再将10×Fast RT Buffer 2.0μL，RTEnzyme Mix 1.0μL，FQ-RT Primer Mix 2.0μL，去RNA酶ddH₂O 5.0μL，混合后加入上述试管中一起混合，水浴锅中42℃加热15min，95℃加热3min，合成的cDNA需要长期保存时，请于-20℃或更低温度保存。For genomic DNA removal reaction, add 5×DNA Buffer 2.0μl, RNA 1μg to the test tube, add RNase-removing ddH₂ O to make the total volume to 10μl, heat in a water bath at 42°C for 3min, then add 10×Fast RT Buffer 2.0μL, RTEnzyme Mix 1.0μL, FQ-RT Primer Mix 2.0μL, RNase-removing ddH₂ O 5.0μL, after mixing, add to the above test tube and mix together, heat in a water bath at 42°C for 15min, then at 95°C for 3min, the synthesized cDNA needs to be stored for a long time , please store at -20°C or lower.

4、QPCR4. QPCR

根据Genbank中FAM189A2基因和GAPDH基因的编码序列设计QPCR扩增引物，具体引物序列如表1所示。The QPCR amplification primers were designed according to the coding sequences of the FAM189A2 gene and the GAPDH gene in Genbank, and the specific primer sequences are shown in Table 1.

表1引物序列Table 1 Primer Sequence

按照表2配制PCR反应体系，其中，SYBR Green聚合酶链式反应体系购自TIANGEN公司(货号FP205)。The PCR reaction system was prepared according to Table 2, wherein the SYBR Green polymerase chain reaction system was purchased from TIANGEN Company (Product No. FP205).

表2 PCR反应体系Table 2 PCR reaction system

试剂Reagent体积volume正向引物forward primer0.6μl0.6μl反向引物reverse primer0.6μl0.6μl2×SuperReal PreMix Plus2×SuperReal PreMix Plus10μl10μlDNA模板DNA template2μl2μl50×Reference Dye^△50×Reference Dye^△2μl2μl蒸馏水distilled water补足至20μlMake up to 20μl

PCR反应条件：95°15min，(95℃10s，55℃30s，72℃32s)×40个循环。以SYBR Green作为荧光标记物，在ABI 7300型荧光定量PCR仪上进行PCR反应，通过融解曲线分析和电泳确定目的条带，2^-ΔΔCT法进行相对定量。PCR reaction conditions: 95°C for 15min, (95°C for 10s, 55°C for 30s, 72°C for 32s) x 40 cycles. Using SYBR Green as a fluorescent marker, the PCR reaction was carried out on the ABI 7300 fluorescent quantitative PCR instrument, the target band was determined by melting curve analysis and electrophoresis, and the relative quantification was carried out by the 2^-ΔΔCT method.

5、结果5. Results

结果如图1所示，与正常人血液相比，FAM189A2基因在帕金森病患者血液中的表达显著上调，差异具有统计学意义。FAM189A2在76例样本中表达上调，其中在帕金森患者中表达上调的有69人，在正常人中表达上调的有7人。以表达上调视为阳性(+)，无显著变化或者下调视为阴性(-)，具体统计情况如表3所示。The results are shown in Figure 1. Compared with normal human blood, the expression of FAM189A2 gene in the blood of patients with Parkinson's disease was significantly up-regulated, and the difference was statistically significant. The expression of FAM189A2 was up-regulated in 76 samples, among which 69 were up-regulated in Parkinson's patients, and 7 were up-regulated in normal subjects. Up-regulation of expression was regarded as positive (+), and no significant change or down-regulation was regarded as negative (-). The specific statistics are shown in Table 3.

表3基因在样本中的表达情况表Table 3 Gene expression in samples

实施例2 FAM189A2的沉默检测Example 2 Silencing detection of FAM189A2

1、细胞培养1. Cell culture

SH-SY5Y细胞以含10％胎牛血清和1％青链霉素混液的DMEM/F12培养基于5％CO₂的37℃恒温培养箱内常规培养，使用倒置显微镜定期观察，2d换一次培养液，待细胞铺满瓶底80％时，使用胰蛋白酶消化传代，采用生长状况良好对数期细胞进行实验。SH-SY5Y cells were cultured in DMEM/F12 containing 10% fetal bovine serum and 1% penicillin-streptomycin mixed solution, and were routinely cultured in a constant temperature incubator at 37°C based on 5% CO₂ , observed regularly with an inverted microscope, and the culture medium was changed every 2 days , when the cells covered 80% of the bottom of the flask, they were digested and passaged with trypsin, and the logarithmic phase cells in good growth condition were used for experiments.

2、转染2. Transfection

将SH-SY5Y细胞消化后接种于6孔板中，待细胞融合度约70％时，严格按Lipofectamine^TM2000说明书操作将siRNA-NC、siRNA-FAM189A2转染至SH-SY5Y细胞中，转染后细胞分为空白对照组、siRNA-NC组和siRNA-FAM189A2组。Digest SH-SY5Y cells and inoculate them in 6-well plates. When the cell confluence is about 70%, transfect siRNA-NC and siRNA-FAM189A2 into SH-SY5Y cells strictly according to the instructions of Lipofectamine^TM 2000. After transfection, Cells were divided into blank control group, siRNA-NC group and siRNA-FAM189A2 group.

其中，siRNA-NC为由上海吉码制药技术有限公司提供的通用阴性对照，siRNA-FAM189A2的序列如下所示：Among them, siRNA-NC is a universal negative control provided by Shanghai Jima Pharmaceutical Technology Co., Ltd., and the sequence of siRNA-FAM189A2 is as follows:

正义链为5’-UGCAUUUACUCCUGAAUGCCC-3’(SEQ ID NO.5)，The sense strand is 5'-UGCAUUUACUCCUGAAUGCCC-3' (SEQ ID NO.5),

反义链为5’-GCAUUCAGGAGUAAAUGCACA-3’(SEQ ID NO.6)。The antisense strand is 5'-GCAUUCAGGAGUAAAUGCACA-3' (SEQ ID NO.6).

3、QPCR检测FAM189A2的表达水平3. QPCR detection of the expression level of FAM189A2

各组细胞转染培养48h后，使用Trizol法提取细胞总RNA，按照实施例1中的方法进行逆转录以及实时定量PCR检测。After the cells of each group were transfected and cultured for 48 hours, the total RNA of the cells was extracted using the Trizol method, and reverse transcription and real-time quantitative PCR detection were performed according to the method in Example 1.

4、统计学方法4. Statistical methods

计量资料以均数±标准差表示，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。The measurement data were expressed as mean ± standard deviation, the difference between the two was tested by t test, and it was considered statistically significant when P<0.05.

5、结果5. Results

以FAM189A2在空白对照组的表达水平作为基准定为1，转染结果显示如表4所示，实验组的siRNA对FAM189A2具有较好的沉默效果。Taking the expression level of FAM189A2 in the blank control group as the benchmark as 1, the transfection results showed that as shown in Table 4, the siRNA in the experimental group had a better silencing effect on FAM189A2.

表4 FAM189A2的相对表达量Table 4 Relative expression of FAM189A2

实施例3 FAM189A2的功能验证Example 3 Function verification of FAM189A2

将生长状态良好的SH-SY5Y细胞接种到96孔板上，待细胞贴壁。经细胞分为四组分别处理，空白组(不处理)，模型组(4mM的MPP⁺)，阴性对照组(4mM的MPP⁺，转染siRNA-NC，)，治疗组(4mM的MPP⁺，转染siRNA-FAM189A2)。The SH-SY5Y cells in good growth state were seeded on 96-well plates and waited for the cells to adhere to the wall. The cells were divided into four groups and treated respectively, blank group (no treatment), model group (4mM MPP⁺ ), negative control group (4mM MPP⁺ , transfected with siRNA-NC), treatment group (4mM MPP⁺ , transfected with siRNA-FAM189A2).

于培养箱内培养48h后，弃培养液加入150μl(浓度为0.5mg/ml)的MTT溶液于培养箱内反应4h，弃上清液后，加入二甲基亚砜150μl振荡反应10min使MTT结晶充分溶解，采用酶标仪检测各组细胞的吸光度值，检测波长为490nm，计算细胞存活率。计算公式如下:细胞活性百分比％＝实验组OD值/对照组OD值*100％。After culturing in the incubator for 48 hours, discard the culture solution and add 150 μl (concentration: 0.5 mg/ml) of MTT solution in the incubator to react for 4 hours. After discarding the supernatant, add 150 μl of dimethyl sulfoxide and shake for 10 minutes to crystallize MTT Fully dissolve, use a microplate reader to detect the absorbance value of the cells in each group, the detection wavelength is 490nm, and calculate the cell survival rate. The calculation formula is as follows: % of cell activity=OD value of experimental group/OD value of control group*100%.

所有实验重复3次，计量资料以均数±标准差表示，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。All experiments were repeated 3 times, and the measurement data were expressed as mean ± standard deviation. The difference between the two was tested by t test, and it was considered statistically significant when P<0.05.

结果如表5所示，以空白对照组的细胞存活率作为100％，治疗组的细胞存活率显著增加，说明FAM189A2可应用于帕金森的治疗。The results are shown in Table 5. Taking the cell survival rate of the blank control group as 100%, the cell survival rate of the treatment group increased significantly, indicating that FAM189A2 can be applied to the treatment of Parkinson's disease.

表5细胞存活率Table 5 Cell viability

上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。The description of the above embodiments is only for understanding the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

序列表 sequence listing

<110> 天津医科大学总医院<110> Tianjin Medical University General Hospital

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Claims (11)

Translated fromChinese

1.检测FAM189A2水平的试剂在制备诊断帕金森病的产品中的应用。1. Application of a reagent for detecting the level of FAM189A2 in the preparation of a product for diagnosing Parkinson's disease.2.根据权利要求1所述的应用，其特征在于，所述试剂包括通过RT-PCR、实时定量PCR、免疫检测、原位杂交或芯片检测FAM189A2基因表达水平的试剂。2. The application according to claim 1, wherein the reagents include reagents for detecting the expression level of the FAM189A2 gene by RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization or chips.3.根据权利要求2所述的应用，其特征在于，所述试剂包括特异性针对FAM189A2探针、引物和/或抗体。3. The application according to claim 2, characterized in that the reagents include probes, primers and/or antibodies specific for FAM189A2.4.根据权利要求3所述的应用，其特征在于，特异性针对FAM189A2的引物序列如SEQ IDNO.1～2所示。4. The application according to claim 3, characterized in that the sequences of the primers specific to FAM189A2 are shown in SEQ ID NO.1-2.5.FAM189A2在筛选治疗帕金森的候选药物中的应用。5. Application of FAM189A2 in screening candidate drugs for Parkinson's disease.6.FAM189A2的抑制剂在制备治疗帕金森病药物中的应用。6. The application of the inhibitor of FAM189A2 in the preparation of drugs for treating Parkinson's disease.7.根据权利要求6所述的应用，其特征在于，所述抑制剂为降低FAM189A2的表达水平的试剂。7. The application according to claim 6, characterized in that the inhibitor is a reagent for reducing the expression level of FAM189A2.8.根据权利要求7所述的应用，其特征在于，所述试剂选自核酸分子、脂类、小分子化学药、核酸构建体。8. The application according to claim 7, wherein the reagent is selected from nucleic acid molecules, lipids, small molecule chemicals, and nucleic acid constructs.9.根据权利要求8所述的应用，其特征在于，所述核酸分子为干扰RNA。9. The application according to claim 8, wherein the nucleic acid molecule is an interfering RNA.10.根据权利要求9所述的应用，其特征在于，干扰RNA的序列如SEQ IDNO.5～6所示。10. The application according to claim 9, characterized in that the sequence of the interfering RNA is as shown in SEQ ID NO.5-6.11.根据权利要求6-10任一项所述的应用，其特征在于，所述药物还包括药学上可接受的载体。11. The use according to any one of claims 6-10, characterized in that the medicine further comprises a pharmaceutically acceptable carrier.

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