CN111690732B - GSK-3β作为血液标志物在制备精神障碍早期诊断试剂中的应用 - Google Patents

GSK-3β作为血液标志物在制备精神障碍早期诊断试剂中的应用 Download PDF

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CN111690732B
CN111690732B CN202010507932.0A CN202010507932A CN111690732B CN 111690732 B CN111690732 B CN 111690732B CN 202010507932 A CN202010507932 A CN 202010507932A CN 111690732 B CN111690732 B CN 111690732B
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储丹丹
顾锦华
伍倩
沈鑫
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Abstract

本发明公开了GSK‑3β作为血液标志物在制备精神障碍疾病，特别是孤独症谱系障碍(ASD)诊断试剂中的应用。GSK‑3β可以作为ASD早期诊断试剂，检测时间窗为新生儿0‑3个月。本发明可以仅使用少量血液样品，进行ASD的非侵入性辅助诊断，有利于大规模的婴幼儿ASD筛查，便于ASD的早发现、早干预、早治疗。

Description

GSK-3β作为血液标志物在制备精神障碍早期诊断试剂中的应用
技术领域
本发明属于生物医药领域，具体涉及GSK-3β作为血液标志物在制备精神障碍，特别是孤独症谱系障碍早期诊断试剂中的应用。
背景技术
糖原合成酶激酶3(GSK-3)是一种丝/苏氨酸蛋白激酶，通过Wnt/-catenin等多条信号通路来调节机体的代谢、生长发育和凋亡等过程，是机体生存必不可少的物质。大量研究表明，GSK-3调节异常可以激活特定细胞通路和环路，从而诱发精神障碍，如双向情感障碍、抑郁症、孤独症和精神分裂症等。哺乳动物的GSK-3有两种亚型:GSK-3α和GSK-3β，在脑组织中呈现高水平表达。GSK-3α主要表达于大脑皮层、海马体、纹状体和小脑的浦肯野细胞，而GSK-3β则是几乎在所有脑区中均表达。
孤独症谱系障碍(autism spectrum disorders,ASD)是一类复杂的神经发育障碍疾病。ASD儿童3岁之前发病，核心症状为社会交往能力损伤、行为模式刻板重复，并常伴认知和语言损伤、焦虑/抑郁、注意力不集中/亢奋等症状(Association,A.P.(2013)."Diagnostic and Statistical Manual of Mental Disorders.5th ed."DSM-5.Butler,M.G.,M.J.Dasouki,et al.(2005)."Subset of individuals with autism spectrumdisorders and extreme macrocephaly associated with germline PTEN tumoursuppressor gene mutations."J Med Genet 42(4):318-321.)。我国儿童ASD患病率约为2.4‰-3.5‰(戴琼,徐海青,et al.(2017)."2000-2016年中国儿童孤独症谱系障碍患病率Meta分析."中国儿童保健杂志:1246。石慧峰,张敬旭,et al.(2017)."中国0～6岁儿童孤独症谱系障碍患病率的meta分析."北京大学学报(医学版)(5):56-64.)，呈逐年上升态势，是造成儿童精神残疾的最主要原因(中华人民共和国卫生部(2010)."儿童孤独症诊疗康复指南.")。大量研究和实践表明，早期干预可明显改善患儿预后。但孤独症患儿往往在2-4岁才被发现和诊断，甚至5-6岁或学前才被诊断，错过了最佳的干预时期。其主要原因是临床无特异性生物学指标和客观有效的诊断方法。目前，孤独症的诊断主要依靠量表，主观性强，难以实现早期诊断。寻找快速、有效的孤独症诊断方法是当前孤独症领域的研究热点。
现有技术报道的与精神障碍有关标志物的研究多集中在脑组织，由于血脑屏障和组织差异性的存在，这些相关蛋白在血液中的表达情况并不明确。
发明内容
本发明以VPA诱导的孤独症模型鼠为研究对象，筛查了多个已知与健康人群脑组织中表达存在差异的蛋白在外周血液中的表达情况，发现孕期暴露VPA的新生小鼠(对应人类新生儿)血液中GSK-3β水平出现明显下降，而GSK-3α、pS9-GSK3β和β-catenin水平没有明显变化，提示GSK3β可作为有效的ASD血液标志物。
本发明技术方案具体如下：
GSK-3β作为血液标志物在制备精神障碍疾病诊断试剂中的应用。所述精神障碍疾病选自双向情感障碍、抑郁症、孤独症谱系障碍或精神分裂症。优选孤独症谱系障碍。
本发明所述的应用，所述诊断试剂为孤独症谱系障碍早期诊断试剂，检测时间窗为新生儿0-3个月。
本发明所述的应用，可检测血液样本GSK-3β基因或者蛋白表达水平。
本发明优点：
ASD是神经系统疾病，但尚缺乏有效的检测标志物。PI3K通路在ASD中脑中有激活现象，但对3岁以内的婴幼儿，颅内标志物检测的配合度很低。并且由于检测设备和家长意愿所限，颅内检测也不利于推广至大规模筛查。本发明研究发现GSK-3β可以作为ASD早期血液标志物，使用少量血液样品，进行ASD的非侵入性辅助诊断，有利于大规模的婴幼儿ASD筛查，便于ASD的早发现、早干预、早治疗。
附图说明
图1为ASD新生鼠Western Blotting电泳结果。
图2为ASD新生鼠Western Blotting半定量检测GSK-3α、GSK3β、pS9-GSK3β和β-catenin的相对表达水平。
图3为出生后第五天、第十五天、第三十天的ASD模型鼠Western Blotting电泳结果。
图4为ASD模型鼠Western Blotting半定量检测GSK-3α、GSK3β、pS9-GSK3β和β-catenin的相对表达水平。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不受实施例限制。
在本发明中所使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实例并参照数据进一步详细描述本发明。应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
实施例1
根据文献报道构建ASD模型小鼠(Lieberman,O.J.,V.Cartocci,et al.(2020)."mTOR Suppresses Macroautophagy During Striatal Postnatal Development and IsHyperactive in Mouse Models of Autism Spectrum Disorders."Front Cell Neurosci14:70.)。构建方法：两月龄C57/BL6J小鼠雌雄比例1:1配种。雌鼠怀孕12天腹腔注射600mg/kg VPA。
新生小鼠断头取血200μl，加入5倍体积裂解液(50mM Tris-HCl,pH7.4，8.5％Sucrose，10mM β-ME，2mM EDTA，50mM NaF，1mM Na3VO4和蛋白酶抑制aprotinin,pepstatin,leupeptin各10μg/ml，1.0mM 4-(2-aminoethyl)benzenesulfonyl fluoride hy-drochloride(AEBSF))，于冰水浴中研磨裂解。裂解后，加入等体积2×Laemmli SDS samplebuffer，沸水浴煮5min。冷却后的样品使用PierceTM660nm Protein Assay kit(ThermoFisher Scientific)测定蛋白浓度，调整浓度统一为2mg/ml，冻存于-80度备用。常规方案进行western blot：配制10％的聚丙烯酰胺凝胶，上样20μg进行电泳。电泳结束后，将聚丙烯酰胺凝胶上的蛋白样品转印至PVDF(polyvinylidene fluoride)膜上，于含5％牛奶的TBS(Tris Buffered saline)缓冲液中封闭30分钟。封闭结束后，取新鲜含5％牛奶的TBS，加入相应浓度抗体，4度孵育过夜。抗体信息如下：GSK3α(#4818，cell signalingtechnology，CST，1:1000)，GSK3β(#9315，CST，1:1000)，pS9-GSK3β(#5558，CST，1:1000)，actin(60008-1-Ig，proteintech,1:5000)，β-catenin(#8480，CST,1:250)。TBST(含0.6％Tween-20的TBS缓冲液)清洗膜3次，每次10分钟。取新鲜含5％牛奶的TBS，1:5000加入二抗HRP Goat Anti-Rabbit IgG(#111-035-003，Jackson Lab，1:5000)或HRP Goat Anti-Mouse IgG(#115-035-003，Jackson Lab,1:5000)，与膜室温孵育2小时。之后取出膜，TBST清洗3次，每次10分钟。加上化学发光液PierceTMECL Western Blotting Substrate，暗室显影。
结果如图1和2所示。结果表明在新生ASD模型小鼠的血液中western blot检测GSK-3α、GSK3β、pS9-GSK3β和GSK3β下游效应物之一β-catenin，发现仅GSK3β发生了显著下降，其他指标均无明显改变。该结果提示GSK3β可作为有效的ASD血液标志物。
为确定GSK3β作为ASD血液标志物的有效时间窗，对ASD模型鼠出生后第五天、第十五天、第三十天的GSK-3α、GSK3β、pS9-GSK3β和β-catenin的表达情况进行检测，结果如图3和4所示。结果显示出生后第五天的ASD模型小鼠血液GSK3β显著下降，而在出生十五天和三十天的ASD小鼠血液中GSK3β无差异。鉴于鼠出生后第五天相当于人类婴儿三月龄、第十五天相当于人类九月龄，根据上述研究结果建议以GSK3β含量降低作为ASD的血液标志物进行检测时，其最佳检测时间窗口在新生儿至三月龄之间，而九月龄之后指示效果不佳。

Claims (1)

1.血液标志物GSK-3β的检测试剂在制备孤独症谱系障碍早期诊断试剂中的应用，检测时间窗为新生儿0-3个月，检测血液样本GSK-3β基因或者蛋白表达水平。
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