CN114137192B - 7-甲基黄嘌呤作为检测靶点在制备2型糖尿病高危个体筛查试剂盒中的应用 - Google Patents
7-甲基黄嘌呤作为检测靶点在制备2型糖尿病高危个体筛查试剂盒中的应用 Download PDFInfo
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Abstract
本发明公开了7‑甲基黄嘌呤作为检测靶点在制备2型糖尿病高危个体筛查试剂盒中的应用。7‑甲基黄嘌呤作为检测靶标在制备2型糖尿病高危个体血清/血浆辅助筛查试剂中的应用。定量检测7‑甲基黄嘌呤的试剂在制备2型糖尿病血清/血浆辅助筛查试剂中的应用。本发明发现7‑甲基黄嘌呤增加是与未来2型糖尿病发病高度相关的高特异性和敏感性的代谢分子,其单独预测2型糖尿病的AUC为0.768;传统危险因素预测2型糖尿病的AUC为0.797;加入代谢物7‑甲基黄嘌呤后AUC上升至0.880,对2型糖尿病发生具有很好预测价值;为2型糖尿病高危个体的早期筛查提供技术支持。
Description
技术领域
本发明属于代谢组学及公共卫生与预防医学领域,涉及7-甲基黄嘌呤作为检测靶点在制备2型糖尿病高危个体筛查试剂盒中的应用。
背景技术
糖尿病属严重影响人民生命健康的重大慢性病,发病、死亡人数不断增加,且呈年轻化趋势,我国18岁及以上成人患病率从1980年的0.67%飙升至2017年的11.2%;年龄标化后死亡率呈上升趋势。糖尿病会引起脑卒中、冠心病、失明、足坏疽等严重并发症,致残、致死率高。糖尿病中,90%以上为2型糖尿病(Type 2Diabetes)。如能早期发现2型糖尿病高危个体并进行干预,可有效降低2型糖尿病及其严重并发症发生,降低该病给患者及其家庭、社会带来的疾病痛苦和经济负担。然而,目前为止在防控实践中,2型糖尿病高危个体筛查技术有限,只能基于年龄、肥胖、糖尿病家族史、血糖、血脂等传统危险因素,存在灵敏度和特异度低、信息偏倚、信息滞后等不足。
代谢组学(Metabolomics)是一门新兴的组学技术,其研究的是代谢物,被称为最接近表型的组学。近年来代谢组学成功应用于各类疾病的早期预测,成为转化医学疾病敏感预测标志物筛选的备受关注的组学平台,代谢组学用于2型糖尿病等重大慢病的筛查及发病机制研究已成为一个热门的新兴领域。2型糖尿病属于慢性代谢性疾病,在血糖、血脂异常前,由代谢紊乱对机体造成的危害就已发生。因此,研究探寻2型糖尿病发病相关的早期生物标志物 (Biomarker),可为高危个体的筛查和实施干预提供有效手段,对于遏制糖尿病发病、死亡不断上升势头具有重大的科学意义。
发明内容
本发明的首要目的是针对上述技术问题,提供一种与2型糖尿病高危个体筛查相关的血清/血浆代谢分子标志物。
本发明第二个目的是为将血清/血浆代谢分子标志物用于2型糖尿病高危个体筛查提供基于UPLC-Q exactive MS的应用。
本发明第三个目的是提供用于2型糖尿病高危个体早期筛查的试剂盒。
本发明的目的是通过下列技术方案实现的:
7-甲基黄嘌呤作为检测靶标在制备2型糖尿病高危个体血清/血浆辅助筛查试剂中的应用。
定量检测7-甲基黄嘌呤的试剂在制备2型糖尿病血清/血浆辅助筛查试剂中的应用。
一种2型糖尿病高危个体血清/血浆辅助筛查试剂盒,该试剂盒含有7-甲基黄嘌呤标准品。
作为本发明的一种优选,所述的辅助诊断试剂盒含有7-甲基黄嘌呤标准品,配套Hypersil GOLD C18色谱柱(100mm×2.1mm,粒径1.9um Thermo Scientific)、试剂A(沉淀蛋白用,含100%甲醇)、试剂B(流动相用,含0.1%的甲酸的水)、试剂C(流动相用,含0.1%的甲酸的乙腈),试剂D(复溶用,100%超纯水)。
本发明的发明点在于发现7-甲基黄嘌呤可以作为2型糖尿病高危个体辅助筛查相关的血清/血浆代谢分子标志物。具体地说,7-甲基黄嘌呤作为血清/血浆代谢分子标志物通过以下方法筛选得到:(1)建立统一标准的标本库和数据库:以标准操作程序(SOP)采集符合标准的血液样本,***收集完整的人口学资料和临床资料,对研究对象临床结局进行随访。(2)2型糖尿病患者血清代谢组学分析:选择12年随访期间新发的2型糖尿病病例、与病例年龄匹配的健康对照,采用UPLC-Q exactive MS方法检测病例及对照组,基线时血清中各代谢物的含量,分析2型糖尿病病例和健康女性对照间代谢物共性和特性,筛选差异表达代谢物。(3)联合7-甲基黄嘌呤和人口社会学及临床特征信息,构建2型糖尿病发病风险预测模型。
本发明的有益效果:
本发明提供的血清/血浆代谢分子标志物7-甲基黄嘌呤作为2型糖尿病高危个体筛查标志物的优越性在于:
本发明基于巢式病例-对照研究,于12年前以标准操作程序(SOP)采集了符合研究对象标准的血液样本,同时***收集有完整的流行病学调查信息、临床信息和随访信息;采用基于UPLC-Q exactive MS的代谢组学方法,通过研究新发2 型糖尿病病例和与其年龄匹配的健康对照基线时血清中代谢物的差异,发现7-甲基黄嘌呤增加是与未来2型糖尿病发病高度相关的高特异性和敏感性的代谢分子。本发明发现7- 甲基黄嘌呤单独预测2型糖尿病的AUC为0.768;传统危险因素(年龄、性别、体质指数、腰围、吸烟、饮酒、家族史、收缩压、舒张压、高密度脂蛋白胆固醇、甘油三酯、空腹血糖)预测2型糖尿病的AUC为0.797;加入代谢物7-甲基黄嘌呤后AUC上升至0.880(如附图),对2型糖尿病发生具有很好预测价值;为2型糖尿病高危个体的早期筛查提供技术支持。
本发明在前瞻性队列人群中,采用巢式病例对照研究,以随访12年中新发的2型糖尿病病例和匹配健康对照为研究对象,检测比较基线时血清代谢物,发现7-甲基黄嘌呤增加与未来2型糖尿病发病相关,可用于早期识别、筛查2型糖尿病高危个体。以上方法和策略的应用加速和保证了血清/血浆代谢分子标志物7-甲基黄嘌呤筛查试剂盒的应用,也为其他疾病生物标志物的研制提供方法和策略上的借鉴。
本发明获得了2型糖尿病发病特异性血清/血浆代谢分子标志物;通过血清/血浆代谢分子标志物7-甲基黄嘌呤检测试剂盒的研制和应用,可使得2型糖尿病筛查更加方便易行,为及早筛查出2型糖尿病高危个体实施精准干预奠定基础,并为发现具有潜在治疗价值的新型小分子药物靶标提供帮助。
附图说明
图1 7-甲基黄嘌呤对2型糖尿病发病风险预测价值,,AUC:0.768(95%CI:0.720-0.817)
图2传统危险因素对2型糖尿病发病风险预测价值,AUC:0.797(95%CI:0.756-0.840)
图3 7-甲基黄嘌呤+传统危险因素联合对2型糖尿病发病风险预测价值,AUC:0.880 (95%CI:0.849-0.913)
本发明发现7-甲基黄嘌呤单独预测2型糖尿病的AUC为0.768(95%CI: 0.720-0.817)。传统危险因素(年龄、性别、体质指数、腰围、吸烟、饮酒、家族史、收缩压、舒张压、高密度脂蛋白胆固醇、甘油三酯、空腹血糖)预测2型糖尿病的AUC为0.797(95%CI:0.756-0.840);代谢物7-甲基黄嘌呤和传统危险因素(年龄、性别、体质指数、腰围、吸烟、饮酒、家族史、收缩压、舒张压、高密度脂蛋白胆固醇、甘油三酯、空腹血糖)共同预测2型糖尿病的AUC上升至 0.880(95%CI:0.849-0.913),两者之间差异有统计学意义(P=1.74×10-6)。
具体实施方式
实施例1研究样本的收集和资料整理
发明人于2007年4-6月,从无锡市梁溪区江海社区和北大街社区中收集了10867例35岁及以上本地居民晨起空腹血样,采集后立即4000r/min离心,取1.5ml血清分装至冻存管保存于-80℃冰箱;同期完成流行病学问卷调查(包括人口社会学信息、行为和生活方式、疾病史和家族史)、体格检查(测量身高、体重、腰围、血压)。通过开展被动随访和主动随访,了解是否发生2型糖尿病。
(1)2型糖尿病病例组:基线调查时空腹血糖小于7.0mmol/L,无糖尿病、心脑血管疾病、肿瘤病史,随访期间被诊断为2型糖尿病(按WHO诊断标准),随机选取220例纳入研究。
(2)健康对照组:基线调查和随访期间未发生糖尿病、心脑血管疾病和肿瘤,2019年健康档案体检空腹血糖小于7.0mmol/L,按年龄(±5岁)、性别与病例匹配,共220例。
实施例2 2型糖尿病病例与匹配对照差异代谢谱分析
将上述符合条件的220例2型糖尿病病例和220例健康对照经代谢组学检测获得相关结果。具体实验方法如下:
1.1仪器
UPLC Ultimate 3000system(Dionex)高效液相色谱仪;Q-Exactive三重四极杆串联质谱仪;TraceFinder 3.1(Thermo Fisher Scientific);Simca P 13.0(瑞典Umetrics公司);XW-80A 漩涡混合器(上海青浦沪西仪器厂);电子天平(MettlerAE240型);KQ3200B型超声波清洗器(昆山市超声仪器有限公司)。
1.2试剂
1579种代谢物标准品(≥98.0%,Sigma–Aldrich,St.Louis,MO,USA);甲醇、乙腈(≥99.9%, Merck,German);甲酸(≥98.0%,Sigma–Aldrich,St.Louis,MO,USA);正己烷(色谱级,≥98.0%;氯仿(≥99.9%);超纯水(PURELAB Ultra纯水仪);液氮(≥99.9%)。
1.3标准品溶液的制备和标准品库建立
准确称取各代谢物标准品适量,以甲醇,正己烷或者氯仿溶解,得到浓度为1mg/mL的标准品贮备液,贮备液经过稀释得到100μg/mL的标准品工作液。将所有溶解的单个标准品贮备液混成1μg/mL混合标准品贮备液,混合标准品贮备液经过稀释得到100ng/mL的混合标准品工作液,并于-20℃冰箱中保存。
1.4代谢物提取
取100μL血清置于EP管中,加入300μL质谱级甲醇沉淀蛋白,涡旋震荡,冰浴静置5min, 15000rpm、4℃离心10min,取一定量的上清转移至1.5ml EP管,并将上清液在室温条件下离心浓缩干燥,用200μL超纯水复溶,进样液相质谱进行代谢物检测。从每个实验样本中取等体积样本混匀作为QC样本。blank样本为含0.1%甲酸的53%甲醇水溶液代替实验样本,前处理过程与实验样本相同,以去除背景离子。
1.5超高效液相色谱串联质谱分析
1.5.1色谱条件:Hypersil GOLD C18色谱柱(100mm×2.1mm,粒径1.9μm ThermoScientific,Germany),流动相A为乙腈(含0.1%甲酸)和流动相B为超纯水(含0.1%甲酸),流速为0.40ml/min,采用梯度洗脱的方式,流动相梯度变化见Table 1.1,柱温:40℃,进样量: 5μL。
表1流动相梯度
1.5.2质谱条件:采用加热电喷雾电离方式(HESI),正离子模式喷雾电压:3.5kV;负离子模式喷雾电压:2.5kV;两种模式下毛细管温度:250℃,加热器温度:425℃,鞘气气流:50AU,辅助气气流:13AU,反吹气气流:0AU;透镜电压:60V。采用全扫模式,扫描范围:70to 1050m/z;分辨率:70000。
1.6物质鉴定
采用TraceFinder 3.1(Thermo Fisher Scientific)软件通过与代谢物标准品精确分子量和保留时间比较,对代谢物进行定性,并相对定量。
1.7代谢组学分析结果
用多因素Logistic回归分析来评估这些代谢物与2型糖尿病的发病风险。运用受试者工作特征曲线下面积(AUC)评价代谢物用于发病风险预测的价值。我们在220例2型糖尿病病例和220例健康对照中运用UPLC-Q exactive MS的方法初步筛选出79个代谢物,其中经多重比较后有5个代谢物具有显著差异且7-甲基黄嘌呤差异最为显著。7-甲基黄嘌呤增加是与2 型糖尿病发病高度相关的高特异性和敏感性的代谢分子(AUC为0.768);运用传统危险因素(年龄、性别、体质指数、腰围、吸烟、饮酒、家族史、收缩压、舒张压、高密度脂蛋白胆固醇、甘油三酯、空腹血糖)对上述样本进行2型糖尿病的发病风险分析,ROC曲线AUC为0.797,联合传统危险因素(年龄、性别、体质指数、腰围、吸烟、饮酒、家族史、收缩压、舒张压、高密度脂蛋白胆固醇、甘油三酯、空腹血糖)和代谢物7-甲基黄嘌呤,ROC曲线AUC 上升至0.880。
实施例3用于2型糖尿病高危个体筛查7-甲基黄嘌呤检测试剂盒的制作
此试剂盒包括7-甲基黄嘌呤检测用试剂和耗材,其中定性和定量采用7-甲基黄嘌呤的标准品。其它还有用于UPLC色谱分离的配套反向色谱柱(Hypersil GOLD C18色谱柱,100 mm×2.1mm,粒径1.9μm)、用于沉淀血浆蛋白的试剂(100%甲醇),用于流动相的试剂(含 0.1%的甲酸的水和含0.1%甲酸的乙腈),用于提取7-甲基黄嘌呤的试剂(100%超纯水)。此试剂盒的价值在于只需要100μl血清,即可检测7-甲基黄嘌呤的含量,再通过含量筛选2型糖尿病高危个体。
具体试剂盒组成如下:
7-甲基黄嘌呤标准品
色谱柱(Thermo 100mm×2.1mm,粒径1.9μm,Hypersil GOLD C18色谱柱)
试剂A(含100%甲醇)
试剂B(含0.1%的甲酸的水)
试剂C(含0.1%的甲酸的乙腈)
试剂D(100%超纯水)。
Claims (2)
1.7-甲基黄嘌呤作为标准品在制备2 型糖尿病高危个体筛查试剂盒中的应用。
2.定量检测7-甲基黄嘌呤的试剂在制备2 型糖尿病血清/血浆辅助筛查试剂中的应用;
该辅助筛查试剂中含有UPLC-Q exactive MS 方法检测7-甲基黄嘌呤的其他试剂;
该辅助筛查试剂中含有7-甲基黄嘌呤标准品,配套Hypersil GOLD C18 色谱柱,规格为100mm×2.1mm, 粒径1.9um ThermoScientific,试剂A:含100%甲醇,试剂B:含0.1%的甲酸的水,试剂C:含0.1%甲酸的乙腈,试剂D:超纯水。
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