尿液或尿液外泌体中肌肉来源6磷酸果糖激酶及其多肽片段在糖尿病中的应用

技术领域

本发明涉及尿液或尿液外泌体中肌肉来源6磷酸果糖激酶及其多肽片段的新用途，具体涉及尿液或尿液外泌体中肌肉来源6磷酸果糖激酶及其多肽片段在糖尿病诊断、鉴别诊断、病情程度判断、治疗效果评价、监测、预后评估及机理研究等制剂的应用。

背景技术

糖尿病是一种以葡萄糖和脂肪代谢紊乱、血浆葡萄糖水平增高为特征的，由遗传与环境因素共同作用而产生的代谢性内分泌疾病。近年来，全球人口糖尿病发病率增长迅速，已成为21世纪最具挑战的世界性健康问题。随着生活水平的提高和生活方式的转变，我国糖尿病的发病率呈快速上升趋势，统计结果表明目前我国糖尿病患者数量位居世界第二，预计至2040年国内糖尿病人口将高达6.42亿。

糖尿病高血糖可以导致碳水化合物、蛋白质以及脂肪的代谢紊乱，从而引起神经、肾脏及心血管等多系统并发症，这些都为公众健康问题带来了巨大的隐患。糖尿病发病较为隐匿，多数患者发现不及时，往往因出现并发症的临床表现才得以确诊。除此之外，糖尿病患者病程较长，需长期甚至终身监测血糖，反复抽血有创检查给患者造成诸多不便。但是，糖尿病患者若未给予及时诊治，随着病情进展常出现全身多个脏器损害，严重者甚至因并发症而致残和死亡。综上所述，糖尿病不仅给患者造成不便和痛苦，也会给患者家庭与社会带来沉重的负担。为提高糖尿病患者的生活质量，解决患者的切实问题，研究一种无创、方便、快捷、易重复的糖尿病检查方法极为必要。

肌肉来源6磷酸果糖激酶 (ATP-PFK )是糖代谢中的关键酶，在机体糖代谢中起着至关重要的作用，是人体糖酵解中的重要一步。在本研究中糖尿病患者尿液或尿液外泌体中肌肉来源6磷酸果糖激酶含量较健康人组出现表达下调，含量降低。

与常用的临床血液样本相比，尿液可以完全无创、连续、大量收集；没有稳态调节，可累积更多种类、更大幅度的变化，机体的很多病理生理变化可能体现在尿液中。一些激素和细胞因子等分子量相对较小的蛋白多肽入血后，会很快被排泄进入尿液，这些蛋白和多肽在尿液中被检测到的概率比在血中大很多；尿液收集之前，尿中可能的蛋白降解过程已经完成，所以尿蛋白可在较长时间内保持稳定。为减轻糖尿病患者多次采血的痛苦，本实验在前期方法学摸索的基础上，期望通过尿液蛋白或多肽研究，实现用无痛、方便、快捷、易重复的尿液检测辅助糖尿病患者的诊断及病情监测，也为进一步尿液多肽检测试剂盒的研究奠定基础。

发明内容

本发明的目的在于提供一种尿液或尿液外泌体中肌肉来源6磷酸果糖激酶及其多肽片段在制备用于糖尿病诊断、鉴别诊断、病情程度判断、治疗效果评价、监测、预后评估及机理研究等制剂的应用。

优选地，所述尿液或尿液外泌体中肌肉来源6磷酸果糖激酶的氨基酸序列包括SEQID NO.1所示（MTHEEHHAAK TLGIGKAIAV LTSGGDAQGM NAAVRAVVRV GIFTGARVFF VHEGYQGLVDGGDHIKEATW ESVSMMLQLG GTVIGSARCK DFREREGRLR AAYNLVKRGI TNLCVIGGDG SLTGADTFRSEWSDLLSDLQ KAGKITDEEA TKSSYLNIVG LVGSIDNDFC GTDMTIGTDS ALHRIMEIVD AITTTAQSHQRTFVLEVMGR HCGYLALVTS LSCGADWVFI PECPPDDDWE EHLCRRLSET RTRGSRLNII IVAEGAIDKNGKPITSEDIK NLVVKRLGYD TRVTVLGHVQ RGGTPSAFDR ILGSRMGVEA VMALLEGTPD TPACVVSLSGNQAVRLPLME CVQVTKDVTK AMDEKKFDEA LKLRGRSFMN NWEVYKLLAH VRPPVSKSGS HTVAVMNVGAPAAGMNAAVR STVRIGLIQG NRVLVVHDGF EGLAKGQIEE AGWSYVGGWT GQGGSKLGTK RTLPKKSFEQISANITKFNI QGLVIIGGFE AYTGGLELME GRKQFDELCI PFVVIPATVS NNVPGSDFSV GADTALNTICTTCDRIKQSA AGTKRRVFII ETMGGYCGYL ATMAGLAAGA DAAYIFEEPF TIRDLQANVE HLVQKMKTTVKRGLVLRNEK CNENYTTDFI FNLYSEEGKG IFDSRKNVLG HMQQGGSPTP FDRNFATKMG AKAMNWMSGKIKESYRNGRI FANTPDSGCV LGMRKRALVF QPVAELKDQT DFEHRIPKEQ WWLKLRPILK ILAKYEIDLDTSDHAHLEHI TRKRSGEAAV）、SEQ ID NO.2所示 (MTHEEHHAAK TLGIGKAIAV LTSGGDAQGMNAAVRAVVRV GIFTGARVFF VHEGYQGLVD GGDHIKEATW ESVSMMLQLG GTVIGSARCK DFREREGRLRAAYNLVKRGI TNLCVIGGDG SLTGADTFRS EWSDLLSDLQ KAGKITDEEA TKSSYLNIVG LVGSIDNDFCGTDMTIGTDS ALHRIMEIVD AITTTAQSHQ RTFVLEVMGR HCGYLALVTS LSCGADWVFI PECPPDDDWEEHLCRRLSET RTRGSRLNII IVAEGAIDKN GKPITSEDIK NGSRMGVEAV MALLEGTPDT PACVVSLSGNQAVRLPLMEC VQVTKDVTKA MDEKKFDEAL KLRGRSFMNN WEVYKLLAHV RPPVSKSGSH TVAVMNVGAPAAGMNAAVRS TVRIGLIQGN RVLVVHDGFE GLAKGQIEEA GWSYVGGWTG QGGSKLGTKR TLPKKSFEQISANITKFNIQ GLVIIGGFEA YTGGLELMEG RKQFDELCIP FVVIPATVSN NVPGSDFSVG ADTALNTICTTCDRIKQSAA GTKRRVFIIE TMGGYCGYLA TMAGLAAGAD AAYIFEEPFT IRDLQANVEH LVQKMKTTVKRGLVLRNEKC NENYTTDFIF NLYSEEGKGI FDSRKNVLGH MQQGGSPTPF DRNFATKMGA KAMNWMSGKIKESYRNGRIF ANTPDSGCVL GMRKRALVFQ PVAELKDQTD FEHRIPKEQW WLKLRPILKI LAKYEIDLDTSDHAHLEHIT RKRSGEAAV)、SEQ ID NO.3所示 (MHKDEFHLKF FMCVIQSRQL VRTPQRTAGEASTSSMLIPK PPPKTDILKS LDTMDDPDTV GSIPVFKTEW IMTHEEHHAA KTLGIGKAIA VLTSGGDAQGMNAAVRAVVR VGIFTGARVF FVHEGYQGLV DGGDHIKEAT WESVSMMLQL GGTVIGSARC KDFREREGRLRAAYNLVKRG ITNLCVIGGD GSLTGADTFR SEWSDLLSDL QKAGKITDEE ATKSSYLNIV GLVGSIDNDFCGTDMTIGTD SALHRIMEIV DAITTTAQSH QRTFVLEVMG RHCGYLALVT SLSCGADWVF IPECPPDDDWEEHLCRRLSE TRTRGSRLNI IIVAEGAIDK NGKPITSEDI KNLVVKRLGY DTRVTVLGHV QRGGTPSAFDRILGSRMGVE AVMALLEGTP DTPACVVSLS GNQAVRLPLM ECVQVTKDVT KAMDEKKFDE ALKLRGRSFMNNWEVYKLLA HVRPPVSKSG SHTVAVMNVG APAAGMNAAV RSTVRIGLIQ GNRVLVVHDG FEGLAKGQIEEAGWSYVGGW TGQGGSKLGT KRTLPKKSFE QISANITKFN IQGLVIIGGF EAYTGGLELM EGRKQFDELCIPFVVIPATV SNNVPGSDFS VGADTALNTI CTTCDRIKQS AAGTKRRVFI IETMGGYCGY LATMAGLAAGADAAYIFEEP FTIRDLQANV EHLVQKMKTT VKRGLVLRNE KCNENYTTDF IFNLYSEEGK GIFDSRKNVLGHMQQGGSPT PFDRNFATKM GAKAMNWMSG KIKESYRNGR IFANTPDSGC VLGMRKRALV FQPVAELKDQTDFEHRIPKE QWWLKLRPIL KILAKYEIDL DTSDHAHLEH ITRKRSGEAA )；或由SEQ ID NO:1~3所示的氨基酸序列衍生的，且与SEQ ID NO:1~3所示的氨基酸序列具有相同功能的氨基酸序列。

优选地，所述制剂为糖尿病患者尿液或尿液外泌体中肌肉来源6磷酸果糖激酶及其多肽片段检测试剂盒。

优选地，所述试剂盒包括抗原抗体反应的免疫方法及其试剂盒如能够特异性结合肌肉来源6磷酸果糖激酶及其多肽片段的适配体抗体或抗体片段中的一种或多种。

优选地，所述检测方法包括直接检测肌肉来源6磷酸果糖激酶及其多肽片段的质谱等方法及其相关试剂盒。

优选地，所述检测方法包括直接检测肌肉来源6磷酸果糖激酶及其多肽片段的相关核酸检测等方法及其相关试剂盒。

优选地，所述试剂盒还包括选自下组的成分：固相载体，稀释液，对照品，标准品，质控品，检测抗体，第二抗体、第二抗体稀释液，发光试剂，洗涤液、显色液、终止液中的任意一种或几种的组合。

优选地，所述标准品包括肌肉来源6磷酸果糖激酶标准品、人源化标签抗体标准品；较佳地，所述质控品包括：肌肉来源6磷酸果糖激酶质控品、人源化标签抗体质控品；较佳地，所述固相载体包括：微粒、微球、玻片、试纸条、塑料珠、液相芯片、微孔板或亲和膜等以及同等功能的其他载体。

优选地，所述固相载体的材质为聚氯乙烯、聚苯乙烯、聚丙酰胺、纤维素中的任意一种及具有类似功能的载体。

发明人首先收集了健康人、糖尿病患者的尿液标本，参照人类尿液蛋白质组计划(Human Urine Proteome Project，HKUPP)和欧洲肾脏和尿液蛋白质组（European Kidneyand Urine Proteomics, EuroKUP）标准操作程序对尿液外泌体的提取，通过透射电子显微镜、外泌体纳米微粒追踪检测及蛋白免疫印迹对尿液外泌体进行鉴定，通过液相二级质谱技术（liquid chromatography tandem mass spectrometry，LC-MS/MS)，采用数据非依赖性扫描模式（Data-independent acquisition，DIA），通过定量值校正/计算绝对定量值、差异分析及目标蛋白全局聚类分析对数据进行处理。将糖尿病组和正常对照组在质谱中得到的数据进行定量计算，最终获得差异蛋白肌肉来源6磷酸果糖激酶。

本发明通过研究证实与健康人相比，尿液或尿液外泌体中肌肉来源6磷酸果糖激酶及其多肽片段在糖尿病患者中呈低表达，与临床诊断有较好的一致性。从而提出检测尿液或尿液外泌体中肌肉来源6磷酸果糖激酶及其多肽片段可用于糖尿病的诊断、鉴别诊断、病情程度判断、治疗效果评价、监测、预后评估及机理研究等制剂的应用。

本发明发挥尿液标本获取无创、可大规模重复取样、保存方便的优势，利用尿液标本检测尿液或尿液外泌体中肌肉来源6磷酸果糖激酶及其多肽片段。

为让本发明的上述和其它目的、特征和优点能更明显易懂，下文特举较佳实施例，并配合附图，作详细说明如下。

附图说明

图1是尿液或尿液外泌体中肌肉来源6磷酸果糖激酶及其多肽片段在糖尿病组及健康对照组中含量图。

图2是尿液或尿液外泌体中肌肉来源6磷酸果糖激酶及其多肽片段在糖尿病组及健康对照组中ELISA验证结果图。

具体实施方式

实施例1

选取糖尿病患者作为糖尿病组，选取同期健康体检者作为正常对照组。收集各组研究对象入院后的新鲜晨尿样本，不能正常排尿者收集其早晨导尿管中尿液，置于干燥、洁净的容器内。参考HKUPP和EuroKUP (http://www.eurokup.org)的标准操作程序，提取浓缩后尿液样本中的外泌体，重悬在预冷的PBS中，保存于-80℃冰箱。通过透射电子显微镜、外泌体纳米微粒追踪检测与蛋白免疫印迹对尿液外泌体进行鉴定。

实施例2

对尿液样品进行蛋白提取及酶解脱盐，对提取的蛋白浓度进行测定。通过先进的液相二级质谱技术（LC-MS/MS)，采用数据非依赖性扫描模式（DIA），构建不同年龄段不同性别正常人群尿液外泌体蛋白质图谱。

实施例3

所有实验均进行三次，所有数值以平均值±标准偏差表示。Fold change 1.5倍，得到差异蛋白的分析结果，

与健康人相比，尿液或尿液外泌体中肌肉来源6磷酸果糖激酶在糖尿病患者中低表达，其在正常对照组和糖尿病患者组中含量如图1所示，表达具有显著性差异。

实施例4

使用酶联免疫吸附试验(ELISA)对健康人群及糖尿病患者尿液或尿液外泌体中肌肉来源6磷酸果糖激酶进行检测，结果如图2所示。尿液或尿液外泌体中肌肉来源6磷酸果糖激酶在糖尿病患者中含量降低，差异具有统计学意义（

虽然本发明已以较佳实施例披露如上，然其并非用以限定本发明，任何所属技术领域的技术人员，在不脱离本发明的精神和范围内，当可作些许的更动与改进，因此本发明的保护范围当视权利要求所界定者为准。

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<110> 张 曼

<120> 尿液肌肉来源6磷酸果糖激酶及其多肽片段在糖尿病中的应用

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Thr Ala Leu Asn Thr Ile Cys Thr Thr Cys Asp Arg Ile Lys Gln Ser

515 520 525

Ala Ala Gly Thr Lys Arg Arg Val Phe Ile Ile Glu Thr Met Gly Gly

530 535 540

Tyr Cys Gly Tyr Leu Ala Thr Met Ala Gly Leu Ala Ala Gly Ala Asp

545 550 555 560

Ala Ala Tyr Ile Phe Glu Glu Pro Phe Thr Ile Arg Asp Leu Gln Ala

565 570 575

Asn Val Glu His Leu Val Gln Lys Met Lys Thr Thr Val Lys Arg Gly

580 585 590

Leu Val Leu Arg Asn Glu Lys Cys Asn Glu Asn Tyr Thr Thr Asp Phe

595 600 605

Ile Phe Asn Leu Tyr Ser Glu Glu Gly Lys Gly Ile Phe Asp Ser Arg

610 615 620

Lys Asn Val Leu Gly His Met Gln Gln Gly Gly Ser Pro Thr Pro Phe

625 630 635 640

Asp Arg Asn Phe Ala Thr Lys Met Gly Ala Lys Ala Met Asn Trp Met

645 650 655

Ser Gly Lys Ile Lys Glu Ser Tyr Arg Asn Gly Arg Ile Phe Ala Asn

660 665 670

Thr Pro Asp Ser Gly Cys Val Leu Gly Met Arg Lys Arg Ala Leu Val

675 680 685

Phe Gln Pro Val Ala Glu Leu Lys Asp Gln Thr Asp Phe Glu His Arg

690 695 700

Ile Pro Lys Glu Gln Trp Trp Leu Lys Leu Arg Pro Ile Leu Lys Ile

705 710 715 720

Leu Ala Lys Tyr Glu Ile Asp Leu Asp Thr Ser Asp His Ala His Leu

725 730 735

Glu His Ile Thr Arg Lys Arg Ser Gly Glu Ala Ala Val

740 745

<210> 3

<211> 850

<212> PRT

<213> Human Urine

<400> 3

Met His Lys Asp Glu Phe His Leu Lys Phe Phe Met Cys Val Ile Gln

1 5 1015

Ser Arg Gln Leu Val Arg Thr Pro Gln Arg Thr Ala Gly Glu Ala Ser

202530

Thr Ser Ser Met Leu Ile Pro Lys Pro Pro Pro Lys Thr Asp Ile Leu

354045

Lys Ser Leu Asp Thr Met Asp Asp Pro Asp Thr Val Gly Ser Ile Pro

505560

Val Phe Lys Thr Glu Trp Ile Met Thr His Glu Glu His His Ala Ala

65707580

Lys Thr Leu Gly Ile Gly Lys Ala Ile Ala Val Leu Thr Ser Gly Gly

859095

Asp Ala Gln Gly Met Asn Ala Ala Val Arg Ala Val Val Arg Val Gly

100 105 110

Ile Phe Thr Gly Ala Arg Val Phe Phe Val His Glu Gly Tyr Gln Gly

115 120 125

Leu Val Asp Gly Gly Asp His Ile Lys Glu Ala Thr Trp Glu Ser Val

130 135 140

Ser Met Met Leu Gln Leu Gly Gly Thr Val Ile Gly Ser Ala Arg Cys

145 150 155 160

Lys Asp Phe Arg Glu Arg Glu Gly Arg Leu Arg Ala Ala Tyr Asn Leu

165 170 175

Val Lys Arg Gly Ile Thr Asn Leu Cys Val Ile Gly Gly Asp Gly Ser

180 185 190

Leu Thr Gly Ala Asp Thr Phe Arg Ser Glu Trp Ser Asp Leu Leu Ser

195 200 205

Asp Leu Gln Lys Ala Gly Lys Ile Thr Asp Glu Glu Ala Thr Lys Ser

210 215 220

Ser Tyr Leu Asn Ile Val Gly Leu Val Gly Ser Ile Asp Asn Asp Phe

225 230 235 240

Cys Gly Thr Asp Met Thr Ile Gly Thr Asp Ser Ala Leu His Arg Ile

245 250 255

Met Glu Ile Val Asp Ala Ile Thr Thr Thr Ala Gln Ser His Gln Arg

260 265 270

Thr Phe Val Leu Glu Val Met Gly Arg His Cys Gly Tyr Leu Ala Leu

275 280 285

Val Thr Ser Leu Ser Cys Gly Ala Asp Trp Val Phe Ile Pro Glu Cys

290 295 300

Pro Pro Asp Asp Asp Trp Glu Glu His Leu Cys Arg Arg Leu Ser Glu

305 310 315 320

Thr Arg Thr Arg Gly Ser Arg Leu Asn Ile Ile Ile Val Ala Glu Gly

325 330 335

Ala Ile Asp Lys Asn Gly Lys Pro Ile Thr Ser Glu Asp Ile Lys Asn

340 345 350

Leu Val Val Lys Arg Leu Gly Tyr Asp Thr Arg Val Thr Val Leu Gly

355 360 365

His Val Gln Arg Gly Gly Thr Pro Ser Ala Phe Asp Arg Ile Leu Gly

370 375 380

Ser Arg Met Gly Val Glu Ala Val Met Ala Leu Leu Glu Gly Thr Pro

385 390 395 400

Asp Thr Pro Ala Cys Val Val Ser Leu Ser Gly Asn Gln Ala Val Arg

405 410 415

Leu Pro Leu Met Glu Cys Val Gln Val Thr Lys Asp Val Thr Lys Ala

420 425 430

Met Asp Glu Lys Lys Phe Asp Glu Ala Leu Lys Leu Arg Gly Arg Ser

435 440 445

Phe Met Asn Asn Trp Glu Val Tyr Lys Leu Leu Ala His Val Arg Pro

450 455 460

Pro Val Ser Lys Ser Gly Ser His Thr Val Ala Val Met Asn Val Gly

465 470 475 480

Ala Pro Ala Ala Gly Met Asn Ala Ala Val Arg Ser Thr Val Arg Ile

485 490 495

Gly Leu Ile Gln Gly Asn Arg Val Leu Val Val His Asp Gly Phe Glu

500 505 510

Gly Leu Ala Lys Gly Gln Ile Glu Glu Ala Gly Trp Ser Tyr Val Gly

515 520 525

Gly Trp Thr Gly Gln Gly Gly Ser Lys Leu Gly Thr Lys Arg Thr Leu

530 535 540

Pro Lys Lys Ser Phe Glu Gln Ile Ser Ala Asn Ile Thr Lys Phe Asn

545 550 555 560

Ile Gln Gly Leu Val Ile Ile Gly Gly Phe Glu Ala Tyr Thr Gly Gly

565 570 575

Leu Glu Leu Met Glu Gly Arg Lys Gln Phe Asp Glu Leu Cys Ile Pro

580 585 590

Phe Val Val Ile Pro Ala Thr Val Ser Asn Asn Val Pro Gly Ser Asp

595 600 605

Phe Ser Val Gly Ala Asp Thr Ala Leu Asn Thr Ile Cys Thr Thr Cys

610 615 620

Asp Arg Ile Lys Gln Ser Ala Ala Gly Thr Lys Arg Arg Val Phe Ile

625 630 635 640

Ile Glu Thr Met Gly Gly Tyr Cys Gly Tyr Leu Ala Thr Met Ala Gly

645 650 655

Leu Ala Ala Gly Ala Asp Ala Ala Tyr Ile Phe Glu Glu Pro Phe Thr

660 665 670

Ile Arg Asp Leu Gln Ala Asn Val Glu His Leu Val Gln Lys Met Lys

675 680 685

Thr Thr Val Lys Arg Gly Leu Val Leu Arg Asn Glu Lys Cys Asn Glu

690 695 700

Asn Tyr Thr Thr Asp Phe Ile Phe Asn Leu Tyr Ser Glu Glu Gly Lys

705 710 715 720

Gly Ile Phe Asp Ser Arg Lys Asn Val Leu Gly His Met Gln Gln Gly

725 730 735

Gly Ser Pro Thr Pro Phe Asp Arg Asn Phe Ala Thr Lys Met Gly Ala

740 745 750

Lys Ala Met Asn Trp Met Ser Gly Lys Ile Lys Glu Ser Tyr Arg Asn

755 760 765

Gly Arg Ile Phe Ala Asn Thr Pro Asp Ser Gly Cys Val Leu Gly Met

770 775 780

Arg Lys Arg Ala Leu Val Phe Gln Pro Val Ala Glu Leu Lys Asp Gln

785 790 795 800

Thr Asp Phe Glu His Arg Ile Pro Lys Glu Gln Trp Trp Leu Lys Leu

805 810 815

Arg Pro Ile Leu Lys Ile Leu Ala Lys Tyr Glu Ile Asp Leu Asp Thr

820 825 830

Ser Asp His Ala His Leu Glu His Ile Thr Arg Lys Arg Ser Gly Glu

835 840 845

Ala Ala

850