CN114814217A - 磷脂酰肌醇聚糖特异性磷脂酶d及其多肽片段在淋巴水肿中的应用 - Google Patents

磷脂酰肌醇聚糖特异性磷脂酶d及其多肽片段在淋巴水肿中的应用 Download PDF

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CN114814217A
CN114814217A CN202110070161.8A CN202110070161A CN114814217A CN 114814217 A CN114814217 A CN 114814217A CN 202110070161 A CN202110070161 A CN 202110070161A CN 114814217 A CN114814217 A CN 114814217A
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张曼
孟倩
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Beijing Shijitan Hospital
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Abstract

本发明提供一种磷脂酰肌醇聚糖特异性磷脂酶D（Phosphatidylinositol‑glycan‑specific phospholipase D）及其多肽片段在尿液和血液中的应用，具体为尿液、血液中的磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段在原发、继发淋巴水肿疾病诊断、鉴别诊断、治疗和预后评估及机理研究中的应用。淋巴水肿是由淋巴系统发育不良或严重损伤而引起的一种慢性疾病，在世界卫生组织对致残类疾病排列中列为第２位。本发明通过研究证实，与健康人（正常对照组）相比，尿液、血液中的磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段在原发淋巴水肿患者中表达降低，在继发淋巴水肿中表达无差异，可用于原发、继发淋巴水肿疾病的诊断、鉴别诊断、治疗和预后评估，并且为进一步机理研究提供了依据。

Description

磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段在淋巴水肿中 的应用
技术领域
本发明涉及尿液、血液磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段的新用途，具体涉及尿液、血液磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段在原发、继发淋巴水肿诊断、鉴别诊断、治疗和预后评估及机理研究中的应用。
背景技术
淋巴水肿是由淋巴系统发育不良或严重损伤而引起的一种慢性疾病，其显著特点为淋巴液生成和引流失衡，大量组织液在组织间隙滞留，最终导致组织广泛水肿。淋巴水肿为高致残类疾病，在世界卫生组织对致残类疾病排列中列为第２位。主要原因是由于该病在发病初期往往得不到重视，疾病由肢体浅层软组织内体液积聚逐渐进展，经历纤维结缔组织增生、脂肪硬化，筋膜增厚及整个肢体变粗的不可逆病程。因皮肤增厚，表皮过度角化，皮下组织增生．其中包括大量增生的纤维成分，使晚期的肢体病变组织坚硬如象皮，又称为象皮肿。病人除肢体增粗外，还常伴有丹毒发作、皮肤赘疣样增生及溃疡等．部分会致残而丧失劳动能力。基于病因不同，淋巴水肿分为原发性和继发性2大类。患者约10％属于先天性淋巴系统缺陷引起的原发性淋巴水肿，起病隐匿，无明显诱因，不易诊断。其余90%是由于丝虫病、局部感染、外伤，肿瘤切除手术后所导致的继发性病变。原发性、继发性淋巴水肿的诊断与鉴别诊断目前在结合临床体征、病史的基础上，主要依赖影像学检查，但各种影像学检查手段都具有一定的局限性。目前针对该疾病，没有能够辅助疾病诊断与鉴别诊断、可作为常规早期筛查的有效的疾病标志物应用于临床
磷脂酰肌醇聚糖特异性磷脂酶D能特异性水解糖基化磷脂酰肌醇（GPI）释放出锚定蛋白而调节锚定蛋白的功能，参与胆固醇代谢、内皮细胞迁徙、造血干细胞迁移、软骨细胞分化等多种生物过程。本研究发现，与健康人（正常对照组）相比，尿液、血液中磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段在原发淋巴水肿患者中表达降低，在继发淋巴水肿中表达无差异，可用于淋巴水肿疾病的诊断与鉴别诊断，治疗和预后评估，也为机理研究提供依据。
血液是临床检测常用的样本类型，而尿液可以完全无创、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。本实验在前期方法学摸索的基础上，期望通过尿液、血液蛋白或多肽研究，实现用方便、快捷、易重复的尿液、血液检测辅助原发、继发淋巴水肿患者的诊断及鉴别诊断，治疗和预后评估，也为进一步尿液、血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、血液磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段在制备用于原发、继发淋巴水肿疾病诊断、鉴别诊断、治疗和预后评估及机理研究等制剂的应用。
优选地，所述尿液、血液磷脂酰肌醇聚糖特异性磷脂酶D的氨基酸序列如SEQ IDNO.1所示(KNDFHRNLTT SLTESVDRNI NYTERGVFFS VNSWTPDSMS FIYKALERNI RTMFIGGSQL 360
SQKHVSSPLA SYFLSFPYAR LGWAMTSADL NQDGHGDLVV GAPGYSRPGH IHIGRVYLIY 420
GNDLGLPPVD LDLDKEAHRI LEGFQPSGRF GSALAVLDFN VDGVPDLAVG APSVGSEQLT 480
YKGAVYVYFG SKQGGMSSSP NITISCQDIY CNLGWTLLAA DVNGDSEPDL VIGSPFAPGG 540
GKQKGIVAAF YSGPSLSDKE KLNVEAANWT VRGEEDFSWF GYSLHGVTVD NRTLLLVGSP 600
TWKNASRLGH LLHIRDEKKS LGRVYGYFPP NGQSWFTISG DKAMGKLGTS LSSGHVLMNG 660
TLKQVLLVGA PTYDDVSKVA FLTVTLHQGG ATRMYALTSD AQPLLLSTFS GDRRFSRFGG 720
VLHLSDLDDD GLDEIIMAAP LRIADVTSGL IGGEDGRVYV YNGKETTLGD MTGKCKSWIT 780
PCPEEKAQYV LISPEASSRF GSSLITVRSK AKNQVVIAAG RSSLGARLSG ALHVYSLGSD 840
）；或由SEQ ID NO.1所示的氨基酸序列衍生的，且与SEQ ID NO.1所示的氨基酸序列具有相同功能的氨基酸序列。
优选地，所述制剂为淋巴水肿患者尿液、血液磷脂酰肌醇聚糖特异性磷脂酶D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磷脂酰肌醇聚糖特异性磷脂酶D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磷脂酰肌醇聚糖特异性磷脂酶D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磷脂酰肌醇聚糖特异性磷脂酶D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：固相载体或液相载体，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，所述标准品包括磷脂酰肌醇聚糖特异性磷脂酶D标准品、人源化标签抗体标准品；较佳地，所述质控品包括：磷脂酰肌醇聚糖特异性磷脂酶D质控品、人源化标签抗体质控品；较佳地，所述载体包括：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固相载体或液相载体。
优选地，所述载体的材质为聚氯乙烯、聚苯乙烯、聚丙酰胺、纤维素、磁珠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、原发和继发淋巴水肿患者的尿液、血液标本，尿液标本4000r/min离心5min后，吸取上清液，采用Bradford法测定提取的蛋白浓度，进行SDS-PAGE酶解。处理后的尿液、血液样本进行Label-free质谱分析。将原发、继发淋巴水肿组和正常对照组在质谱中得到的数据进行定量计算。以蛋白表达量差异在1.2倍以上且经统计检验P<0.05作为参考标准筛选差异性多肽。然后发明人对具有统计学意义的差异性多肽进行鉴定，利用数据库检索得到差异蛋白磷脂酰肌醇聚糖特异性磷脂酶D。
本发明通过研究证实与健康人相比，磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段在原发淋巴水肿患者中表达降低，在继发淋巴水肿中表达无差异，从而提出检测尿液、血液磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段可用于原发、继发淋巴水肿的辅助诊断、鉴别诊断、治疗和预后评估。
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是尿液磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段在原发、继发淋巴水肿组及健康对照组中含量图。
图2是血液磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段在原发、继发淋巴水肿组及健康对照组中含量图。
具体实施方式
实施例1 尿液、血液标本的收集与处理
选取原发、继发淋巴水肿患者作为实验组，选取同期健康体检者作为正常对照组。收集各组研究对象的血液样本1ml、新鲜晨尿样本30ml，将收集的尿液标本4000r/min离心5min后，吸取上清液，每管2ml分装，所有样本-80℃冰箱保存。
实施例2 质谱分析和尿液、血液多肽的筛选
对尿液、血液样品进行蛋白提取，并对提取的蛋白浓度进行测定。尿液、血液样本进行Label-free质谱分析。将实验组和正常对照组在质谱中得到的数据进行定量计算。组间比较采用t-test进行差异分析，以蛋白表达量差异在1.2倍以上且经统计检验P<0.05作为参考标准筛选差异表达蛋白。
与健康人相比，磷脂酰肌醇聚糖特异性磷脂酶D在原发淋巴水肿患者中表达降低，在继发淋巴水肿中表达无差异，分别如图1，2所示，磷脂酰肌醇聚糖特异性磷脂酶D在正常对照和继发组与原发淋巴水肿组尿液、血液中的表达具有显著性差异。
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。
SEQUENCE LISTING
<110> 首都医科大学附属北京世纪坛医院
<120> 磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段在淋巴水肿中的应用
<130> 1
<140> 21PGPLD1-CN
<141> 2021-1-18
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<170> PatentIn version 3.5
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<213> Phosphatidylinositol-glycan-specific phospholipase D
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Asn Tyr Thr Glu Arg Gly Val Phe Phe Ser Val Asn Ser Trp Thr Pro
325 330 335
Asp Ser Met Ser Phe Ile Tyr Lys Ala Leu Glu Arg Asn Ile Arg Thr
340 345 350
Met Phe Ile Gly Gly Ser Gln Leu Ser Gln Lys His Val Ser Ser Pro
355 360 365
Leu Ala Ser Tyr Phe Leu Ser Phe Pro Tyr Ala Arg Leu Gly Trp Ala
370 375 380
Met Thr Ser Ala Asp Leu Asn Gln Asp Gly His Gly Asp Leu Val Val
385 390 395 400
Gly Ala Pro Gly Tyr Ser Arg Pro Gly His Ile His Ile Gly Arg Val
405 410 415
Tyr Leu Ile Tyr Gly Asn Asp Leu Gly Leu Pro Pro Val Asp Leu Asp
420 425 430
Leu Asp Lys Glu Ala His Arg Ile Leu Glu Gly Phe Gln Pro Ser Gly
435 440 445
Arg Phe Gly Ser Ala Leu Ala Val Leu Asp Phe Asn Val Asp Gly Val
450 455 460
Pro Asp Leu Ala Val Gly Ala Pro Ser Val Gly Ser Glu Gln Leu Thr
465 470 475 480
Tyr Lys Gly Ala Val Tyr Val Tyr Phe Gly Ser Lys Gln Gly Gly Met
485 490 495
Ser Ser Ser Pro Asn Ile Thr Ile Ser Cys Gln Asp Ile Tyr Cys Asn
500 505 510
Leu Gly Trp Thr Leu Leu Ala Ala Asp Val Asn Gly Asp Ser Glu Pro
515 520 525
Asp Leu Val Ile Gly Ser Pro Phe Ala Pro Gly Gly Gly Lys Gln Lys
530 535 540
Gly Ile Val Ala Ala Phe Tyr Ser Gly Pro Ser Leu Ser Asp Lys Glu
545 550 555 560
Lys Leu Asn Val Glu Ala Ala Asn Trp Thr Val Arg Gly Glu Glu Asp
565 570 575
Phe Ser Trp Phe Gly Tyr Ser Leu His Gly Val Thr Val Asp Asn Arg
580 585 590
Thr Leu Leu Leu Val Gly Ser Pro Thr Trp Lys Asn Ala Ser Arg Leu
595 600 605
Gly His Leu Leu His Ile Arg Asp Glu Lys Lys Ser Leu Gly Arg Val
610 615 620
Tyr Gly Tyr Phe Pro Pro Asn Gly Gln Ser Trp Phe Thr Ile Ser Gly
625 630 635 640
Asp Lys Ala Met Gly Lys Leu Gly Thr Ser Leu Ser Ser Gly His Val
645 650 655
Leu Met Asn Gly Thr Leu Lys Gln Val Leu Leu Val Gly Ala Pro Thr
660 665 670
Tyr Asp Asp Val Ser Lys Val Ala Phe Leu Thr Val Thr Leu His Gln
675 680 685
Gly Gly Ala Thr Arg Met Tyr Ala Leu Thr Ser Asp Ala Gln Pro Leu
690 695 700
Leu Leu Ser Thr Phe Ser Gly Asp Arg Arg Phe Ser Arg Phe Gly Gly
705 710 715 720
Val Leu His Leu Ser Asp Leu Asp Asp Asp Gly Leu Asp Glu Ile Ile
725 730 735
Met Ala Ala Pro Leu Arg Ile Ala Asp Val Thr Ser Gly Leu Ile Gly
740 745 750
Gly Glu Asp Gly Arg Val Tyr Val Tyr Asn Gly Lys Glu Thr Thr Leu
755 760 765
Gly Asp Met Thr Gly Lys Cys Lys Ser Trp Ile Thr Pro Cys Pro Glu
770 775 780
Glu Lys Ala Gln Tyr Val Leu Ile Ser Pro Glu Ala Ser Ser Arg Phe
785 790 795 800
Gly Ser Ser Leu Ile Thr Val Arg Ser Lys Ala Lys Asn Gln Val Val
805 810 815
Ile Ala Ala Gly Arg Ser Ser Leu Gly Ala Arg Leu Ser Gly Ala Leu
820 825 830
His Val Tyr Ser Leu Gly Ser Asp
835 840

Claims (9)

1.尿液、血液磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段在制备用于原发、继发淋巴水肿疾病诊断、鉴别诊断、治疗和预后评估及机理研究等制剂的应用。
2.根据权利要求1所述的应用，其特征在于，所述尿液、血液磷脂酰肌醇聚糖特异性磷脂酶D的氨基酸序列如SEQ ID NO.1所示；或由SEQ ID NO.1所示的氨基酸序列衍生的，且与SEQ ID NO.1所示的氨基酸序列具有相同功能的氨基酸序列。
3.根据权利要求1所述的应用，其特征在于，所述制剂为原发、继发淋巴水肿患者尿液、血液磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段检测试剂盒。
4.根据权利要求3所述的应用，其特征在于，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磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段的适配体抗体或抗体片段中的一种或多种。
5.根据权利要求3所述的应用，其特征在于，所述检测方法包括直接检测磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段的质谱等方法及其相关试剂盒。
6.根据权利要求3所述的应用，其特征在于，所述检测方法包括直接检测磷脂酰肌醇聚糖特异性磷脂酶D及其多肽片段或其相关核酸检测等方法及其相关试剂盒。
7.根据权利要求3所述的应用，其特征在于，所述试剂盒还包括选自下组的成分：固相载体或液相载体，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。
8.根据权利要求7所述的应用，其特征在于，所述标准品包括磷脂酰肌醇聚糖特异性磷脂酶D标准品、人源化标签抗体标准品；较佳地，所述质控品包括：磷脂酰肌醇聚糖特异性磷脂酶D控品、人源化标签抗体质控品；较佳地，所述载体包括：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固相载体或液相载体。
9.根据权利要求8所述的应用，其特征在于，所述载体的材质为聚氯乙烯、聚苯乙烯、聚丙酰胺、纤维素、磁珠等中的任意一种及具有类似功能的其他载体。
CN202110070161.8A 2021-01-19 2021-01-19 磷脂酰肌醇聚糖特异性磷脂酶d及其多肽片段在淋巴水肿中的应用 Pending CN114814217A (zh)

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