CN113759123A - 尿液软骨间层蛋白及其多肽片段在烧伤中的应用 - Google Patents
尿液软骨间层蛋白及其多肽片段在烧伤中的应用 Download PDFInfo
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Abstract
本发明提供一种尿液软骨间层蛋白（Cartilage intermediate‑layer protein）及其多肽片段的应用，具体为尿液软骨间层蛋白及其多肽片段在制备用于烧伤诊断、鉴别诊断、烧伤面积及程度评价、治疗效果评价、监测、预后评估及机理研究等制剂的应用。烧伤是日常生活中常见的重要创伤，每年每100万人中约有5000～100000人烧伤，据世界卫生组织统计，每年全球死于烧伤患者超过30万人，严重烧伤救治存活率仍处于较低水平。本发明通过研究证实，与健康人（正常对照组）相比，尿液软骨间层蛋白及其多肽片段在烧伤患者中表达下调，并随烧伤程度加重而降低。可用于烧伤患者的辅助诊断及病情监测。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软骨间层蛋白及其多肽片段。
Description
技术领域
本发明涉及尿液软骨间层蛋白及其多肽片段的新用途，具体涉及尿液软骨间层蛋白及其多肽片段在于烧伤诊断、鉴别诊断、烧伤面积及程度评价、治疗效果评价、监测、预后评估及机理研究等的应用。
背景技术
烧伤是指由于火焰、高温气体、灼热的固体或液体、放射线、电能、强酸强碱等化学物质所引起的人体皮肤、组织甚至深层脏器的损伤，是一种全身性综合性疾病。烧伤后创面组织大量坏死、感染、休克及凝血功能障碍等反应可引起机体发生一系列的病理生理学变化。不同程度的烧伤对人体有着不同的影响，严重的烧伤可对人体内环境造成破坏，使烧伤患者出现各系统复杂性的病理生理变化，相关的检测指标也会随着烧伤严重程度的不同随之出现相应的改变。及时检测这些指标的变化可为临床医生对于疾病的诊断，病情的判断，治疗方法的选择以及患者预后的评估等多方面提供有价值的参考依据。
但是，严重烧伤的病人皮肤完整性较差，血液学检测作为皮肤有创性检查在此类患者的临床应用中存在着诸多困难，反复的抽血检测还会加剧患者痛苦。尿液作为血液的超滤液蕴含着丰富的生物学信息，其采集过程具有无创便捷等优势，这在烧伤患者的检测中尤为明显。在尿液中寻找有助于烧伤诊断及反映病情变化的生物标志物，可提高烧伤患者的生活质量和依从性，减轻多次采血的痛苦，更好的为临床医生提供有利于疾病诊断和治疗的参考依据。
软骨间层蛋白(cartilage Intermediate layer Protein，CILP)是人类关节软骨的基质组成成分．因其分布只局限于软骨组织特别是在软骨中间层分布丰富而得名。但目前在半月板、肌腱、韧带、滑膜及椎间盘均发现其存在，目前可对该蛋白的研究较少。本研究烧伤患者尿液中软骨间层蛋白较健康人组出现表达下调，且与烧伤程度呈一定相关性，烧伤程度越严重该蛋白含量越低。
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。为减轻烧伤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烧伤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软骨间层蛋白及其多肽片段在制备用于烧伤诊断、鉴别诊断、烧伤面积及程度评价、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，所述尿液软骨间层蛋白的氨基酸序列如SEQ ID NO.1所示(MVGTKAWVFSFLVLEVTSVL GRQTMLTQSV RRVQPGKKNP SIFAKPADTL ESPGEWTTWF NIDYPGGKGD YERLDAIRFYYGDRVCARPL RLEARTTDWT PAGSTGQVVH GSPREGFWCL NREQRPGQNC SNYTVRFLCP PGSLRRDTERIWSPWSPWSK CSAACGQTGV QTRTRICLAE MVSLCSEASE EGQHCMGQDC TACDLTCPMG QVNADCDACMCQDFMLHGAV SLPGGAPASG AAIYLLTKTP KLLTQTDSDG RFRIPGLCPD GKSILKITKV KFAPIVLTMPKTSLKAATIK AEFVRAETPY MVMNPETKAR RAGQSVSLCC KATGKPRPDK YFWYHNDTLL DPSLYKHESKLVLRKLQQHQ AGEYFCKAQS DAGAVKSKVA QLIVIASDET PCNPVPESYL IRLPHDCFQN ATNSFYYDVGRCPVKTCAGQ QDNGIRCRDA VQNCCGISKT EEREIQCSGY TLPTKVAKEC SCQRCTETRS IVRGRVSAADNGEPMRFGHV YMGNSRVSMT GYKGTFTLHV PQDTERLVLT FVDRLQKFVN TTKVLPFNKK GSAVFHEIKMLRRKKPITLE AMETNIIPLG EVVGEDPMAE LEIPSRSFYR QNGEPYIGKV KASVTFLDPR NISTATAAQTDLNFINDEGD TFPLRTYGMF SVDFRDEVTS EPLNAGKVKV HLDSTQVKMP EHISTVKLWS LNPDTGLWEEEGDFKFENQR RNKREDRTFL VGNLEIRERR LFNLDVPESR RCFVKVRAYR SERFLPSEQI QGVVISVINLEPRTGFLSNP RAWGRFDSVI TGPNGACVPA FCDDQSPDAY SAYVLASLAG EELQAVESSP KFNPNAIGVPQPYLNKLNYR RTDHEDPRVK KTAFQISMAK PRPNSAEESN GPIYAFENLR ACEEAPPSAA HFRFYQIEGDRYDYNTVPFN EDDPMSWTED YLAWWPKPME FRACYIKVKI VGPLEVNVRS RNMGGTHRQT VGKLYGIRDVRSTRDRDQPN VSAACLEFKC SGMLYDQDRV DRTLVKVIPQ GSCRRASVNP MLHEYLVNHL PLAVNNDTSEYTMLAPLDPL GHNYGIYTVT DQDPRTAKEI ALGRCFDGTS DGSSRIMKSN VGVALTFNCV ERQVGRQSAFQYLQSTPAQS PAAGTVQGRV PSRRQQRASR GGQRQGGVVA SLRFPRVAQQ PLIN）；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，所述制剂为烧伤患者尿液软骨间层蛋白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软骨间层蛋白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软骨间层蛋白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软骨间层蛋白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，所述标准品包括软骨间层蛋白标准品、人源化标签抗体标准品；较佳地，所述质控品包括：软骨间层蛋白质控品、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、不同烧伤程度患者的尿液标本， 4000r/min离心5min后，吸取上清，采用Bradford法测定提取的蛋白浓度，进行SDS-PAGE酶解。尿液样本的Label-free质谱分析由OrbitrapFusion型质谱完成。将烧伤组和正常对照组在质谱中得到的数据采用Maxquant算法中的Labelfree算法进行非标记定量计算。以蛋白表达量差异在1.5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软骨间层蛋白。
本发明通过研究证实与健康人相比，软骨间层蛋白及其多肽片段在烧伤患者的尿液中呈低表达，并随烧伤程度的加重而降低，与临床诊断有较好的一致性。从而提出检测尿液软骨间层蛋白及其多肽片段可用于烧伤的辅助诊断或病情监测。
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软骨间层蛋白及其多肽片段。
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是尿液软骨间层蛋白及其多肽片段在烧伤组及健康对照组中含量图。
图2是软骨间层蛋白参与主要生物过程示意图。
具体实施方式
实施例1 尿液标本的收集与处理
选取烧伤患者作为烧伤组，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30ml，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。将收集的尿液标本4000r/min离心5min后，吸取上清，每管2ml分装，-80℃冰箱保存。
实施例2 质谱分析和尿液多肽的筛选
对尿液样品蛋白提取，并对提取的蛋白浓度进行测定。尿液样本的质谱分析由OrbitrapFusion型质谱完成。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1.5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。
实施例3 差异多肽的鉴定及分析
本次使用的数据库为Uniprot_Homo数据库，产生的质谱原始文件采用MaxQuant软件处理，检索参数设置见表1。
与健康人相比，软骨间层蛋白在烧伤患者的尿液中低表达，如图1所示，其参与的主要生物过程如图2所示，软骨间层蛋白在正常对照组和烧伤组尿液中的表达有显著性差异，并随烧伤程度的加重而降低。
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。
序列表
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<120> 尿液软骨间层蛋白及其多肽片段在烧伤中的应用
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1045 1050 1055
Asp Thr Ser Glu Tyr Thr Met Leu Ala Pro Leu Asp Pro Leu Gly His
1060 1065 1070
Asn Tyr Gly Ile Tyr Thr Val Thr Asp Gln Asp Pro Arg Thr Ala Lys
1075 1080 1085
Glu Ile Ala Leu Gly Arg Cys Phe Asp Gly Thr Ser Asp Gly Ser Ser
1090 1095 1100
Arg Ile Met Lys Ser Asn Val Gly Val Ala Leu Thr Phe Asn Cys Val
1105 1110 1115 1120
Glu Arg Gln Val Gly Arg Gln Ser Ala Phe Gln Tyr Leu Gln Ser Thr
1125 1130 1135
Pro Ala Gln Ser Pro Ala Ala Gly Thr Val Gln Gly Arg Val Pro Ser
1140 1145 1150
Arg Arg Gln Gln Arg Ala Ser Arg Gly Gly Gln Arg Gln Gly Gly Val
1155 1160 1165
Val Ala Ser Leu Arg Phe Pro Arg Val Ala Gln Gln Pro Leu Ile Asn
1170 1175 1180
Claims (9)
1.尿液软骨间层蛋白及其多肽片段在制备用于烧伤诊断、鉴别诊断、烧伤面积及程度评价、治疗效果评价、监测、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，所述尿液软骨间层蛋白的氨基酸序列如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，所述制剂为烧伤患者尿液软骨间层蛋白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软骨间层蛋白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软骨间层蛋白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软骨间层蛋白及其多肽片段的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，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，所述标准品包括软骨间层蛋白标准品、人源化标签抗体标准品；较佳地，所述质控品包括：软骨间层蛋白控品、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。
9.根据权利要求8所述的应用，其特征在于，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。
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| WO2007045197A2 (de) * | 2005-10-21 | 2007-04-26 | Justus Liebig Universität Giessen | Erfindung betreffend expressionsprofile zur vorhersage von septischen zuständen |
Non-Patent Citations (1)
| Title |
|---|
| 赵乔妹;刘丹;雷婷;张曼;: "不同程度烧伤患者凝血功能变化及意义", 标记免疫分析与临床, no. 05, 25 May 2019 (2019-05-25), pages 36 - 40 * |
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