基于干旱环境中大鼠泪腺差异表达蛋白的筛选初探“燥伤清窍”机理

Preliminarily explore on the mechanism of dryness damaging upper orifices based on the screening of differential expression proteins from rat lacrimal gland in drought envi⁃ronment

目的通过筛选干旱环境中大鼠泪腺的差异表达蛋白,探讨与“燥伤清窍”病机相关的蛋白质或信号通路,从分子水平认识外燥证的本质。方法20只SD雌鼠随机均分为2组,分别为在常温常湿环境中饲养的对照组(CG),以及采用人工气候箱和砂尘试验箱模拟干旱环境(西北燥证的主要病因)干预的干旱风沙组(DD),均饲养48 d。随后摘取大鼠左侧泪腺,采用串联质谱标签(TMT)技术筛选差异表达蛋白,并进行生物信息分析。结果(1)差异表达蛋白:筛选出DD组大鼠泪腺中差异表达蛋白共505个,其中上调蛋白有210个,下调蛋白有295个。(2)基因本论(GO)功能注释:差异表达蛋白主要影响细胞过程和单生物过程,主要定位在细胞和细胞器,分子层面主要影响蛋白的结合和催化活性。(3)GO富集和京都基因与基因组百科全书(KEGG)通路富集:上调蛋白多与免疫功能相关,主要富集在抗原加工呈递通路;下调蛋白多与离子的转运和肌肉的收缩相关,主要富集在与氧化、磷酸化相关的通路。(4)候选靶标蛋白:S100钙结合蛋白A9(S100A9)、α-1-酸性糖蛋白(AGP1)、趋化因子C-X3-C基元配体1(CX3CL1)、髓过氧化物酶(MPO)、泛醇-细胞色素C还原酶铰链蛋白(UQCRH)、T-激肽原1(KNT1)、蛋白酶体亚基β-8(PSMB8)、前列腺素D合酶(PGDS)、免疫球蛋白G(IgG)、热休克蛋白27(HSP27)、血清转铁蛋白(TF)、酪蛋白激酶Ⅱα'亚基(CSNK2A2)、泛素羧基末端水解酶L3(UCHL3)为探索“燥伤清窍”微观机制的候选靶标蛋白。结论基于眼部症状,或可立足免疫功能(尤指炎症反应与变态反应)和细胞凋亡,选择相应的信号通路探索“燥伤清窍”的微观机制。

OBJECTIVE To explore proteins or signal pathways related to the pathogenesis of dryness damaging upper orifices by screening differential expression proteins from rat lacrimal gland in drought environment,and to understand the essence of external dryness syndrome at the molecular level.METHODS A total of 20 female SD rats were randomly divided into two groups,control group(CG)that was fed in normal temperature and humidity environment,and dryness&sand dust group(DD)that was intervened in drought environment(the main pathogenic factors of northwest dryness syndrome)simulated by using artificial climate chamber and sand-dust test chamber,all of which were fed for 48 days.Subsequently,the left lacrimal glands of rats were excised for screening the differential expression proteins by using tandem mass tag(TMT)technique and was analyzed by bioinformatics analysis techniques.RESULTS(1)The differential expression proteins:A total of 505 differential expression proteins were screened out in group DD,including 210 up-regulated proteins and 295 down-regulated proteins.(2)Gene ontology(GO)functional annotations:Differential expression proteins mainly influenced cellular process and single-organism process,mainly located in cells and organelles,and mainly affected the binding and catalytic activity of proteins at the molecular level.(3)GO enrichment and kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment:The up-regulated proteins were mainly related to immune function and enriched in antigen processing and presentation pathway.Down-regulated proteins were mainly associated with ion transport and muscle contraction and enriched in oxidative and phosphorylation pathway.(4)Candidate target proteins:S100 calbindin A9(S100A9),alpha-1-acid glycoprotein(AGP1),chemokine C-X3-C motif ligand 1(CX3CL1),myeloperoxidase(MPO),ubiquinol-cytochrome c reductase hinge protein(UQCRH),T-kininogen 1(KNT1),proteasome subunitβtype-8(PSMB8),prostaglandin D synthase(PGDS),immunoglobulin G(IgG),heat shock protein 27(HSP27),serotransferrin(TF),casein kinase 2 alpha 2(CSNK2A2)and ubiquitin carboxyl-terminal hydrolase L3(UCHL3)were the candidate target proteins for exploring the micro-mechanism of dryness damaging upper orifices.CONCLUSIONS According to eye symptoms,it is possible to explore the micro-mechanism of dryness damaging upper orifice by selecting the corresponding signal pathways based on immunological function(especially inflammation and allergic reaction)and apoptosis.