脉络膜脱离型孔源性视网膜脱离玻璃体分子标志物的筛选及发病机制研究

发布时间：2018-11-07 18:37

【摘要】：目的采用代谢组学技术及蛋白质组学技术筛选并分析脉络膜脱离型孔源性视网膜脱离(Rhegmatogenous retinal detachment associated with choroidal detachment, RRDCD)患者与孔源性视网膜脱离(Rhegmatogenous retinal detachment, RRD)患者玻璃体液中差异代谢物及蛋白质,以期发现能够揭示RRDCD发病机制的分子标志物。方法在代谢组学研究中,采用液相色谱-四极飞行时间质谱仪(Liquid chromatography-quadrupole-time-of-flight/mass spectrometer, LC-Q-TOF/MS)检测15名原发性RRDCD患者与14名原发性RRD患者玻璃体液中的代谢产物含量。采用SIMCA-P对数据进行多因素分析比较,并对结果进行Bonferroni校正的单因素方差统计分析。所用数据库为Biofluid Metabolites数据库和人类代谢组数据库。对16个玻璃体液样本(8个RRDCD、8个RRD)的蛋白质组学分析中,先对样本进行酶解、iTRAQ肽段标记后进行强阳离子交换柱(Strong cation exchange, SCX)分级,最后运用毛细管高效液相色谱分离及电喷雾离子阱质谱鉴定(Electrospray ion trap mass spectrometry, ESI-MS)进行质谱分析,将原始数据通过Maxquant软件(1.3.0.5)软件进行数据库检索、GO分析、KEGG通路分析等生物信息学分析。筛选标准为RRDCD/RRD差异倍数Ratio+/-1.2且P0.05。结果通过代谢组学研究方法对比发现RRD和RRDCD患者玻璃体液之间存在265种差异离子。通过搜索MS和MS/MS片段以及在Biofluid Metabolites数据库和Human Metabolome数据库中的比对,最终确定了24种差异代谢物(23个正离子,1个负离子)。蛋白质组学中,质谱定性到肽段总数2510个,共计蛋白质750个,其中以筛选标准RRDCD/RRD差异倍数Ratio+/-1.2且P0.05筛选的差异表达蛋白质数目并排除不典型蛋白质后,两组间差异蛋白数103个(上调49个,下调54个)。GO分析显示定性到的差异蛋白质主要位于细胞外间隙。KEGG通路分析表明RRDCD的差异蛋白质在补体及凝血级联系统中显著富集。结论本研究成功运用代谢组学及蛋白质组学技术对RRDCD及RRD患者玻璃体液进行研究,两种技术能够敏感鉴定两组玻璃体液样本间的差异,揭示了RRDCD的病程需要消耗更多的能量完成各种生物学功能并具有更显著的增殖反应,同时补体-凝血级联反应通路在RRDCD的发病中扮演重要角色。
[Abstract]:Objective to screen and analyze the patients with choroidal detachment type rhegmatogenous retinal detachment (Rhegmatogenous retinal detachment associated with choroidal detachment, RRDCD) and rhegmatogenous retinal detachment (Rhegmatogenous retinal detachment,) by using metabonomics and proteomics techniques. In order to find the molecular markers which can reveal the pathogenesis of RRDCD, the differential metabolites and proteins in vitreous body fluid of patients with RRD. Methods liquid chromatography-quadrupole time-of-flight mass spectrometer (Liquid chromatography-quadrupole-time-of-flight/mass spectrometer,) was used in the study of metabolomics. The contents of metabolites in vitreous fluid of 15 patients with primary RRDCD and 14 patients with primary RRD were detected by LC-Q-TOF/MS. The data were analyzed and compared by SIMCA-P, and the results were analyzed by single factor variance of Bonferroni correction. The databases used are Biofluid Metabolites database and human metabolic group database. In proteomic analysis of 16 vitreous body fluid samples (8 RRDCD, 8 RRD), the samples were first hydrolyzed by enzyme, then labeled with iTRAQ peptide and then classified by (Strong cation exchange, SCX) with strong cation exchange column. Finally, (Electrospray ion trap mass spectrometry, ESI-MS was analyzed by capillary high performance liquid chromatography and electrospray ion-trap mass spectrometry. The original data were searched by Maxquant software (1.3.0.5) and analyzed by GO. KEGG pathway analysis and other bioinformatics analysis. The screening criteria were RRDCD/RRD differential multiple Ratio /-1.2 and P0.05. Results there were 265 different ions in vitreous fluid of patients with RRD and RRDCD. By searching for MS and MS/MS fragments and comparing them in Biofluid Metabolites database and Human Metabolome database, 24 different metabolites (23 positive ions and 1 negative ion) were identified. In proteomics, the total number of peptides identified by mass spectrometry was 2510, with 750 proteins, among which the number of differentially expressed proteins screened by the standard RRDCD/RRD differential multiple Ratio / -1.2 and P05 was excluded, and atypical proteins were excluded. The number of differential proteins between the two groups was 103 (49 up-regulated and 54 down-regulated by). GO). KEGG pathway analysis showed that the differential proteins of RRDCD were significantly enriched in complement and coagulation cascade systems. Conclusion Metabolomics and proteomics techniques were successfully used to study the vitreous fluid of patients with RRDCD and RRD. The two techniques can sensitively identify the differences between the two groups of vitreous fluid samples. It is revealed that the course of RRDCD needs more energy to complete various biological functions and has a more significant proliferative response, and the complement coagulation cascade reaction pathway plays an important role in the pathogenesis of RRDCD.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.12

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