【摘要】：目的采用代謝組學(xué)技術(shù)及蛋白質(zhì)組學(xué)技術(shù)篩選并分析脈絡(luò)膜脫離型孔源性視網(wǎng)膜脫離(Rhegmatogenous retinal detachment associated with choroidal detachment, RRDCD)患者與孔源性視網(wǎng)膜脫離(Rhegmatogenous retinal detachment, RRD)患者玻璃體液中差異代謝物及蛋白質(zhì),以期發(fā)現(xiàn)能夠揭示RRDCD發(fā)病機(jī)制的分子標(biāo)志物。方法在代謝組學(xué)研究中,采用液相色譜-四極飛行時(shí)間質(zhì)譜儀(Liquid chromatography-quadrupole-time-of-flight/mass spectrometer, LC-Q-TOF/MS)檢測15名原發(fā)性RRDCD患者與14名原發(fā)性RRD患者玻璃體液中的代謝產(chǎn)物含量。采用SIMCA-P對數(shù)據(jù)進(jìn)行多因素分析比較,并對結(jié)果進(jìn)行Bonferroni校正的單因素方差統(tǒng)計(jì)分析。所用數(shù)據(jù)庫為Biofluid Metabolites數(shù)據(jù)庫和人類代謝組數(shù)據(jù)庫。對16個(gè)玻璃體液樣本(8個(gè)RRDCD、8個(gè)RRD)的蛋白質(zhì)組學(xué)分析中,先對樣本進(jìn)行酶解、iTRAQ肽段標(biāo)記后進(jìn)行強(qiáng)陽離子交換柱(Strong cation exchange, SCX)分級(jí),最后運(yùn)用毛細(xì)管高效液相色譜分離及電噴霧離子阱質(zhì)譜鑒定(Electrospray ion trap mass spectrometry, ESI-MS)進(jìn)行質(zhì)譜分析,將原始數(shù)據(jù)通過Maxquant軟件(1.3.0.5)軟件進(jìn)行數(shù)據(jù)庫檢索、GO分析、KEGG通路分析等生物信息學(xué)分析。篩選標(biāo)準(zhǔn)為RRDCD/RRD差異倍數(shù)Ratio+/-1.2且P0.05。結(jié)果通過代謝組學(xué)研究方法對比發(fā)現(xiàn)RRD和RRDCD患者玻璃體液之間存在265種差異離子。通過搜索MS和MS/MS片段以及在Biofluid Metabolites數(shù)據(jù)庫和Human Metabolome數(shù)據(jù)庫中的比對,最終確定了24種差異代謝物(23個(gè)正離子,1個(gè)負(fù)離子)。蛋白質(zhì)組學(xué)中,質(zhì)譜定性到肽段總數(shù)2510個(gè),共計(jì)蛋白質(zhì)750個(gè),其中以篩選標(biāo)準(zhǔn)RRDCD/RRD差異倍數(shù)Ratio+/-1.2且P0.05篩選的差異表達(dá)蛋白質(zhì)數(shù)目并排除不典型蛋白質(zhì)后,兩組間差異蛋白數(shù)103個(gè)(上調(diào)49個(gè),下調(diào)54個(gè))。GO分析顯示定性到的差異蛋白質(zhì)主要位于細(xì)胞外間隙。KEGG通路分析表明RRDCD的差異蛋白質(zhì)在補(bǔ)體及凝血級(jí)聯(lián)系統(tǒng)中顯著富集。結(jié)論本研究成功運(yùn)用代謝組學(xué)及蛋白質(zhì)組學(xué)技術(shù)對RRDCD及RRD患者玻璃體液進(jìn)行研究,兩種技術(shù)能夠敏感鑒定兩組玻璃體液樣本間的差異,揭示了RRDCD的病程需要消耗更多的能量完成各種生物學(xué)功能并具有更顯著的增殖反應(yīng),同時(shí)補(bǔ)體-凝血級(jí)聯(lián)反應(yīng)通路在RRDCD的發(fā)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R774.12

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