本文選題：眼軸 + 年齡　； 參考：《第四軍醫(yī)大學(xué)》2014年博士論文

【摘要】：白內(nèi)障是發(fā)展中國家最主要的視力損害和致盲原因,隨著白內(nèi)障患者年輕化,白內(nèi)障和近視的相關(guān)性逐漸引起了人們的重視。在眾多危險因素中,已證實不同人種、不同地區(qū)情況下近視與核性白內(nèi)障的發(fā)生發(fā)展有非常密切的關(guān)系。高度近視誘導(dǎo)的核性白內(nèi)障,其特征不同與年齡相關(guān)性白內(nèi)障:玻璃體液化、晶狀體核硬度明顯增加。目前在我國,近視患者的比例呈逐年上升的趨勢,白內(nèi)障手術(shù)年齡呈現(xiàn)年輕化的趨勢,使我國現(xiàn)有的防盲任務(wù)更加艱巨。眼軸與玻璃體液化引起晶狀體核硬化的機制已成為本領(lǐng)域研究的熱點。目前高度近視并發(fā)核性白內(nèi)障的研究主要基于流行病學(xué)調(diào)查和臨床研究,尚無理想的動物模型用于發(fā)病機制的研究。近視并發(fā)核性白內(nèi)障成因十分復(fù)雜,進一步探索并揭示高度近視核性白內(nèi)障的發(fā)病機制是非常重要的。隨著生物質(zhì)譜技術(shù)和生物信息學(xué)迅猛發(fā)展,蛋白質(zhì)組學(xué)研究成為生命科學(xué)的前沿。因此在蛋白質(zhì)水平上發(fā)現(xiàn)關(guān)鍵靶蛋白,對我們揭示眼軸相關(guān)性核性白內(nèi)障的形成機制以及潛在治療靶點有重要意義,可為防盲工作提供更多依據(jù)。[目的]1通過超聲彈性成像技術(shù)檢測不同年齡、不同眼軸在體人晶狀體核硬度,探討其在眼軸相關(guān)性核性白內(nèi)障研究方面的價值。2通過收集人白內(nèi)障晶狀體標本,利用定量蛋白質(zhì)組學(xué)聯(lián)合質(zhì)譜技術(shù),篩選和鑒定與眼軸及年齡相關(guān)的核性白內(nèi)障晶狀體核的相關(guān)蛋白質(zhì)。3驗證相關(guān)蛋白在臨床病例中的表達,為探索與眼軸及年齡相關(guān)核性白內(nèi)障發(fā)病機制,尋找能夠作為治療的新靶點提供重要理論依據(jù)。[方法]1年齡和眼軸相關(guān)性核性白內(nèi)障晶狀體核硬度的研究(1)超聲彈性成像檢測年齡相關(guān)性晶狀體核硬度觀察48例雙眼晶狀體透明或輕度混濁的臨床受試者,根據(jù)年齡分為A,B,C組,每例任選一眼檢測。A組16例16眼,女8眼,男8眼,均齡81±5.5歲。B組16例16眼,女6眼,男10眼,均齡44±3.2歲;C組16例16眼,女6眼,男10眼,均齡21±2.5歲。三組平均眼軸23.4±0.44(23~24mm),角膜地形圖排除圓錐角膜對眼軸的影響,眼壓:平均15.6±1.46 mm Hg(14mm Hg~19 mm Hg),排除全身疾病及眼部其他疾患,UCVA≥0.5,觀察者知情同意。儀器采用Hitachi EUB 7500型彩色多普勒超聲診斷儀(探頭型號:EUP2L54M,7L,頻率8~10MHz)。在超聲彈性圖中,綠色代表彈性成像感興趣區(qū)(ROI)內(nèi)組織的平均硬度,紅色代表應(yīng)變性較大,說明組織較軟;藍色表示應(yīng)變性較小,組織硬度大。每例連續(xù)測量3次,取平均值。評價觀察者晶狀體彈性應(yīng)變率與年齡變化的關(guān)系。(2)超聲彈性成像檢測屈光參差晶狀體核硬度觀察14例(28眼)單性高度近視性屈光參差、復(fù)性近視性屈光參差的臨床病例,男5眼,女9眼,均齡62±3.3歲,平均眼壓17.11±1.46 mm Hg(14mm Hg~20 mm Hg);角膜地形圖排除圓錐角膜對眼軸的影響,排除全身疾病及眼部其他疾患;患者知情同意。每位患者長眼軸檢測數(shù)據(jù)納入A組(14眼),相對短眼軸檢測數(shù)據(jù)納入B組(14眼),超聲彈性檢測方法同上。觀察長眼軸組與相對短眼軸組晶狀體彈性應(yīng)變率與眼軸的關(guān)系。2不同年齡、眼軸相關(guān)性核性白內(nèi)障晶狀體蛋白質(zhì)組學(xué)研究(1)二維液相色譜聯(lián)合i TRAQ標記法串聯(lián)質(zhì)譜鑒定、篩選不同年齡和眼軸晶狀體核差異蛋白質(zhì)通過白內(nèi)障囊外摘除(ECCE)手術(shù)收集48例白內(nèi)障患者的晶狀體核,根據(jù)年齡和眼軸分為6組,每組8例:A組(平均眼軸28.7±1.5mm,平均年齡79.8±1.9歲),B組(平均眼軸28.7±1.4 mm,平均年齡58.0±4.0歲),C組(平均眼軸23.0±0.4 mm,平均年齡80.3±4.5歲),D組(平均眼軸23.0±0.3 mm,平均年齡56.9±4.2歲),E組(平均眼軸19.9±0.5 mm,平均年齡為75.1±2.5歲),F組(平均眼軸20.7±0.6 mm,平均年齡為57.6±5.3歲)。G組為透明晶狀體對照組(平均眼軸23.5±0.6 mm,平均年齡為34.7±4.2歲),取自角膜移植供體的7枚透明晶狀體核。每組提取晶狀體水溶性,水不溶尿素溶性、水不溶尿素不溶性蛋白,每組蛋白質(zhì)肽段混合,經(jīng)過除鹽、變性、還原、烷基化、酶解后分別進行穩(wěn)定同位素114,115,116,117,118,119,121 i TRAQ標記、二維液相色譜分離,然后用串聯(lián)質(zhì)譜進行檢測。所得質(zhì)譜數(shù)據(jù)在IPIv3.45蛋白數(shù)據(jù)庫進行檢索鑒定,并進行差異蛋白質(zhì)數(shù)據(jù)分析。實驗技術(shù)重復(fù)兩次,選擇重復(fù)出現(xiàn)且表達趨勢一致的蛋白質(zhì)作為差異蛋白質(zhì)進行分析。(2)Western blot法驗證差異蛋白質(zhì)收集新的白內(nèi)障囊外摘除晶狀體核組織20例,分4組,每組5例。A組(平均眼軸29.1±1.2mm,平均年齡59.0±3.0歲),B組(平均眼軸23.1±0.3mm,平均年齡55.8±3.5歲),C組(平均眼軸28.8±1.2mm,平均年齡78.4±1.9歲),D組(平均眼軸23.0±0.6 mm,平均年齡79.3±3.5歲)。提取晶狀體水溶性,水不溶尿素溶性、水不溶尿素不溶性蛋白質(zhì)組分(方法同前)。利用Western blot法對質(zhì)譜檢測中有意義的差異表達蛋白質(zhì)進行驗證。[結(jié)果]1晶狀體超聲彈性成像數(shù)據(jù)顯示晶狀體核硬度與年齡變化的關(guān)系在高齡組(A組),晶狀體核區(qū)的應(yīng)變率最小(0.02±0.08);在低齡組(C組),晶狀體核區(qū)的應(yīng)變率最大(1.95±0.87);中齡組(B組)晶狀體核區(qū)的應(yīng)變率為(0.69±0.12)。通過單因素方差分析顯示:A組與B組,A組與C組,B組與C組比較均有統(tǒng)計學(xué)意義(P㩳0.005)。高齡組晶狀體核應(yīng)變率小,低齡組晶狀體核應(yīng)變率大。2晶狀體超聲彈性成像顯示屈光參差患者晶狀體核硬度與眼軸變化的關(guān)系長眼軸組晶狀體核區(qū)的應(yīng)變率為(0.16±0.08),相對短眼軸組晶狀體核區(qū)的應(yīng)變率為(0.54±0.16),獨立樣本t檢驗分析顯示:兩組間比較P㩳0.005,有統(tǒng)計學(xué)意義。長眼軸組晶狀體核應(yīng)變率小,相對短眼軸組晶狀體核應(yīng)變率大。3用i TRAQ技術(shù)篩選出了6個與眼軸相關(guān)的差異蛋白質(zhì),9個與年齡相關(guān)的差異蛋白質(zhì)。第一次i TRAQ分析鑒定出148種蛋白質(zhì),第二次i TRAQ分析鑒定出103種蛋白質(zhì)。選擇兩次結(jié)果的交集,共發(fā)現(xiàn)80種蛋白質(zhì)。顯著差異表達蛋白界值設(shè)定為:p值小于0.05,且差異倍數(shù)大于1.2倍或小于0.8倍的蛋白質(zhì)。控制年齡因素后,80個蛋白質(zhì)中分析得出6種蛋白質(zhì)與軸性核性白內(nèi)障密切相關(guān)。其中3種蛋白質(zhì)在長眼軸組中上調(diào),在短眼軸組中含量少,分別是γ-烯醇酶(Gamma-enolase),丙酮酸激酶同工酶M1/M2(Pyruvate kinase isozymes M1/M2)和山梨醇脫氫酶(Sorbitol dehydrogenase);另3種蛋白質(zhì)在長眼軸組中表達下調(diào),在短眼軸組中含量增高,分別是縫隙連接蛋白3(Gap junction alpha-3 protein),βB2-晶狀體蛋白(Beta-crystallin B2),T-復(fù)合物多肽1(T-complex protein 1 subunit beta)。控制眼軸因素后,分析得出9種蛋白與年齡相關(guān)性核性白內(nèi)障密切相關(guān),其中2個蛋白質(zhì)在高齡組中含量上調(diào),在低齡組中含量少,分別是脂肪酸結(jié)合蛋白(Fatty acid-binding protein),蝶呤-4-α-甲醇胺脫水酶(Pterin-4-alpha-carbinolamine dehydratase);另7個蛋白質(zhì)在高齡組中含量下調(diào),在低齡組中含量增高,分別是αB-晶狀體蛋白(Alpha-crystallin B chain),甜菜堿高半胱氨酸甲基轉(zhuǎn)移酶(Betaine--homocysteine S-methyltransferase 1),血影蛋白β鏈(腦型1)(Spectrin beta chain-brain 1),晶狀體蛋白(Phakinin),γC-晶狀體蛋白(Gamma-crystallin C),磷酸甘油酸激酶(Phosphoglycerate kinase 1),谷胱甘肽合成酶(glutathione synthetase)。4 Westen blot驗證結(jié)果顯示有2種與眼軸相關(guān)的蛋白質(zhì)表達與質(zhì)譜分析結(jié)果一致,分別為βB2-晶狀體蛋白和山梨醇脫氫酶。驗證了與年齡相關(guān)的2個蛋白質(zhì):αB-晶狀體蛋白和谷胱甘肽合成酶,Western blot結(jié)果顯示其表達均與質(zhì)譜分析結(jié)果一致。[結(jié)論]1晶狀體超聲彈性成像可以成功獲得在體晶狀體彈性分布的定量信息。通過分析彈性應(yīng)變率,可以了解在體晶狀體的硬度情況,對晶狀體核硬度的臨床分級提供有效的補充。同時,可為白內(nèi)障術(shù)中超聲乳化能量的設(shè)置提供參考。2晶狀體超聲彈性成像顯示:長眼軸晶狀體核較短眼軸晶狀體核硬。超聲彈性成像對眼軸相關(guān)核性白內(nèi)障的研究提供了核硬度定量的參考數(shù)據(jù)。3基于i TRAQ的比較蛋白質(zhì)組學(xué)是一種有效的篩選眼軸相關(guān)核性白內(nèi)障潛在差異蛋白質(zhì)的研究手段。我們首次采用i TRAQ質(zhì)譜技術(shù)鑒定出βB2-晶狀體蛋白和山梨醇脫氫酶可能作為重要潛在的生物標志物,參與眼軸相關(guān)性核性白內(nèi)障的發(fā)生發(fā)展。αB-晶狀體蛋白和谷胱甘肽合成酶可能參與了年齡相關(guān)性核性白內(nèi)障的形成。
[Abstract]:Cataracts are the leading cause of visual impairment and blindness in developing countries. The correlation of cataract and myopia has been paid more attention as cataract patients are younger. Among the many risk factors, there is a very close relationship between myopia and the development of nuclear cataract in different populations and different areas. The characteristics of cataract induced by myopia are different from the age related cataract: vitreous liquefaction, the hardness of the lens nucleus is obviously increased. In China, the proportion of myopia is increasing year by year, the age of cataract surgery is becoming younger, which makes the task of preventing blindness more arduous in our country. Eye axis and vitreous liquefaction lead. The mechanism of nuclear sclerosis has become a hot spot in this field. At present, the study of nuclear cataract with high myopia is mainly based on epidemiological investigation and clinical study. There is no ideal animal model for the study of pathogenesis. The cause of nuclear cataract with myopia is very complicated, and the high myopia nucleus is further explored and revealed. The pathogenesis of sexual cataract is very important. With the rapid development of biological mass spectrometry and bioinformatics, proteomics has become the forefront of life science. Therefore, the discovery of key target proteins at the protein level is of great significance to reveal the formation mechanism of ocular axis related nuclear cataract and potential therapeutic targets. [Objective]1 to provide more evidence for the prevention of blindness. [Objective] to detect the hardness of the lens nucleus of the body and lens in different ages by ultrasonic elastography, and to explore its value in the study of ocular axis related nuclear cataract..2 through collecting human cataract lens specimens, using the combined mass spectrometry technology of fixed quantity proteomics, screening and identification. The expression of related protein.3 related protein in the nucleus of ocular axis and age-related nuclear cataracts in clinical cases can provide an important theoretical basis for exploring the pathogenesis of ocular axis and age-related nuclear cataract and finding new targets for treatment. [square method]1 age and ocular axis related nuclear cataract crystal form) Study of the hardness of body nucleus (1) the clinical subjects of age related lens nuclear hardness detected by ultrasonic elastography in 48 cases of transparent or mild opacities in the binocular lens, according to age, group A, B, C, each case was selected in group.A 16 cases, 16 eyes, 8 women, 8 eyes, 81 + 5.5 years old.B group 16 eyes, 6 eyes, male 10 eyes, average age 44 + 3.2 years old; C There were 16 cases of 16 eyes, 6 eyes of women and 10 eyes of male, 21 + 2.5 years old. The average axial axis of the three groups was 23.4 + 0.44 (23~24mm). Corneal topography excluded the influence of keratoconus on the eye axis, intraocular pressure: average 15.6 + 1.46 mm Hg (14mm Hg~19 mm Hg), excluding systemic diseases and other diseases of the eye, UCVA > 0.5, and the observer informed consent. The instrument used Hitachi EUB 7500 color multi. The puller ultrasound diagnostic instrument (EUP2L54M, 7L, frequency 8~10MHz). In the ultrasound elastography, the green represents the average hardness of the tissue in the region of interest (ROI). Red represents a larger strain, indicating that the tissue is softer; the blue indicates that the strain is smaller and the tissue is very hard. Each case is measured 3 times continuously, and the average value is taken. Evaluate the crystal shape of the observer. The relationship between body elastic strain rate and age change. (2) ultrasonic elastography detection of anisometropic lens nucleus hardness observation 14 cases (28 eyes) of unilateral high myopia anisometropia, complex myopic anisometropia, male 5 eyes, 9 eyes, age 62 + 3.3 years, average ocular pressure 17.11 + 1.46 mm Hg (14mm Hg~20 mm Hg); corneal topography exclusion The effect of keratoconus on the eye axis, excluding systemic disease and other ocular diseases, patients' informed consent. The long eye axis detection data of each patient were included in group A (14 eyes), and the relative short ocular axis detection data were included in group B (14 eyes), and the method of ultrasonic elastic detection was same. 2 different age, ocular axis related nuclear cataract lens proteomics (1) two dimensional liquid chromatography combined with I TRAQ tandem mass spectrometry identification, screening different age and ocular axis lens nucleus difference protein through cataract extraction (ECCE) surgery to collect 48 cases of cataract patients' lens nucleus, according to age and eye axis of 6 Group 8: group A (average axial axis 28.7 + 1.5mm, average age 79.8 + 1.9 years), group B (average axial axis 28.7 + 1.4 mm, average age 58 + 4 years), C group (average axial axis 23 + 0.4 mm, average age 80.3 + 4.5 years), D group (average axis 23 + mm, average age of years of age), E group (average axial axis mm, mean age of mean years of age), F Groups (average axial axis 20.7 + 0.6 mm, average age 57.6 + 5.3 years old) group.G were transparent lens control group (average axial axis 23.5 + 0.6 mm, average age 34.7 + 4.2 years old), from 7 lens nucleus of cornea transplantation donor. Each group extracted water solubility, water insoluble urine solubility, water insoluble urea insoluble protein, each group of protein peptide segment After mixing, after desalination, denaturation, reduction, alkylation and enzymatic hydrolysis, stable isotopes 114115116117118119121 I TRAQ were labeled, separated by two-dimensional liquid chromatography, and then detected by tandem mass spectrometry. The mass spectrometry data were retrieved and identified in the IPIv3.45 protein database, and the analysis of differential protein data was carried out. Experimental techniques were repeated. The two time, the protein was selected as the differential protein to repeat and express the same trend. (2) the Western blot method verified 20 cases of cataract extraction and extraction of lens nuclear tissue from the new cataract capsule, 4 groups of 5.A groups in each group (average axial axis 29.1 + 1.2mm, average age 59 + 3 years old), B group (average axial axis 23.1 + 0.3mm, mean, average) Age 55.8 + 3.5 years old), group C (average axis of eye axis 28.8 + 1.2mm, average age 78.4 + 1.9 years), D group (average axis 23 + 0.6 mm, average age 79.3 + 3.5 years). Extract lens water solubility, water insoluble urea solubility, water insoluble urea insoluble protein components (method of the same before). Using Western blot method to express significant difference in mass spectrometry detection of differential expression of eggs The white matter was verified. [results]1 lens ultrasound elastography data showed that the relationship between nuclear hardness and age changes in the age group (group A), the strain rate of the lens nucleus was the smallest (0.02 + 0.08), and the strain rate of the lens nucleus was the largest in the low age group (group C) (1.95 + 0.87), and the strain rate of the middle age group (group B) was (0.69 + 0.12). The analysis of single factor ANOVA showed that A group and B group, A group and C group, B group and C group were statistically significant (P? 0.005). The strain rate of lens nucleus in the elderly group was small, and the strain rate of the nucleus lens nucleus of the younger age group and the large.2 lens ultrasound elastography showed the relationship between the nucleus stiffness and the ocular axis in the patients with anisometropia. The strain rate was (0.16 + 0.08) and the strain rate was (0.54 + 0.16) in the lens nucleus of the relative short eye axis. The independent sample t test analysis showed that the two groups were compared with P? 0.005. The strain rate of the lens nucleus of the long ocular axis was small, and the strain rate of the lens nucleus in the relative short ocular axis group was large.3 using I TRAQ technology to screen out 6 differential eggs related to the ocular axis. White matter, 9 age related differential proteins. 148 proteins were identified by the first I TRAQ analysis and 103 proteins were identified by second I TRAQ analysis. A total of 80 proteins were found in the intersection of two results. The significant differential expression protein boundary values were set as the p value less than 0.05, and the differential multiple was more than 1.2 times or less than 0.8 times the protein. After controlling the age, the 80 proteins showed that 6 proteins were closely related to axial nuclear cataracts. Among them, 3 proteins were up up in the long eye axis group and in the short eye axis group, they were gamma enolase (Gamma-enolase), pyruvate kinase isoenzyme M1/M2 (Pyruvate kinase isozymes M1/M2) and sorbitol dehydrogenase (Sorbit). Ol dehydrogenase); the other 3 proteins were down regulated in the long eye axis group and increased in the short eye axis group, which were gap connexin 3 (Gap junction Alpha-3 protein), beta B2- crystallin (Beta-crystallin B2), T- complex polypeptide 1 (T-complex protein 1). After controlling the eye axis factor, 9 kinds of protein and year were analyzed. Age related nuclear cataract is closely related, 2 of which are up regulated in the age group, and in the lower age group, the content is less, which are fatty acid binding protein (Fatty acid-binding protein) and methotrexate -4- alpha methamine dehydrase (Pterin-4-alpha-carbinolamine dehydratase); the other 7 proteins are down down in the age group and in the lower age group. The content of the group was increased, which were alpha B- crystallin (Alpha-crystallin B chain), betaine homocysteine methyltransferase (Betaine--homocysteine S-methyltransferase 1), blood shadow protein beta chain (brain type 1) (Spectrin beta chain-brain 1), crystallin (Phakinin), gamma C- lens protein (Gamma-crystallin), phosphate glyoacid excitation The results of enzyme (Phosphoglycerate kinase 1), glutathione synthetase (glutathione synthetase).4 Westen blot showed that 2 kinds of protein expressions associated with ocular axis were in accordance with the results of mass spectrometric analysis, respectively, beta B2- crystallin and sorbitol dehydrogenase, respectively. 2 proteins associated with age were verified: alpha B- crystallin and Gu Guanggan The results of peptide synthetase, Western blot, show that the expression of the enzyme is in agreement with the results of mass spectrometry. [conclusion]1 lens ultrasound elastography can successfully obtain quantitative information of the elastic distribution of the body lens. By analyzing the elastic strain rate, the hardness of the body lens can be understood and the clinical classification of the hardness of the lens can be effectively supplemental. At the same time, we can provide a reference.2 lens ultrasound elastography for the setting of phacoemulsification energy during cataract surgery: the nucleus of the lens nucleus of the long eye axis is more hard than the short axis of the lens. The quantitative reference number of the nuclear hardness of the eye axis related nuclear cataract is provided by the ultrasound elastography. The comparative proteomics of the.3 based on I TRAQ is a kind of comparative proteomics. Effective screening of potential differential proteins for ocular axis related nuclear cataracts. We first identified the beta B2- crystallin and sorbitol dehydrogenase as an important potential biomarker by I TRAQ mass spectrometry, involved in the development of ocular axis related nuclear cataracts. Alpha B- crystallin and glutathione combination Enzyme production may be involved in the formation of age-related nuclear cataract.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R776.1

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