不同年龄和眼轴人晶状体硬度的在体检测及基于iTRAQ的蛋白质组学研究

发布时间：2018-06-26 11:28

本文选题：眼轴 + 年龄　；参考：《第四军医大学》2014年博士论文

【摘要】：白内障是发展中国家最主要的视力损害和致盲原因,随着白内障患者年轻化,白内障和近视的相关性逐渐引起了人们的重视。在众多危险因素中,已证实不同人种、不同地区情况下近视与核性白内障的发生发展有非常密切的关系。高度近视诱导的核性白内障,其特征不同与年龄相关性白内障:玻璃体液化、晶状体核硬度明显增加。目前在我国,近视患者的比例呈逐年上升的趋势,白内障手术年龄呈现年轻化的趋势,使我国现有的防盲任务更加艰巨。眼轴与玻璃体液化引起晶状体核硬化的机制已成为本领域研究的热点。目前高度近视并发核性白内障的研究主要基于流行病学调查和临床研究,尚无理想的动物模型用于发病机制的研究。近视并发核性白内障成因十分复杂,进一步探索并揭示高度近视核性白内障的发病机制是非常重要的。随着生物质谱技术和生物信息学迅猛发展,蛋白质组学研究成为生命科学的前沿。因此在蛋白质水平上发现关键靶蛋白,对我们揭示眼轴相关性核性白内障的形成机制以及潜在治疗靶点有重要意义,可为防盲工作提供更多依据。[目的]1通过超声弹性成像技术检测不同年龄、不同眼轴在体人晶状体核硬度,探讨其在眼轴相关性核性白内障研究方面的价值。2通过收集人白内障晶状体标本,利用定量蛋白质组学联合质谱技术,筛选和鉴定与眼轴及年龄相关的核性白内障晶状体核的相关蛋白质。3验证相关蛋白在临床病例中的表达,为探索与眼轴及年龄相关核性白内障发病机制,寻找能够作为治疗的新靶点提供重要理论依据。[方法]1年龄和眼轴相关性核性白内障晶状体核硬度的研究(1)超声弹性成像检测年龄相关性晶状体核硬度观察48例双眼晶状体透明或轻度混浊的临床受试者,根据年龄分为A,B,C组,每例任选一眼检测。A组16例16眼,女8眼,男8眼,均龄81±5.5岁。B组16例16眼,女6眼,男10眼,均龄44±3.2岁;C组16例16眼,女6眼,男10眼,均龄21±2.5岁。三组平均眼轴23.4±0.44(23~24mm),角膜地形图排除圆锥角膜对眼轴的影响,眼压: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,观察者知情同意。仪器采用Hitachi EUB 7500型彩色多普勒超声诊断仪(探头型号:EUP2L54M,7L,频率8~10MHz)。在超声弹性图中,绿色代表弹性成像感兴趣区(ROI)内组织的平均硬度,红色代表应变性较大,说明组织较软;蓝色表示应变性较小,组织硬度大。每例连续测量3次,取平均值。评价观察者晶状体弹性应变率与年龄变化的关系。(2)超声弹性成像检测屈光参差晶状体核硬度观察14例(28眼)单性高度近视性屈光参差、复性近视性屈光参差的临床病例,男5眼,女9眼,均龄62±3.3岁,平均眼压17.11±1.46 mm Hg(14mm Hg~20 mm Hg);角膜地形图排除圆锥角膜对眼轴的影响,排除全身疾病及眼部其他疾患;患者知情同意。每位患者长眼轴检测数据纳入A组(14眼),相对短眼轴检测数据纳入B组(14眼),超声弹性检测方法同上。观察长眼轴组与相对短眼轴组晶状体弹性应变率与眼轴的关系。2不同年龄、眼轴相关性核性白内障晶状体蛋白质组学研究(1)二维液相色谱联合i TRAQ标记法串联质谱鉴定、筛选不同年龄和眼轴晶状体核差异蛋白质通过白内障囊外摘除(ECCE)手术收集48例白内障患者的晶状体核,根据年龄和眼轴分为6组,每组8例:A组(平均眼轴28.7±1.5mm,平均年龄79.8±1.9岁),B组(平均眼轴28.7±1.4 mm,平均年龄58.0±4.0岁),C组(平均眼轴23.0±0.4 mm,平均年龄80.3±4.5岁),D组(平均眼轴23.0±0.3 mm,平均年龄56.9±4.2岁),E组(平均眼轴19.9±0.5 mm,平均年龄为75.1±2.5岁),F组(平均眼轴20.7±0.6 mm,平均年龄为57.6±5.3岁)。G组为透明晶状体对照组(平均眼轴23.5±0.6 mm,平均年龄为34.7±4.2岁),取自角膜移植供体的7枚透明晶状体核。每组提取晶状体水溶性,水不溶尿素溶性、水不溶尿素不溶性蛋白,每组蛋白质肽段混合,经过除盐、变性、还原、烷基化、酶解后分别进行稳定同位素114,115,116,117,118,119,121 i TRAQ标记、二维液相色谱分离,然后用串联质谱进行检测。所得质谱数据在IPIv3.45蛋白数据库进行检索鉴定,并进行差异蛋白质数据分析。实验技术重复两次,选择重复出现且表达趋势一致的蛋白质作为差异蛋白质进行分析。(2)Western blot法验证差异蛋白质收集新的白内障囊外摘除晶状体核组织20例,分4组,每组5例。A组(平均眼轴29.1±1.2mm,平均年龄59.0±3.0岁),B组(平均眼轴23.1±0.3mm,平均年龄55.8±3.5岁),C组(平均眼轴28.8±1.2mm,平均年龄78.4±1.9岁),D组(平均眼轴23.0±0.6 mm,平均年龄79.3±3.5岁)。提取晶状体水溶性,水不溶尿素溶性、水不溶尿素不溶性蛋白质组分(方法同前)。利用Western blot法对质谱检测中有意义的差异表达蛋白质进行验证。[结果]1晶状体超声弹性成像数据显示晶状体核硬度与年龄变化的关系在高龄组(A组),晶状体核区的应变率最小(0.02±0.08);在低龄组(C组),晶状体核区的应变率最大(1.95±0.87);中龄组(B组)晶状体核区的应变率为(0.69±0.12)。通过单因素方差分析显示:A组与B组,A组与C组,B组与C组比较均有统计学意义(P㩳0.005)。高龄组晶状体核应变率小,低龄组晶状体核应变率大。2晶状体超声弹性成像显示屈光参差患者晶状体核硬度与眼轴变化的关系长眼轴组晶状体核区的应变率为(0.16±0.08),相对短眼轴组晶状体核区的应变率为(0.54±0.16),独立样本t检验分析显示:两组间比较P㩳0.005,有统计学意义。长眼轴组晶状体核应变率小,相对短眼轴组晶状体核应变率大。3用i TRAQ技术筛选出了6个与眼轴相关的差异蛋白质,9个与年龄相关的差异蛋白质。第一次i TRAQ分析鉴定出148种蛋白质,第二次i TRAQ分析鉴定出103种蛋白质。选择两次结果的交集,共发现80种蛋白质。显著差异表达蛋白界值设定为:p值小于0.05,且差异倍数大于1.2倍或小于0.8倍的蛋白质。控制年龄因素后,80个蛋白质中分析得出6种蛋白质与轴性核性白内障密切相关。其中3种蛋白质在长眼轴组中上调,在短眼轴组中含量少,分别是γ-烯醇酶(Gamma-enolase),丙酮酸激酶同工酶M1/M2(Pyruvate kinase isozymes M1/M2)和山梨醇脱氢酶(Sorbitol dehydrogenase);另3种蛋白质在长眼轴组中表达下调,在短眼轴组中含量增高,分别是缝隙连接蛋白3(Gap junction alpha-3 protein),βB2-晶状体蛋白(Beta-crystallin B2),T-复合物多肽1(T-complex protein 1 subunit beta)。控制眼轴因素后,分析得出9种蛋白与年龄相关性核性白内障密切相关,其中2个蛋白质在高龄组中含量上调,在低龄组中含量少,分别是脂肪酸结合蛋白(Fatty acid-binding protein),蝶呤-4-α-甲醇胺脱水酶(Pterin-4-alpha-carbinolamine dehydratase);另7个蛋白质在高龄组中含量下调,在低龄组中含量增高,分别是αB-晶状体蛋白(Alpha-crystallin B chain),甜菜碱高半胱氨酸甲基转移酶(Betaine--homocysteine S-methyltransferase 1),血影蛋白β链(脑型1)(Spectrin beta chain-brain 1),晶状体蛋白(Phakinin),γC-晶状体蛋白(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验证结果显示有2种与眼轴相关的蛋白质表达与质谱分析结果一致,分别为βB2-晶状体蛋白和山梨醇脱氢酶。验证了与年龄相关的2个蛋白质:αB-晶状体蛋白和谷胱甘肽合成酶,Western blot结果显示其表达均与质谱分析结果一致。[结论]1晶状体超声弹性成像可以成功获得在体晶状体弹性分布的定量信息。通过分析弹性应变率,可以了解在体晶状体的硬度情况,对晶状体核硬度的临床分级提供有效的补充。同时,可为白内障术中超声乳化能量的设置提供参考。2晶状体超声弹性成像显示:长眼轴晶状体核较短眼轴晶状体核硬。超声弹性成像对眼轴相关核性白内障的研究提供了核硬度定量的参考数据。3基于i TRAQ的比较蛋白质组学是一种有效的筛选眼轴相关核性白内障潜在差异蛋白质的研究手段。我们首次采用i TRAQ质谱技术鉴定出βB2-晶状体蛋白和山梨醇脱氢酶可能作为重要潜在的生物标志物,参与眼轴相关性核性白内障的发生发展。αB-晶状体蛋白和谷胱甘肽合成酶可能参与了年龄相关性核性白内障的形成。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R776.1

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