SIRT1基因表达对人晶状体上皮细胞的保护作用及其机制研究

发布时间：2018-05-08 19:12

本文选题：年龄相关性白内障 + 晶状体上皮细胞　；参考：《复旦大学》2011年博士论文

【摘要】：年龄相关性白内障(age-related cataract, ARC)是世界首位致盲性眼病,其防治工作已成为本世纪防盲治盲工作的重点。研究ARC的发病机制,通过预防白内障发生或延缓其发展的途径征服白内障,己成为一项全球性的社会和经济问题。目前已公认氧化损伤是ARC发生的主要危险因素和始动环节。这一过程的病理学基础,主要表现在晶状体上皮细胞(lens epithelial cells, LECs)的凋亡和晶状体蛋白改变两方面。其中,LECs的凋亡又是晶状体蛋白改变的发生因素之一。因此,ARC的发生发展与LECs的状态变化密切相关。Li等于1995年首次提出,LECs的凋亡是各型非先天性白内障共同的细胞学基础。基于以上背景,LECs的凋亡相关研究成为ARC发病机理研究中的热点问题,寻找避免LECs凋亡的保护途径、或激活机体自身的抗凋亡保护体系,无疑具有重要的干预意义。沉默信息调节因子相关酶1(sirtuin type 1, SIRT1)即为机体自身抗衰老、抗凋亡体系的重要一员。SIRT1是一种细胞代谢辅酶NAD+依赖的111类组蛋白去乙酰化酶,具有延长低等生物寿命和延缓多种年龄相关性疾病发展的作用。它主要通过组蛋白脱乙酰基作用调节P53和又头转录因子(Forkhead box O, FOXOs)等转录因子的活性,在抵抗氧化应激、对抗细胞凋亡等活动中发挥重要作用。SIRT1已成为治疗年龄相关性疾病的颇具潜力的药物靶点。遗憾的是,在眼科最常见的年龄相关性疾病ARC相关研究中,尚未对SIRT1有任何涉猎。因此,本研究将观察人LECs (human LECs, HLECs)中SIRT1基因的表达,及其在ARC的发生及氧化损伤环境中的表达变化和对HLECs的保护作用,并进一步研究SIRT1通过其下游P53通路对HLECs的保护机制。第一部分人年龄相关性白内障发生中SIRT1基因及其下游通路蛋白的表达变化目的观察HLECs中SIRT1基因及其下游P53及FOXOs通路蛋白表达在ARC发生中的表达改变,初步分析SIRT1在ARC发生中的保护作用和可能的作用途径。方法眼库取材晶状体前囊膜分为：(1)青年组：正常青年人晶状体前囊膜(20-40岁)；(2)老年组：正常老年人晶状体前囊膜(50-70岁)；(3)ARC组：ARC老年人晶状体前囊膜(50-70岁且伴有ARC),各82例。各组样本行SIRT1 mRNA复合逆转录聚合酶链反应(real-time quantitative reverse transcription, RT-PCR)检测,SIRT1、p53、乙酰化p53、FOXO3a、FOXO4、p27、p130、Bim蛋白的蛋白免疫印迹(western blot, WB)检测,SIRT1免疫荧光染色、原位末端转移酶标记技术(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, TUNEL)染色。结果SIRT1 mRNA转录水平RT-PCR检测2(△△Ct)值为青年组(1.000±0.143),老年组(0.451±0.065),ARC组(0.715±0.171),差异有统计学意义。SIRT1蛋白WB检测及免疫荧光染色均为青年组最强,ARC组居中,老年组最弱。P53通路检测显示,P53蛋白表达为青年组最低,老年组次之,ARC组最高,但具有凋亡相关活性的乙酰化p53为ARC组低于老年组。FOXOs通路检测显示,FOXO3a、FOXO4、p27kip1、p130蛋白表达均为ARC组最高,凋亡相关Bim蛋白表达为ARC组最低,差异有统计学意义。TUNEL检测发现,各组HLECs的凋亡百分比分别为：青年组(0.8±0.7)%,老年组(8.5±2.7)%,ARC组(25.0±9.8)%,差异有统计学意义。结论SIRT1基因在人晶状体上皮细胞中的表达随年龄增长而减少,但在ARC发生中呈保护性上调。下游通路蛋白检测提示,上调的SIRT1可能通过p53通路与FOXOs通路诱导细胞周期停滞、抑制细胞凋亡,在ARC发生中发挥保护作用。第二部分增强与抑制SIRT1蛋白功能对氧化环境下HLECs凋亡的影响目的观察SIRT1活性改变对氧化环境下培养的HLECs凋亡的影响,分析SIRT1对HLECs的保护作用。方法体外培养HLECs系SRA01/04,分为4个大组：(1)正常对照组：常规培养；(2)H2O2组：加入H2O2终浓度为400μmol/L; (3) SIRT1激活剂白藜芦醇(resveratrol, RES)组,分设4个RES浓度组：加入H2O2及RES溶液,H2O2终浓度为400μmol/L, RES终浓度分别为5,10,20,40μmol/L; (4) SIRT1抑制剂烟酰胺(Nicotinamide, NAM)组,分设4个NAM浓度组：加入H2O2及NAM溶液,H2O2终浓度为400μmol/L, NAM终浓度分别为25,50,100,200μmol/L。各组进行以上干预并孵育24h后,行SIRT1蛋白免疫荧光染色,SIRT1及下游通路P53、乙酰化P53蛋白WB检测、倒置显微镜形态学观察、HLECs的增殖活性MTT比色法检测、HLECs凋亡水平TUNEL染色检测。结果正常对照组仅有少量SIRT1蛋白免疫荧光染色,H2O2组则明显增加,4个RES浓度组荧光进一步增强且随RES浓度上升而增加；4个NAM浓度组荧光与H2O2组比较未见明显变化。SIRT1 Western Blot检测结果与SIRT1免疫荧光染色结果一致。乙酰化P53蛋白水平随着添加RES浓度的增加而逐渐下降,随着添加NAM浓度的增加而逐渐升高。倒置显微镜观察发现,正常对照组HLECs呈较规则六角形或椭圆形,细胞密度较高；H2O2组细胞密度明显下降,少数细胞呈长梭形：加入RES后,长梭形细胞明显减少,细胞密度回升且随RES浓度上升而逐渐增加；随着添加NAM浓度的增加,细胞形态的不规则性明显增加,并出现大量长梭形细胞甚至互相联结呈网状。HLECs的MTT比色OD值随RES浓度增加而上升,RES浓度为10,20和40μmol/L时与单纯添加H2O2组比较有统计学差异(P0.05),但RES浓度由20μmol/L上升为40μmol/L时OD值无统计学差异(P0.05)；添加NAM浓度为50,100,200μmol/L时与单纯添加H2O2组比较MTT值明显下降(P0.05)。TUNEL检测发现,RES浓度为10,20,40μmol/L时细胞凋亡百分比与H2O2组比较显著降低(P0.05)。添加NAM浓度为25,50,100,200μmol/L时细胞凋亡百分与H2O2组比较则显著增加(P0.05)。结论在氧化损伤境下,HLECs中的SIRT1基因表达反应性上调；RES可通过上调SIRT1活性,减少H2O2导致的HLECs的凋亡；NAM则通过抑制SIRT1的活性,进一步加剧了HLECs的凋亡,提示SIRT1对HLECs具有抗凋亡的保护作用。第三部分在HLECs中阻断P53通路对SIRT1保护作用的影响目的应用pifithrin-a (p-fifty three inhibitor, PFT-α)阻断P53通路,观察SIRT1对HLECs的保护作用的影响,分析P53通路是否为SIRT1在HLECs中发挥保护作用的下游通路。方法体外培养HLECs SRA01/04,分为4个大组：(1)正常对照组：常规培养；(2)H202组：加入H202,终浓度为400μmol/L; (3) NAM组：加入H202及NAM溶液,H202终浓度为400μmol/L, NAM终浓度为100μmol/L; (4) PFT-α组,分设3个PFT-α浓度组：加入H202、NAM及PFT-α溶液,H202终浓度为400μmol/L, NAM终浓度为100μmol/L, PFT-α终浓度分别为2.5,5,10μmol/L。各组进行以上干预并孵育24h后,行SIRT1蛋白免疫荧光染色,SIRT1及下游通路P53、乙酰化P53蛋白WB检测、倒置显微镜形态学观察、HLECs的增殖活性MTT比色法检测、HLECs凋亡水平TUNEL染色检测。结果正常对照组仅有少量SIRT1蛋白免疫荧光染色,H202组则明显增加。NAM组及3个PFT-α浓度组的SIRT1荧光染色与H202组比较均未见明显变化。SIRT1 WB检测结果与SIRT1免疫荧光染色结果一致。与H202组比较,NAM组及3个PFT-α浓度组乙酰化P53均升高,但在这4组间无明显变化。倒置显微镜观察发现,正常对照组HLECs呈较规则六角形或椭圆形,细胞密度较高；H202组细胞密度明显下降,少数细胞呈长梭形；加入NAM后,细胞形态的不规则性明显增加,出现大量长梭形细胞;加入PFT-α后,细胞密度回升且随PFT-α浓度上升而逐渐增加,长梭形细胞明显减少,细胞形态渐趋正常。在H202培养条件下加入NAM后,HLECs的MTT比色OD值下降,但随着添加PFT-α浓度增加而回升,添加3个PFT-α浓度时与NAM组比较均有统计学差异(P0.05)。TUNEL检测发现,在H202培养条件下添加NAM后HLECs凋亡百分比上升,随着添加PFT-α浓度的上升,细胞凋亡百分比逐渐降低(P0.05)。结论PFT-α具有抑制HLECs细胞凋亡的作用,用PFT-α阻断P53通路可消除NAM对SIRT1保护作用的抑制,提示P53通路是SIRT1发挥保护功能的重要通路。
[Abstract]:Age-related cataract (ARC) is the first blind eye disease in the world. Its prevention and control work has become the focus of blindness prevention in this century. The study of the pathogenesis of ARC and the conquest of cataract by preventing the occurrence of cataract or postponing its development has become a global social and economic problem.
It is now recognized that oxidative damage is the main risk factor and initiation link of ARC. The pathological basis of this process is mainly manifested in the two aspects of apoptosis and change of lens protein in the lens epithelial cells (lens epithelial cells, LECs). Among them, the apoptosis of LECs is one of the factors that occur in the change of the crystalline body protein. Therefore, ARC The occurrence and development are closely related to the state changes of LECs..Li is first proposed in 1995. The apoptosis of LECs is the common cytological basis of various types of non congenital cataract. Based on the above background, the apoptosis related research of LECs has become a hot issue in the study of the pathogenesis of ARC, looking for the protection way to avoid LECs apoptosis, or activating the body's own resistance. The protective system of apoptosis is of great importance for intervention.
The silencing information regulating factor related enzyme 1 (sirtuin type 1, SIRT1) is an important member of the body itself against aging, and an important member of the anti apoptotic system.SIRT1 is a 111 class of histone deacetylase that is dependent on the cell metabolism coenzyme NAD+. It has the role of prolonging low life life and retarding the development of many age-related diseases. It mainly through histone Deacetylation regulates the activity of transcriptional factors such as P53 and Forkhead box O (FOXOs), and plays an important role in resisting oxidative stress and antagonism to cell apoptosis..SIRT1 has become a potential drug target for the treatment of age-related diseases. Unfortunately, the most common age related disease, ARC, in the ophthalmology. In the related study, SIRT1 has not been dabbled. Therefore, this study will observe the expression of SIRT1 gene in human LECs (human LECs, HLECs), and its expression in the occurrence of ARC and the changes in the oxidative damage environment and the protection of HLECs, and further study the protection mechanism of SIRT1 through its downstream P53 pathway.
Part one: expression changes of SIRT1 gene and its downstream pathway proteins in human age-related cataract
Objective To observe the expression of SIRT1 gene in HLECs and its expression in the downstream P53 and FOXOs pathway protein in the occurrence of ARC, and to preliminarily analyze the protective effect and possible pathway of SIRT1 in the occurrence of ARC.
Methods the anterior capsule of ocular lens was divided into: (1) young group: the anterior capsule of normal young people (20-40 years old); (2) the elderly group: the anterior capsule (50-70 years old) of the normal aged people (50-70 years); (3) ARC group: the anterior capsule of the ARC elderly (50-70 years old and ARC), each of the 82 cases. The SIRT1 mRNA compound reverse transcription polymerase chain reaction in each group (real-time quantitative reverse transcription, RT-PCR) detection, SIRT1, p53, acetylated p53, FOXO3a, FOXO4, p27, p130, protein immunoblotting detection, immunofluorescence staining, in situ terminal transferase labeling technique NEL) dyed.
Results the value of SIRT1 mRNA transcriptional level RT-PCR Detection 2 (delta delta Ct) was in young group (1 + 0.143), aged group (0.451 + 0.065) and ARC group (0.715 + 0.171). The difference was statistically significant,.SIRT1 protein WB detection and immunofluorescence staining were the strongest in young group, ARC group was in the middle, and the weakest.P53 pathway in old year group showed that P53 protein expression was the lowest in young group. Group ARC was the highest in the aged group, but the acetylation p53 with apoptosis related activity was lower than that of the old group. The expression of FOXO3a, FOXO4, p27kip1, p130 protein was the highest in the ARC group, and the expression of apoptosis related Bim protein was the lowest in the ARC group, and the difference was statistically significant for the.TUNEL detection. The percentage of apoptosis in each group was respectively. For young group (0.8 + 0.7)%, aged group (8.5 + 2.7)%, group ARC (25 + 9.8)%, the difference was statistically significant.
Conclusion the expression of SIRT1 gene in human lens epithelial cells decreases with age, but it is up regulated in the occurrence of ARC. Downstream pathway protein detection suggests that up regulation SIRT1 may induce cell cycle stagnation through the p53 pathway and FOXOs pathway, inhibit cell apoptosis and play a protective role in the occurrence of ARC.
The second part is to enhance and inhibit the function of SIRT1 protein on the apoptosis of HLECs in oxidative environment.
Objective To observe the effect of SIRT1 activity on the apoptosis of HLECs cultured in oxidative environment, and to analyze the protective effect of SIRT1 on HLECs.
Methods in vitro culture HLECs line SRA01/04, divided into 4 large groups: (1) normal control group: normal culture; (2) H2O2 group: H2O2 terminal concentration was 400 mu mol/L; (3) SIRT1 activator, resveratrol (resveratrol, RES) group, divided into 4 RES concentration groups: H2O2 and RES solution, 400 micron end concentration, respectively. Ol/L (4) SIRT1 inhibitor nicotinamide (Nicotinamide, NAM) group, divided into 4 NAM concentration groups: H2O2 and NAM solution, H2O2 terminal concentration is 400 u, NAM end concentration is 25,50100200 micron each intervention and incubation. Morphological observation of inverted microscope showed that the proliferation activity of HLECs was detected by MTT colorimetric assay, and the apoptosis level of HLECs was detected by TUNEL staining.
Results only a small amount of SIRT1 protein immunofluorescence staining was found in the normal control group, and the H2O2 group increased significantly. The fluorescence of the 4 RES concentration groups was further enhanced and increased with the increase of RES concentration. The fluorescence of the 4 NAM concentration group had no obvious changes in the.SIRT1 Western Blot detection results and the SIRT1 immunofluorescence staining results. Acetylation P53 eggs. The white level gradually decreased with the increase of RES concentration, and gradually increased with the increase of the concentration of NAM. The inverted microscope showed that the normal control group HLECs was more regular hexagonal or oval, the cell density was higher, the cell density in the H2O2 group decreased obviously, and the small cell was long shuttle shape: after RES, the long spindle cells were obvious. The density of cells increased and increased with the increase of RES concentration. With the increase of the concentration of NAM, the irregularity of cell morphology increased obviously, and a large number of long spindle cells and even interlinked.HLECs MTT colorimetric OD values increased with the increase of RES concentration. RES concentration was 10,20 and 40 u mol/L with the simple addition of H2O2 group. There was a statistically significant difference (P0.05), but there was no significant difference in the OD value when the concentration of RES increased from 20 to 40 mol/L (P0.05). When the concentration of NAM was 50100200 mu mol/L, the MTT value decreased significantly compared with that of the addition of H2O2 group (P0.05).TUNEL detection. (P0.05) when NAM concentration was 25,50100200 mol/L, the percentage of apoptotic cells increased significantly compared with H2O2 group (P0.05).
Conclusion the SIRT1 gene expression in HLECs is up-regulated under oxidative stress, and RES can reduce the apoptosis of HLECs induced by H2O2 by up regulation of SIRT1 activity, and NAM further aggravates the apoptosis of HLECs by inhibiting the activity of SIRT1, suggesting that SIRT1 has the protective effect of anti apoptosis on HLECs.
The third part is to block the effect of P53 pathway on SIRT1 protection in HLECs.
Objective to use pifithrin-a (p-fifty three inhibitor, PFT- alpha) to block the P53 pathway and to observe the effect of SIRT1 on the protection of HLECs, and to analyze whether the P53 pathway is a downstream pathway for SIRT1 to play a protective role in HLECs.
Methods in vitro culture HLECs SRA01/04, divided into 4 large groups: (1) normal control group: normal culture, (2) group H202: H202, final concentration of 400 mu mol/L; (3) NAM group: H202 and NAM solution, H202 final concentration of 400 mu mol/L, NAM concentration of 100 micron mol/L; (4) group of 3 alpha concentration groups: join together, 3, and alpha solutions, The final concentration of H202 was 400 mu mol/L, the final concentration of NAM was 100 mu mol/L, and the final concentration of PFT- alpha was 2.5,5,10 mu mol/L. in each group, and 24h was incubated with the SIRT1 protein immunofluorescence staining, SIRT1 and downstream pathway P53, acetylated P53 protein detection, inverted microscope morphological observation. Dead level TUNEL staining.
Results there was only a small amount of SIRT1 protein immunofluorescence staining in the normal control group, and in the group H202, the SIRT1 fluorescence staining of the.NAM group and the 3 PFT- alpha concentration group had no obvious changes in the H202 group. The results of.SIRT1 WB detection were the same as those of the SIRT1 immunofluorescence staining. Compared with the H202 group, the NAM group and the 3 PFT- alpha concentration groups were all increased. However, there was no obvious change between the 4 groups. The inverted microscope showed that the normal control group HLECs showed a regular hexagonal or oval shape, the cell density was higher, the cell density of the H202 group decreased obviously, and the few cells showed long spindle shape. After adding NAM, the irregular shape of cell morphology was obviously increased, and a large number of long spindle cells appeared. After adding PFT- alpha, cells were added to the cells. With the increase of density and the increase of PFT- alpha concentration, the long spindle cells obviously decreased and the cell morphology gradually became normal. After adding NAM to the H202 culture, the MTT ratio of HLECs decreased, but as the concentration of PFT- alpha was increased, there was a statistical difference between the addition of 3 PFT- alpha concentration and NAM group (P0.05).TUNEL detection, The percentage of HLECs apoptosis increased with the addition of NAM in H202 culture condition, and the percentage of apoptosis decreased gradually with the increase of PFT- alpha concentration (P0.05).
Conclusion PFT- alpha can inhibit the apoptosis of HLECs cells. Blocking the P53 pathway by PFT- alpha can eliminate the inhibition of the protective effect of NAM to SIRT1, suggesting that P53 pathway is an important pathway for SIRT1 to play a protective function.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R776.1

【参考文献】