發(fā)布時(shí)間：2018-05-08 19:12

  本文選題：年齡相關(guān)性白內(nèi)障 + 晶狀體上皮細(xì)胞��； 參考：《復(fù)旦大學(xué)》2011年博士論文

【摘要】：年齡相關(guān)性白內(nèi)障(age-related cataract, ARC)是世界首位致盲性眼病,其防治工作已成為本世紀(jì)防盲治盲工作的重點(diǎn)。研究ARC的發(fā)病機(jī)制,通過預(yù)防白內(nèi)障發(fā)生或延緩其發(fā)展的途徑征服白內(nèi)障,己成為一項(xiàng)全球性的社會(huì)和經(jīng)濟(jì)問題。 目前已公認(rèn)氧化損傷是ARC發(fā)生的主要危險(xiǎn)因素和始動(dòng)環(huán)節(jié)。這一過程的病理學(xué)基礎(chǔ),主要表現(xiàn)在晶狀體上皮細(xì)胞(lens epithelial cells, LECs)的凋亡和晶狀體蛋白改變兩方面。其中,LECs的凋亡又是晶狀體蛋白改變的發(fā)生因素之一。因此,ARC的發(fā)生發(fā)展與LECs的狀態(tài)變化密切相關(guān)。Li等于1995年首次提出,LECs的凋亡是各型非先天性白內(nèi)障共同的細(xì)胞學(xué)基礎(chǔ)�；谝陨媳尘�,LECs的凋亡相關(guān)研究成為ARC發(fā)病機(jī)理研究中的熱點(diǎn)問題,尋找避免LECs凋亡的保護(hù)途徑、或激活機(jī)體自身的抗凋亡保護(hù)體系,無(wú)疑具有重要的干預(yù)意義。 沉默信息調(diào)節(jié)因子相關(guān)酶1(sirtuin type 1, SIRT1)即為機(jī)體自身抗衰老、抗凋亡體系的重要一員。SIRT1是一種細(xì)胞代謝輔酶NAD+依賴的111類組蛋白去乙�；�,具有延長(zhǎng)低等生物壽命和延緩多種年齡相關(guān)性疾病發(fā)展的作用。它主要通過組蛋白脫乙�；饔谜{(diào)節(jié)P53和又頭轉(zhuǎn)錄因子(Forkhead box O, FOXOs)等轉(zhuǎn)錄因子的活性,在抵抗氧化應(yīng)激、對(duì)抗細(xì)胞凋亡等活動(dòng)中發(fā)揮重要作用。SIRT1已成為治療年齡相關(guān)性疾病的頗具潛力的藥物靶點(diǎn)。遺憾的是,在眼科最常見的年齡相關(guān)性疾病ARC相關(guān)研究中,尚未對(duì)SIRT1有任何涉獵。因此,本研究將觀察人LECs (human LECs, HLECs)中SIRT1基因的表達(dá),及其在ARC的發(fā)生及氧化損傷環(huán)境中的表達(dá)變化和對(duì)HLECs的保護(hù)作用,并進(jìn)一步研究SIRT1通過其下游P53通路對(duì)HLECs的保護(hù)機(jī)制。 第一部分人年齡相關(guān)性白內(nèi)障發(fā)生中SIRT1基因及其下游通路蛋白的表達(dá)變化 目的觀察HLECs中SIRT1基因及其下游P53及FOXOs通路蛋白表達(dá)在ARC發(fā)生中的表達(dá)改變,初步分析SIRT1在ARC發(fā)生中的保護(hù)作用和可能的作用途徑。 方法眼庫(kù)取材晶狀體前囊膜分為：(1)青年組：正常青年人晶狀體前囊膜(20-40歲)；(2)老年組：正常老年人晶狀體前囊膜(50-70歲)；(3)ARC組：ARC老年人晶狀體前囊膜(50-70歲且伴有ARC),各82例。各組樣本行SIRT1 mRNA復(fù)合逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(real-time quantitative reverse transcription, RT-PCR)檢測(cè),SIRT1、p53、乙�；痯53、FOXO3a、FOXO4、p27、p130、Bim蛋白的蛋白免疫印跡(western blot, WB)檢測(cè),SIRT1免疫熒光染色、原位末端轉(zhuǎn)移酶標(biāo)記技術(shù)(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, TUNEL)染色。 結(jié)果SIRT1 mRNA轉(zhuǎn)錄水平RT-PCR檢測(cè)2(△△Ct)值為青年組(1.000±0.143),老年組(0.451±0.065),ARC組(0.715±0.171),差異有統(tǒng)計(jì)學(xué)意義。SIRT1蛋白WB檢測(cè)及免疫熒光染色均為青年組最強(qiáng),ARC組居中,老年組最弱。P53通路檢測(cè)顯示,P53蛋白表達(dá)為青年組最低,老年組次之,ARC組最高,但具有凋亡相關(guān)活性的乙�；痯53為ARC組低于老年組。FOXOs通路檢測(cè)顯示,FOXO3a、FOXO4、p27kip1、p130蛋白表達(dá)均為ARC組最高,凋亡相關(guān)Bim蛋白表達(dá)為ARC組最低,差異有統(tǒng)計(jì)學(xué)意義。TUNEL檢測(cè)發(fā)現(xiàn),各組HLECs的凋亡百分比分別為：青年組(0.8±0.7)%,老年組(8.5±2.7)%,ARC組(25.0±9.8)%,差異有統(tǒng)計(jì)學(xué)意義。 結(jié)論SIRT1基因在人晶狀體上皮細(xì)胞中的表達(dá)隨年齡增長(zhǎng)而減少,但在ARC發(fā)生中呈保護(hù)性上調(diào)。下游通路蛋白檢測(cè)提示,上調(diào)的SIRT1可能通過p53通路與FOXOs通路誘導(dǎo)細(xì)胞周期停滯、抑制細(xì)胞凋亡,在ARC發(fā)生中發(fā)揮保護(hù)作用。 第二部分增強(qiáng)與抑制SIRT1蛋白功能對(duì)氧化環(huán)境下HLECs凋亡的影響 目的觀察SIRT1活性改變對(duì)氧化環(huán)境下培養(yǎng)的HLECs凋亡的影響,分析SIRT1對(duì)HLECs的保護(hù)作用。 方法體外培養(yǎng)HLECs系SRA01/04,分為4個(gè)大組：(1)正常對(duì)照組：常規(guī)培養(yǎng)；(2)H2O2組：加入H2O2終濃度為400μmol/L; (3) SIRT1激活劑白藜蘆醇(resveratrol, RES)組,分設(shè)4個(gè)RES濃度組：加入H2O2及RES溶液,H2O2終濃度為400μmol/L, RES終濃度分別為5,10,20,40μmol/L; (4) SIRT1抑制劑煙酰胺(Nicotinamide, NAM)組,分設(shè)4個(gè)NAM濃度組：加入H2O2及NAM溶液,H2O2終濃度為400μmol/L, NAM終濃度分別為25,50,100,200μmol/L。各組進(jìn)行以上干預(yù)并孵育24h后,行SIRT1蛋白免疫熒光染色,SIRT1及下游通路P53、乙�；疨53蛋白WB檢測(cè)、倒置顯微鏡形態(tài)學(xué)觀察、HLECs的增殖活性MTT比色法檢測(cè)、HLECs凋亡水平TUNEL染色檢測(cè)。 結(jié)果正常對(duì)照組僅有少量SIRT1蛋白免疫熒光染色,H2O2組則明顯增加,4個(gè)RES濃度組熒光進(jìn)一步增強(qiáng)且隨RES濃度上升而增加；4個(gè)NAM濃度組熒光與H2O2組比較未見明顯變化。SIRT1 Western Blot檢測(cè)結(jié)果與SIRT1免疫熒光染色結(jié)果一致。乙�；疨53蛋白水平隨著添加RES濃度的增加而逐漸下降,隨著添加NAM濃度的增加而逐漸升高。倒置顯微鏡觀察發(fā)現(xiàn),正常對(duì)照組HLECs呈較規(guī)則六角形或橢圓形,細(xì)胞密度較高；H2O2組細(xì)胞密度明顯下降,少數(shù)細(xì)胞呈長(zhǎng)梭形：加入RES后,長(zhǎng)梭形細(xì)胞明顯減少,細(xì)胞密度回升且隨RES濃度上升而逐漸增加；隨著添加NAM濃度的增加,細(xì)胞形態(tài)的不規(guī)則性明顯增加,并出現(xiàn)大量長(zhǎng)梭形細(xì)胞甚至互相聯(lián)結(jié)呈網(wǎng)狀。HLECs的MTT比色OD值隨RES濃度增加而上升,RES濃度為10,20和40μmol/L時(shí)與單純添加H2O2組比較有統(tǒng)計(jì)學(xué)差異(P0.05),但RES濃度由20μmol/L上升為40μmol/L時(shí)OD值無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)；添加NAM濃度為50,100,200μmol/L時(shí)與單純添加H2O2組比較MTT值明顯下降(P0.05)。TUNEL檢測(cè)發(fā)現(xiàn),RES濃度為10,20,40μmol/L時(shí)細(xì)胞凋亡百分比與H2O2組比較顯著降低(P0.05)。添加NAM濃度為25,50,100,200μmol/L時(shí)細(xì)胞凋亡百分與H2O2組比較則顯著增加(P0.05)。 結(jié)論在氧化損傷境下,HLECs中的SIRT1基因表達(dá)反應(yīng)性上調(diào)；RES可通過上調(diào)SIRT1活性,減少H2O2導(dǎo)致的HLECs的凋亡；NAM則通過抑制SIRT1的活性,進(jìn)一步加劇了HLECs的凋亡,提示SIRT1對(duì)HLECs具有抗凋亡的保護(hù)作用。 第三部分在HLECs中阻斷P53通路對(duì)SIRT1保護(hù)作用的影響 目的應(yīng)用pifithrin-a (p-fifty three inhibitor, PFT-α)阻斷P53通路,觀察SIRT1對(duì)HLECs的保護(hù)作用的影響,分析P53通路是否為SIRT1在HLECs中發(fā)揮保護(hù)作用的下游通路。 方法體外培養(yǎng)HLECs SRA01/04,分為4個(gè)大組：(1)正常對(duì)照組：常規(guī)培養(yǎng)；(2)H202組：加入H202,終濃度為400μmol/L; (3) NAM組：加入H202及NAM溶液,H202終濃度為400μmol/L, NAM終濃度為100μmol/L; (4) PFT-α組,分設(shè)3個(gè)PFT-α濃度組：加入H202、NAM及PFT-α溶液,H202終濃度為400μmol/L, NAM終濃度為100μmol/L, PFT-α終濃度分別為2.5,5,10μmol/L。各組進(jìn)行以上干預(yù)并孵育24h后,行SIRT1蛋白免疫熒光染色,SIRT1及下游通路P53、乙�；疨53蛋白WB檢測(cè)、倒置顯微鏡形態(tài)學(xué)觀察、HLECs的增殖活性MTT比色法檢測(cè)、HLECs凋亡水平TUNEL染色檢測(cè)。 結(jié)果正常對(duì)照組僅有少量SIRT1蛋白免疫熒光染色,H202組則明顯增加。NAM組及3個(gè)PFT-α濃度組的SIRT1熒光染色與H202組比較均未見明顯變化。SIRT1 WB檢測(cè)結(jié)果與SIRT1免疫熒光染色結(jié)果一致。與H202組比較,NAM組及3個(gè)PFT-α濃度組乙�；疨53均升高,但在這4組間無(wú)明顯變化。倒置顯微鏡觀察發(fā)現(xiàn),正常對(duì)照組HLECs呈較規(guī)則六角形或橢圓形,細(xì)胞密度較高；H202組細(xì)胞密度明顯下降,少數(shù)細(xì)胞呈長(zhǎng)梭形；加入NAM后,細(xì)胞形態(tài)的不規(guī)則性明顯增加,出現(xiàn)大量長(zhǎng)梭形細(xì)胞;加入PFT-α后,細(xì)胞密度回升且隨PFT-α濃度上升而逐漸增加,長(zhǎng)梭形細(xì)胞明顯減少,細(xì)胞形態(tài)漸趨正常。在H202培養(yǎng)條件下加入NAM后,HLECs的MTT比色OD值下降,但隨著添加PFT-α濃度增加而回升,添加3個(gè)PFT-α濃度時(shí)與NAM組比較均有統(tǒng)計(jì)學(xué)差異(P0.05)。TUNEL檢測(cè)發(fā)現(xiàn),在H202培養(yǎng)條件下添加NAM后HLECs凋亡百分比上升,隨著添加PFT-α濃度的上升,細(xì)胞凋亡百分比逐漸降低(P0.05)。 結(jié)論P(yáng)FT-α具有抑制HLECs細(xì)胞凋亡的作用,用PFT-α阻斷P53通路可消除NAM對(duì)SIRT1保護(hù)作用的抑制,提示P53通路是SIRT1發(fā)揮保護(hù)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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R776.1

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