糖基化终末产物对角膜上皮创伤愈合的影响及机制研究

发布时间：2018-05-05 13:01

本文选题：AGEs + THCE细胞　；参考：《山东大学》2013年博士论文

【摘要】：研究背景随着人民生活水平的提高、生活方式的改变和人口老龄化,我国糖尿病发病率呈明显的逐年增高趋势。糖尿病已经构成严重威胁人类健康的世界性问题。糖尿病患者在角膜外伤或接受角膜手术时极易出现角膜上皮愈合延迟甚至不愈,临床表现为：持续性角膜上皮损失、反复性角膜上皮糜烂、浅层角膜溃疡形成、继发严重角膜感染甚至失明。目前尚没有有效的治疗方法。因此,研究糖尿病角膜上皮创伤愈合延迟的发病机制和防治措施已成为眼科亟待解决的课题。研究发现,持久的高血糖状态可导致糖尿病患者体内多种蛋白质、脂质甚至核酸发生糖基化反应,形成具有结构多样、高度活性的糖基化终末产物(advanced glycation end products, AGEs)。正常情况下体内AGEs的水平随年龄的增长而缓慢增加,而在糖尿病患者体内,病理性高血糖可加速糖基化反应,形成大量的AGEs,并在组织中蓄积。AGEs具有不可逆性,这使其在高血糖被纠正后也不能回复到正常水平。AGEs具有广泛的生物学活性,参与了多种糖尿病并发症的发生、发展。随着对AGEs研究的深入,人们发现AGEs与糖尿病皮肤创面愈合延迟密切相关。近年研究发现,AGEs大量蓄积在糖尿病患者及糖尿病动物模型的角膜上皮及基底膜中,但其是否参与角膜上皮创伤愈合过程而导致创伤愈合延迟?目前尚不清楚。因此,深入研究AGEs在角膜上皮创伤愈合延迟中的作用,将进一步揭示糖尿病角膜上皮创伤愈合延迟的机制,为其防治提供新的理论依据。 AGEs与细胞表面特异性受体(receptor for advanced glycation end products, RAGE)结合,能够增加细胞内活性氧(Reactive oxygen species, ROS)的生成,诱导细胞氧化应激,造成组织氧化损伤。RAGE作为信号转导受体,介导AGEs结合在细胞表面,激活细胞内各种转导信号。研究证实RAGE在正常单核巨噬细胞、血管内皮细胞、肾系膜细胞、神经细胞及平滑肌细胞等细胞中呈低水平表达,但在糖尿病条件下其表达明显增加。AGEs与RAGE表达之间存在正反馈调节机制,AGEs蓄积的病变部位往往伴随着RAGE表达的增加。ROS是指化学性质活跃的氧代谢产物或由其衍生的产物,主要有超氧阴离子、过氧化氢、羟自由基等。ROS生成过多将在细胞内形成氧化应激状态,通过氧化反应破坏许多重要生物大分子的结构和功能,导致疾病的发生、发展。目前关于AGEs对角膜上皮创伤愈合的影响及机制研究在国内外尚未见报导。AGEs是否延迟角膜上皮创伤愈合过程?是否通过受体RAGE及ROS发挥作用?具体机制如何?回答这些问题将从根本上揭示AGEs在糖尿病角膜上皮创伤愈合延迟中的作用及机制,不仅对理解糖尿病角膜上皮创伤愈合延迟病理机制具有重要的意义,也必将为糖尿病角膜上皮创伤愈合延迟的防治提供新理论、新视角和新靶点。因此,本研究以体外制备的糖基化终末产物(AGE-BSA)作为干预因素,观察AGE-BSA对人永生化角膜上皮细胞(Human telomerase-immortalized corneal epithelial cells, THCE) RAGE、ROS表达的影响并研究其机制,探讨AGE-BSA对THCE细胞增殖、迁移以及角膜上皮创伤愈合的影响及机制。全文共分三部分：(一)AGE-BSA对THCE细胞RAGE及ROS表达的影响；(二)AGE-BSA诱导THCE细胞氧化应激机制的研究；(三)AGE-BSA对THCE细胞增殖、迁移及角膜上皮创伤愈合的影响。第一部分糖基化终末产物对角膜上皮细胞糖基化终末产物受体及活性氧表达的影响目的：研究AGEs对THCE细胞RAGE及ROS表达的影响。方法：1.用D-葡萄糖和牛血清白蛋白(Bovine serum albumin, BSA)共同孵育10周制备糖基化终末产物(AGE-BSA)。 2.分别用浓度为50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA处理THCE细胞24h和浓度为2000μg/ml的AGE-BSA分别处理THCE细胞6h、12h、24h、48h。Real-time PCR和Western blot检测ⅠRAGE mRNA和蛋白的表达； 3.分别用浓度为50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA处理THCE细胞12h。预先应用anti-RAGE中和抗体处理THCE细胞1h,再用浓度为200μg/ml的AGE-BSA孵育细胞12h,激光共聚焦显微镜和流式细胞仪检测ROS的表达。结果：1.体外制备的AGE-BSA的荧光强度为55.96荧光单位/mg蛋白,而BSA的荧光强度为1.98荧光单位/mg蛋白。 2.未经干预的THCE细胞表达少量的RAGE mRNA和蛋白,用BSA干预后RAGE mRNA和蛋白的表达与对照组无明显差异。与对照组相比,浓度为50μg/ml的AGE-BSA明显上调THCE细胞RAGE mRNA和蛋白的表达(P0.05),随着AGE-BSA浓度的增加,RAGE mRNA和蛋白的表达逐渐增高,AGE-BSA浓度为20μg/ml时达到峰值(P0.05)。 3.在浓度为200μg/ml的AGE-BSA作用下,AGE-BSA作用THCE细胞6h明显上调RAGE mRNA的表达(P0.05),AGE-BSA作用THCE细胞12h明显上调RAGE蛋白的表达(P0.05),随着AGE-BSA作用时间的延长,RAGE mRNA和蛋白的表达逐渐增高,AGE-BSA作用时间为24h时达到峰值(P0.05)。 4.未经干预的THCE细胞表达极少量的ROS,用BSA干预后ROS的表达与对照组无明显差异。与对照组相比,浓度为100μg/ml的AGE-BSA明显上调THCE细胞ROS的表达(P0.05),随着AGE-BSA浓度的增加,ROS的表达逐渐增高,AGE-BSA浓度为200μg/ml时达到峰值(P0.05)。 5. anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著抑制AGE-BAS对ROS表达的上调作用(P0.05)。结论：1.AGE-BSA显著增加THCE细胞RAGE mRNA、蛋白及ROS的表达。 2. AGE-BSA通过与受体RAGE结合,诱导THCE细胞大量生成ROS,导致角膜上皮细胞氧化损伤,可能参与糖尿病角膜上皮创伤愈合延迟过程。第二部分糖基化终末产物诱导角膜上皮细胞氧化应激机制的研究目的：探讨AGEs诱导THCE细胞氧化应激的机制。方法：1.预先应用NADPH氧化酶抑制齐apocynin及Diphenyleneiodonium (DPI)、线粒体酶复合体Ⅰ抑制齐rotenone、线粒体酶复合体Ⅱ抑制剂thenoyltrifluoroacetone (TTFA)、线粒体酶复合体Ⅲ抑制齐antimycin A、黄嘌呤氧化酶抑制齐allopurinol处理THCE细胞1h后,再用浓度为200μg/m1的AGE-BSA孵育细胞12h,应用流式细胞仪检测ROS的表达。 2.预先应用anti-RAGE中和抗体处理THCE细胞1h,再用浓度为200μg/m1的AGE-BSA孵育细胞12h,应用Real-time PCR检测NADPH氧化酶亚基p22phox、 NOX4mRNA的表达；Western blot检测p22phox、NOX4蛋白的表达。 3.预先应用anti-RAGE中和抗体处理THCE细胞1h,再用浓度为200μg/ml的AGE-BSA呼育细胞12h,检测抗氧化酶超氧化物歧化酶(Superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)的活性及丙二醛(malondialdehyde, MD A)的含量。结果：1.与对照组相比,AGE-BSA显著上调THCE细胞ROS的表达(P0.05),NADPH氧化酶抑制剂apocynin和DPI显著抑制AGE-BAS对ROS表达的上调作用(P0.05),而线粒体酶复合体抑制剂、黄嘌呤氧化酶抑制剂对ROS的表达无明显影响。 2.与对照组相比,AGE-BSA明显上调THCE细胞P22phox、NOX4mRNA和蛋白的表达(P0.05),应用anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著抑制AGE-BAS对p22phox、NOX4mRNA和蛋白表达的上调作用(P0.05)。 3.与对照组相比,AGE-BSA显著降低THCE细胞SOD和CAT的活性(P0.05)；应用anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著减轻AGE-BAS对SOD和CAT活性的抑制作用(P0.05) 4.与对照组相比,AGE-BSA显著增加THCE细胞MDA的含量(P0.05)；应用anti-RAGE中和抗体阻断AGE-BSA与RAGE结合后,显著抑制AGE-BAS对MDA含量的上调作用(P0.05) 结论：1. AGE-BSA通过与受体RAGE结合,诱导THCE细胞NADPH氧化酶亚基p22phox、NOX4mRNA和蛋白高表达,促使NADPH氧化酶激活,生成大量ROS。 2. AGE-BSA通过与受体RAGE结合,降低THCE细胞抗氧化酶SOD口CAT的活性、增加MDA的含量,导致氧化／抗氧化系统失衡,促使角膜上皮细胞处于氧化应激状态。第三部分糖基化终末产物对角膜上皮细胞增殖、迁移及角膜上皮创伤愈合的影响目的：探讨AGEs对THCE细胞增殖、迁移及角膜上皮创伤愈合的影响。方法：1.分别用浓度为50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA与THCE细胞共培养24h,CCK-8法检测THCE细胞增殖能力；刮痕愈合实验检测THCE细胞迁移能力。 2.预先应用anti-RAGE中和抗体、抗氧化剂乙酰半胱胺酸(N-acetyl-L-cysteine, NAC)处理THCE细胞1h,再用浓度为200μg/ml的AGE-BSA孵育细胞24h, CCK-8法检测THCE细胞增殖能力；刮痕愈合实验检测THCE细胞迁移能力。 3.体外培养猪角膜上皮创伤器官模型,在角膜中央制作直径为5mm的上皮损伤区域。预先应用anti-RAGE中和抗体、抗氧化剂NAC处理1h,再用浓度为200μg/ml的AGE-BSA5孵育48h,观察角膜上皮创伤愈合程度。结果：1.与对照组相比,浓度为50μg/ml的AGE-BSA显著抑制THCE细胞的增殖、迁移(P0.05),随着AGE-BSA浓度的增加,细胞增殖、迁移能力逐渐降低,AGE-BSA浓度为200μg/ml寸达到最低。anti-RAGE中和抗体、NAC显著减轻AGE-BAS对细胞增殖、迁移的抑制作用。 2.与对照组相比,浓度为200μg/ml的AGE-BSA显著延迟体外培养猪角膜上皮创伤器官模型中角膜上皮创伤愈合过程(P0.05),anti-RAGE中和抗体、NAC可显著促进角膜上皮创伤的愈合(P0.05) 结论：1. AGE-BSA显著抑制THCE细胞增殖、迁移,并且呈浓度依赖性。 2. AGE-BSA通过与受体RAGE结合,诱导ROS大量生成,引发氧化应激反应,进而抑制THCE细胞增殖、迁移,延迟角膜上皮创伤愈合过程。阻断AGE-BSA与RAGE结合,或清除过多的ROS,可成为防治糖尿病角膜上皮创伤愈合延迟的新途径。
[Abstract]:Research background
With the improvement of the living standard of the people, the change of life style and the aging of the population, the incidence of diabetes in China is obviously increasing year by year. Diabetes has become a serious threat to human health. The performance of the bed is: persistent corneal epithelial loss, recurrent corneal epithelial erosion, superficial corneal ulcer formation, secondary severe corneal infection or even blindness. There is no effective treatment at present. Therefore, the study of the pathogenesis and prevention measures of the delayed healing of diabetic corneal epithelium has become an urgent problem in the ophthalmology.
It has been found that persistent hyperglycemia can lead to glycosylation of various proteins, lipids and even nucleic acids in the body of diabetes, forming a glycosylation end product (advanced glycation end products, AGEs) with structural diversity and high activity. In normal cases, the level of AGEs in the body increases slowly with age. In diabetic patients, pathological hyperglycemia can accelerate glycosylation, form a large number of AGEs and accumulate.AGEs irreversibility in tissue, which can not revert to normal level after the hyperglycemia is corrected to have extensive biological activity and participate in the occurrence and development of a variety of diabetic complications. With the study of AGEs In recent years, people have found that AGEs is closely related to the delayed healing of diabetic skin wounds. In recent years, studies have found that AGEs is accumulated in the corneal epithelium and basement membrane of diabetic and diabetic animal models. But is it involved in the healing process of corneal epithelial wound healing and causing trauma delay? It is still unclear. Therefore, the in-depth study of AG The effect of Es on the delayed healing of corneal epithelial wound will further reveal the mechanism of delayed healing of diabetic corneal epithelial wound healing, and provide a new theoretical basis for its prevention and treatment.
AGEs combined with cell surface specific receptor (receptor for advanced glycation end products, RAGE), which can increase the formation of intracellular reactive oxygen species (Reactive oxygen species, ROS), induce oxidative stress, and cause tissue oxidative damage as a signal transduction receptor. The study confirmed that RAGE was expressed at low level in normal mononuclear macrophages, vascular endothelial cells, renal mesangial cells, nerve cells and smooth muscle cells, but in diabetes, the expression of.AGEs and RAGE expressed a positive feedback regulation mechanism, and the site of the accumulation of AGEs was often accompanied by RAGE expression. The increase of.ROS is a chemically active oxygen metabolite or derived product, which mainly consists of superoxide anion, hydrogen peroxide, hydroxyl radical and so on, which will form oxidative stress in the cells and destroy the structure and function of many important biological macromolecules by oxidation reaction, resulting in the occurrence and development of the disease.
The present study on the effect and mechanism of AGEs on corneal epithelial wound healing has not been reported at home and abroad. Is.AGEs delayed the healing process of corneal epithelial wound healing? Is it possible to play a role in the receptor RAGE and ROS? How is the mechanism? Answers to these questions will fundamentally reveal the role of AGEs in the delayed healing of corneal epithelial wound healing in diabetes And the mechanism is of great significance not only to understand the delayed pathological mechanism of corneal epithelial wound healing in diabetic patients, but also to provide new theories, new perspectives and new targets for the prevention and treatment of delayed healing of corneal epithelial wound in diabetes. Therefore, this study uses AGE-BSA as an intervention factor to observe AGE-BSA to people. The influence and mechanism of Human telomerase-immortalized corneal epithelial cells (THCE) RAGE, ROS expression and its mechanism were studied. The effect and mechanism of AGE-BSA on proliferation, migration and corneal epithelial wound healing of THCE cells were investigated. (1) the effect of AGE-BSA on THCE cells and expression; Two) the mechanism of AGE-BSA induced oxidative stress in THCE cells; (three) the effect of AGE-BSA on THCE cell proliferation, migration and corneal epithelial wound healing.
Part 1 Effects of advanced glycation end products on the expression of receptor for advanced glycation end products and reactive oxygen species in corneal epithelial cells
Objective: To study the effect of AGEs on the expression of RAGE and ROS in THCE cells.
Methods: 1. glycosylation end products (AGE-BSA) were prepared by incubation with D- glucose and Bovine serum albumin (BSA) for 10 weeks.
2. THCE cell 24h was treated with a concentration of 50 mu g/ml, 100 mu g/ml, 200 mu g/ml, and 400 micron g/ml respectively, and THCE cell 6h was treated respectively with the concentration of 2000 micron g/ml.
3. THCE cell 12h. with a concentration of 50 mu g/ml, 100 mu g/ml, 200 mu g/ml and 400 micron g/ml was used to treat THCE cell 1h in advance with anti-RAGE neutralization antibody, and then the AGE-BSA incubated cells with a concentration of 200 mu g/ml were used to detect the expression of 12h..
Results: 1. the fluorescence intensity of AGE-BSA prepared in vitro was 55.96 fluorescent unit /mg protein, while the fluorescence intensity of BSA was 1.98 fluorescence unit /mg protein.
2. untreated THCE cells expressed a small amount of RAGE mRNA and protein. The expression of RAGE mRNA and protein was not significantly different from that of the control group after the intervention of BSA. Compared with the control group, the AGE-BSA with a concentration of 50 mu g/ml significantly increased the THCE cell RAGE mRNA and the expression of protein (P0.05). With the increase of concentration, the expression of the protein and protein increased gradually. When the concentration of AGE-BSA is 20 g/ml, the peak value (P0.05) is high.
3. under the action of AGE-BSA of concentration of 200 g/ml, 6h obviously up regulation of RAGE mRNA expression (P0.05). AGE-BSA action THCE cell 12h obviously up-regulated the expression of RAGE protein.
4. untreated THCE cells expressed a very small amount of ROS. The expression of ROS was not significantly different from that of the control group after the intervention of BSA. Compared with the control group, the AGE-BSA significantly up-regulated the expression of THCE cell ROS (P0.05), and the ROS expression increased with the increase of AGE-BSA concentration, and the AGE-BSA concentration reached a peak of 200 mu.
5. anti-RAGE neutralization antibody blocked the binding of AGE-BSA to RAGE, which significantly inhibited the up regulation effect of AGE-BAS on ROS expression (P0.05).
Conclusion: 1.AGE-BSA significantly increased the expression of RAGE mRNA, protein and ROS in THCE cells.
2. AGE-BSA, by binding to the receptor RAGE, induces a large number of ROS in THCE cells, resulting in oxidative damage of corneal epithelial cells, and may be involved in the delayed healing process of diabetic corneal epithelium.
The second part is the mechanism of oxidative stress induced by advanced glycation end products in corneal epithelial cells.
Objective: To explore the mechanism of oxidative stress induced by AGEs in THCE cells.
Methods: 1. NADPH oxidase was used to inhibit homogeneous apocynin and Diphenyleneiodonium (DPI), mitochondrial enzyme complex I inhibited Qi rotenone, mitochondrial enzyme complex II inhibitor Thenoyltrifluoroacetone (TTFA), mitochondrial enzyme complex III inhibited Qi antimycin A, and xanthine oxidase inhibited Qi allopurinol to treat THCE cell 1H. Cell 12h was incubated with AGE-BSA with a concentration of 200 g/m1, and the expression of ROS was detected by flow cytometry.
2. THCE cell 1H was treated with anti-RAGE neutralization antibody in advance, and 12h was incubated in AGE-BSA with a concentration of 200 g/m1, and Real-time PCR was used to detect the NADPH oxidase subunit p22phox, NOX4mRNA expression.
3. THCE cell 1H was treated with anti-RAGE neutralization antibody in advance, and 12h of AGE-BSA rearing cells with a concentration of 200 mu g/ml was used to detect the activity of antioxidant enzyme superoxide dismutase (Superoxide dismutase, SOD), catalase (catalase, CAT) and the content of malondialdehyde (malondialdehyde).
Results: 1. compared with the control group, AGE-BSA significantly increased the expression of ROS in THCE cells (P0.05), NADPH oxidase inhibitor apocynin and DPI significantly inhibited the up regulation of AGE-BAS on ROS expression (P0.05), but the inhibitor of mitochondrial enzyme complex and xanthine oxidase inhibitor had no significant effect on the expression of ROS.
2. compared with the control group, AGE-BSA obviously up-regulated the expression of P22phox, NOX4mRNA and protein in THCE cells (P0.05). After the combination of anti-RAGE neutralization antibody blocking AGE-BSA and RAGE, the up regulation of AGE-BAS on p22phox, NOX4mRNA and protein expression was significantly inhibited (P0.05).
3. compared with the control group, AGE-BSA significantly reduced the activity of SOD and CAT in THCE cells (P0.05), and the inhibitory effect of AGE-BAS on SOD and CAT activity (P0.05) was significantly reduced after the combination of anti-RAGE neutralizing antibodies blocking AGE-BSA and RAGE.
4. compared with the control group, AGE-BSA significantly increased the content of MDA in THCE cells (P0.05). After the combination of anti-RAGE neutralization antibody blocking AGE-BSA and RAGE, the up regulation of AGE-BAS to MDA content was significantly inhibited (P0.05).
Conclusion: 1. AGE-BSA induces a high expression of NADPH oxidase subunit p22phox, NOX4mRNA and protein by binding to receptor RAGE, which promotes the activation of NADPH oxidase and produces a large number of ROS..
2. AGE-BSA can reduce the activity of CAT in the SOD mouth of THCE cells by binding to the receptor RAGE, and increase the content of MDA, resulting in the imbalance of oxidation / antioxidant system and the oxidative stress of corneal epithelial cells.
The third part is the effect of advanced glycation end products on corneal epithelial cell proliferation, migration and corneal epithelial wound healing.
Objective: To investigate the effects of AGEs on proliferation, migration and corneal epithelial wound healing of THCE cells.
Methods: 1. the 24h was co cultured with AGE-BSA and THCE cells with a concentration of 50 mu g/ml, 100 mu g/ml, 200 mu g/ml, 400 micron g/ml, and CCK-8 assay was used to detect the proliferation ability of THCE cells, and the scratch healing test was used to detect the migration ability of THCE cells.
2. the anti-RAGE neutralization antibody and N-acetyl-L-cysteine (NAC) were used to treat THCE cell 1H, and then the cell 24h was incubated with the AGE-BSA concentration of 200 mu g/ml. The proliferation ability of THCE cells was detected by CCK-8 method, and the migration ability of THCE cells was detected by the scratch healing test.
3. in vitro culture of porcine corneal epithelial wound organ model, the epithelial lesion area of 5mm in the center of the cornea was produced. Anti-RAGE neutralized antibody, antioxidant NAC was used to treat 1H, and then 48h was incubated with AGE-BSA5 with a concentration of 200 mu g/ml. The healing degree of corneal epithelial wound was observed.
Results: 1. compared with the control group, AGE-BSA with a concentration of 50 g/ml significantly inhibited the proliferation of THCE cells, migration (P0.05), with the increase of AGE-BSA concentration, cell proliferation and migration ability gradually decreased, AGE-BSA concentration was 200 mu g/ml to the lowest.Anti-RAGE neutralizing antibody, NAC significantly alleviated the inhibitory effect of AGE-BAS on cell proliferation and migration.
2. compared with the control group, AGE-BSA with a concentration of 200 g/ml significantly delayed the healing process of corneal epithelial wound healing (P0.05), anti-RAGE neutralizing antibody in the cultured porcine corneal epithelial wound organ model in vitro, and NAC could significantly promote the healing of corneal epithelial wound (P0.05).
Conclusion: 1. AGE-BSA significantly inhibited the proliferation and migration of THCE cells in a concentration dependent manner.
2. AGE-BSA can induce the formation of ROS by binding to the receptor RAGE, induce the oxidative stress reaction, and then inhibit the proliferation and migration of THCE cells and delay the healing process of corneal epithelial wound. Blocking the combination of AGE-BSA with RAGE, or removing too much ROS can be a new way to prevent the delayed healing of corneal epithelial wound in diabetes.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R772.2

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