發(fā)布時(shí)間：2018-05-05 13:01

  本文選題：AGEs + THCE細(xì)胞　； 參考：《山東大學(xué)》2013年博士論文

【摘要】：研究背景 隨著人民生活水平的提高、生活方式的改變和人口老齡化,我國(guó)糖尿病發(fā)病率呈明顯的逐年增高趨勢(shì)。糖尿病已經(jīng)構(gòu)成嚴(yán)重威脅人類健康的世界性問題。糖尿病患者在角膜外傷或接受角膜手術(shù)時(shí)極易出現(xiàn)角膜上皮愈合延遲甚至不愈,臨床表現(xiàn)為：持續(xù)性角膜上皮損失、反復(fù)性角膜上皮糜爛、淺層角膜潰瘍形成、繼發(fā)嚴(yán)重角膜感染甚至失明。目前尚沒有有效的治療方法。因此,研究糖尿病角膜上皮創(chuàng)傷愈合延遲的發(fā)病機(jī)制和防治措施已成為眼科亟待解決的課題。 研究發(fā)現(xiàn),持久的高血糖狀態(tài)可導(dǎo)致糖尿病患者體內(nèi)多種蛋白質(zhì)、脂質(zhì)甚至核酸發(fā)生糖基化反應(yīng),形成具有結(jié)構(gòu)多樣、高度活性的糖基化終末產(chǎn)物(advanced glycation end products, AGEs)。正常情況下體內(nèi)AGEs的水平隨年齡的增長(zhǎng)而緩慢增加,而在糖尿病患者體內(nèi),病理性高血糖可加速糖基化反應(yīng),形成大量的AGEs,并在組織中蓄積。AGEs具有不可逆性,這使其在高血糖被糾正后也不能回復(fù)到正常水平。AGEs具有廣泛的生物學(xué)活性,參與了多種糖尿病并發(fā)癥的發(fā)生、發(fā)展。隨著對(duì)AGEs研究的深入,人們發(fā)現(xiàn)AGEs與糖尿病皮膚創(chuàng)面愈合延遲密切相關(guān)。近年研究發(fā)現(xiàn),AGEs大量蓄積在糖尿病患者及糖尿病動(dòng)物模型的角膜上皮及基底膜中,但其是否參與角膜上皮創(chuàng)傷愈合過程而導(dǎo)致創(chuàng)傷愈合延遲?目前尚不清楚。因此,深入研究AGEs在角膜上皮創(chuàng)傷愈合延遲中的作用,將進(jìn)一步揭示糖尿病角膜上皮創(chuàng)傷愈合延遲的機(jī)制,為其防治提供新的理論依據(jù)。 AGEs與細(xì)胞表面特異性受體(receptor for advanced glycation end products, RAGE)結(jié)合,能夠增加細(xì)胞內(nèi)活性氧(Reactive oxygen species, ROS)的生成,誘導(dǎo)細(xì)胞氧化應(yīng)激,造成組織氧化損傷。RAGE作為信號(hào)轉(zhuǎn)導(dǎo)受體,介導(dǎo)AGEs結(jié)合在細(xì)胞表面,激活細(xì)胞內(nèi)各種轉(zhuǎn)導(dǎo)信號(hào)。研究證實(shí)RAGE在正常單核巨噬細(xì)胞、血管內(nèi)皮細(xì)胞、腎系膜細(xì)胞、神經(jīng)細(xì)胞及平滑肌細(xì)胞等細(xì)胞中呈低水平表達(dá),但在糖尿病條件下其表達(dá)明顯增加。AGEs與RAGE表達(dá)之間存在正反饋調(diào)節(jié)機(jī)制,AGEs蓄積的病變部位往往伴隨著RAGE表達(dá)的增加。ROS是指化學(xué)性質(zhì)活躍的氧代謝產(chǎn)物或由其衍生的產(chǎn)物,主要有超氧陰離子、過氧化氫、羥自由基等。ROS生成過多將在細(xì)胞內(nèi)形成氧化應(yīng)激狀態(tài),通過氧化反應(yīng)破壞許多重要生物大分子的結(jié)構(gòu)和功能,導(dǎo)致疾病的發(fā)生、發(fā)展。 目前關(guān)于AGEs對(duì)角膜上皮創(chuàng)傷愈合的影響及機(jī)制研究在國(guó)內(nèi)外尚未見報(bào)導(dǎo)。AGEs是否延遲角膜上皮創(chuàng)傷愈合過程?是否通過受體RAGE及ROS發(fā)揮作用?具體機(jī)制如何?回答這些問題將從根本上揭示AGEs在糖尿病角膜上皮創(chuàng)傷愈合延遲中的作用及機(jī)制,不僅對(duì)理解糖尿病角膜上皮創(chuàng)傷愈合延遲病理機(jī)制具有重要的意義,也必將為糖尿病角膜上皮創(chuàng)傷愈合延遲的防治提供新理論、新視角和新靶點(diǎn)。因此,本研究以體外制備的糖基化終末產(chǎn)物(AGE-BSA)作為干預(yù)因素,觀察AGE-BSA對(duì)人永生化角膜上皮細(xì)胞(Human telomerase-immortalized corneal epithelial cells, THCE) RAGE、ROS表達(dá)的影響并研究其機(jī)制,探討AGE-BSA對(duì)THCE細(xì)胞增殖、遷移以及角膜上皮創(chuàng)傷愈合的影響及機(jī)制。全文共分三部分：(一)AGE-BSA對(duì)THCE細(xì)胞RAGE及ROS表達(dá)的影響；(二)AGE-BSA誘導(dǎo)THCE細(xì)胞氧化應(yīng)激機(jī)制的研究；(三)AGE-BSA對(duì)THCE細(xì)胞增殖、遷移及角膜上皮創(chuàng)傷愈合的影響。 第一部分糖基化終末產(chǎn)物對(duì)角膜上皮細(xì)胞糖基化終末產(chǎn)物受體及活性氧表達(dá)的影響 目的：研究AGEs對(duì)THCE細(xì)胞RAGE及ROS表達(dá)的影響。 方法：1.用D-葡萄糖和牛血清白蛋白(Bovine serum albumin, BSA)共同孵育10周制備糖基化終末產(chǎn)物(AGE-BSA)。 2.分別用濃度為50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA處理THCE細(xì)胞24h和濃度為2000μg/ml的AGE-BSA分別處理THCE細(xì)胞6h、12h、24h、48h。Real-time PCR和Western blot檢測(cè)ⅠRAGE mRNA和蛋白的表達(dá)； 3.分別用濃度為50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA處理THCE細(xì)胞12h。預(yù)先應(yīng)用anti-RAGE中和抗體處理THCE細(xì)胞1h,再用濃度為200μg/ml的AGE-BSA孵育細(xì)胞12h,激光共聚焦顯微鏡和流式細(xì)胞儀檢測(cè)ROS的表達(dá)。 結(jié)果：1.體外制備的AGE-BSA的熒光強(qiáng)度為55.96熒光單位/mg蛋白,而BSA的熒光強(qiáng)度為1.98熒光單位/mg蛋白。 2.未經(jīng)干預(yù)的THCE細(xì)胞表達(dá)少量的RAGE mRNA和蛋白,用BSA干預(yù)后RAGE mRNA和蛋白的表達(dá)與對(duì)照組無明顯差異。與對(duì)照組相比,濃度為50μg/ml的AGE-BSA明顯上調(diào)THCE細(xì)胞RAGE mRNA和蛋白的表達(dá)(P0.05),隨著AGE-BSA濃度的增加,RAGE mRNA和蛋白的表達(dá)逐漸增高,AGE-BSA濃度為20μg/ml時(shí)達(dá)到峰值(P0.05)。 3.在濃度為200μg/ml的AGE-BSA作用下,AGE-BSA作用THCE細(xì)胞6h明顯上調(diào)RAGE mRNA的表達(dá)(P0.05),AGE-BSA作用THCE細(xì)胞12h明顯上調(diào)RAGE蛋白的表達(dá)(P0.05),隨著AGE-BSA作用時(shí)間的延長(zhǎng),RAGE mRNA和蛋白的表達(dá)逐漸增高,AGE-BSA作用時(shí)間為24h時(shí)達(dá)到峰值(P0.05)。 4.未經(jīng)干預(yù)的THCE細(xì)胞表達(dá)極少量的ROS,用BSA干預(yù)后ROS的表達(dá)與對(duì)照組無明顯差異。與對(duì)照組相比,濃度為100μg/ml的AGE-BSA明顯上調(diào)THCE細(xì)胞ROS的表達(dá)(P0.05),隨著AGE-BSA濃度的增加,ROS的表達(dá)逐漸增高,AGE-BSA濃度為200μg/ml時(shí)達(dá)到峰值(P0.05)。 5. anti-RAGE中和抗體阻斷AGE-BSA與RAGE結(jié)合后,顯著抑制AGE-BAS對(duì)ROS表達(dá)的上調(diào)作用(P0.05)。 結(jié)論：1.AGE-BSA顯著增加THCE細(xì)胞RAGE mRNA、蛋白及ROS的表達(dá)。 2. AGE-BSA通過與受體RAGE結(jié)合,誘導(dǎo)THCE細(xì)胞大量生成ROS,導(dǎo)致角膜上皮細(xì)胞氧化損傷,可能參與糖尿病角膜上皮創(chuàng)傷愈合延遲過程。 第二部分糖基化終末產(chǎn)物誘導(dǎo)角膜上皮細(xì)胞氧化應(yīng)激機(jī)制的研究 目的：探討AGEs誘導(dǎo)THCE細(xì)胞氧化應(yīng)激的機(jī)制。 方法：1.預(yù)先應(yīng)用NADPH氧化酶抑制齊apocynin及Diphenyleneiodonium (DPI)、線粒體酶復(fù)合體Ⅰ抑制齊rotenone、線粒體酶復(fù)合體Ⅱ抑制劑thenoyltrifluoroacetone (TTFA)、線粒體酶復(fù)合體Ⅲ抑制齊antimycin A、黃嘌呤氧化酶抑制齊allopurinol處理THCE細(xì)胞1h后,再用濃度為200μg/m1的AGE-BSA孵育細(xì)胞12h,應(yīng)用流式細(xì)胞儀檢測(cè)ROS的表達(dá)。 2.預(yù)先應(yīng)用anti-RAGE中和抗體處理THCE細(xì)胞1h,再用濃度為200μg/m1的AGE-BSA孵育細(xì)胞12h,應(yīng)用Real-time PCR檢測(cè)NADPH氧化酶亞基p22phox、 NOX4mRNA的表達(dá)；Western blot檢測(cè)p22phox、NOX4蛋白的表達(dá)。 3.預(yù)先應(yīng)用anti-RAGE中和抗體處理THCE細(xì)胞1h,再用濃度為200μg/ml的AGE-BSA呼育細(xì)胞12h,檢測(cè)抗氧化酶超氧化物歧化酶(Superoxide dismutase, SOD)、過氧化氫酶(catalase, CAT)的活性及丙二醛(malondialdehyde, MD A)的含量。 結(jié)果：1.與對(duì)照組相比,AGE-BSA顯著上調(diào)THCE細(xì)胞ROS的表達(dá)(P0.05),NADPH氧化酶抑制劑apocynin和DPI顯著抑制AGE-BAS對(duì)ROS表達(dá)的上調(diào)作用(P0.05),而線粒體酶復(fù)合體抑制劑、黃嘌呤氧化酶抑制劑對(duì)ROS的表達(dá)無明顯影響。 2.與對(duì)照組相比,AGE-BSA明顯上調(diào)THCE細(xì)胞P22phox、NOX4mRNA和蛋白的表達(dá)(P0.05),應(yīng)用anti-RAGE中和抗體阻斷AGE-BSA與RAGE結(jié)合后,顯著抑制AGE-BAS對(duì)p22phox、NOX4mRNA和蛋白表達(dá)的上調(diào)作用(P0.05)。 3.與對(duì)照組相比,AGE-BSA顯著降低THCE細(xì)胞SOD和CAT的活性(P0.05)；應(yīng)用anti-RAGE中和抗體阻斷AGE-BSA與RAGE結(jié)合后,顯著減輕AGE-BAS對(duì)SOD和CAT活性的抑制作用(P0.05) 4.與對(duì)照組相比,AGE-BSA顯著增加THCE細(xì)胞MDA的含量(P0.05)；應(yīng)用anti-RAGE中和抗體阻斷AGE-BSA與RAGE結(jié)合后,顯著抑制AGE-BAS對(duì)MDA含量的上調(diào)作用(P0.05) 結(jié)論：1. AGE-BSA通過與受體RAGE結(jié)合,誘導(dǎo)THCE細(xì)胞NADPH氧化酶亞基p22phox、NOX4mRNA和蛋白高表達(dá),促使NADPH氧化酶激活,生成大量ROS。 2. AGE-BSA通過與受體RAGE結(jié)合,降低THCE細(xì)胞抗氧化酶SOD口CAT的活性、增加MDA的含量,導(dǎo)致氧化／抗氧化系統(tǒng)失衡,促使角膜上皮細(xì)胞處于氧化應(yīng)激狀態(tài)。 第三部分糖基化終末產(chǎn)物對(duì)角膜上皮細(xì)胞增殖、遷移及角膜上皮創(chuàng)傷愈合的影響 目的：探討AGEs對(duì)THCE細(xì)胞增殖、遷移及角膜上皮創(chuàng)傷愈合的影響。 方法：1.分別用濃度為50μg/ml、100μg/ml、200μg/ml、400μg/ml的AGE-BSA與THCE細(xì)胞共培養(yǎng)24h,CCK-8法檢測(cè)THCE細(xì)胞增殖能力；刮痕愈合實(shí)驗(yàn)檢測(cè)THCE細(xì)胞遷移能力。 2.預(yù)先應(yīng)用anti-RAGE中和抗體、抗氧化劑乙酰半胱胺酸(N-acetyl-L-cysteine, NAC)處理THCE細(xì)胞1h,再用濃度為200μg/ml的AGE-BSA孵育細(xì)胞24h, CCK-8法檢測(cè)THCE細(xì)胞增殖能力；刮痕愈合實(shí)驗(yàn)檢測(cè)THCE細(xì)胞遷移能力。 3.體外培養(yǎng)豬角膜上皮創(chuàng)傷器官模型,在角膜中央制作直徑為5mm的上皮損傷區(qū)域。預(yù)先應(yīng)用anti-RAGE中和抗體、抗氧化劑NAC處理1h,再用濃度為200μg/ml的AGE-BSA5孵育48h,觀察角膜上皮創(chuàng)傷愈合程度。 結(jié)果：1.與對(duì)照組相比,濃度為50μg/ml的AGE-BSA顯著抑制THCE細(xì)胞的增殖、遷移(P0.05),隨著AGE-BSA濃度的增加,細(xì)胞增殖、遷移能力逐漸降低,AGE-BSA濃度為200μg/ml寸達(dá)到最低。anti-RAGE中和抗體、NAC顯著減輕AGE-BAS對(duì)細(xì)胞增殖、遷移的抑制作用。 2.與對(duì)照組相比,濃度為200μg/ml的AGE-BSA顯著延遲體外培養(yǎng)豬角膜上皮創(chuàng)傷器官模型中角膜上皮創(chuàng)傷愈合過程(P0.05),anti-RAGE中和抗體、NAC可顯著促進(jìn)角膜上皮創(chuàng)傷的愈合(P0.05) 結(jié)論：1. AGE-BSA顯著抑制THCE細(xì)胞增殖、遷移,并且呈濃度依賴性。 2. AGE-BSA通過與受體RAGE結(jié)合,誘導(dǎo)ROS大量生成,引發(fā)氧化應(yīng)激反應(yīng),進(jìn)而抑制THCE細(xì)胞增殖、遷移,延遲角膜上皮創(chuàng)傷愈合過程。阻斷AGE-BSA與RAGE結(jié)合,或清除過多的ROS,可成為防治糖尿病角膜上皮創(chuàng)傷愈合延遲的新途徑。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R772.2

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