本文選題：褪黑素　切入點：糖尿病視網膜病變　出處：《復旦大學》2014年博士論文

【摘要】：隨著生活節(jié)奏和方式的轉變,我國糖尿病的發(fā)病率逐年上升,現已高達9.7%[1],說明該疾病已成為我國主要的公共衛(wèi)生問題之一。糖尿病可導致如白內障、青光眼、視神經病變等各種眼部并發(fā)癥,而其中尤以糖尿病性視網膜病變(Diabetic Retinopathy, DR)最為嚴重,已成為獲得性致盲的主要原因之一。研究表明,DR的發(fā)生發(fā)展涉及多條細胞信號轉導通路,包括多元醇途徑,蛋白激酶C激活,糖基化終末產物的積聚,氨基己糖途徑等[2-3]。但深入研究闡明,氧化損傷是各條通路導致DR發(fā)生的根本機制[4],同時慢性炎癥也是參與DR病程發(fā)展的重要因素[5]。因此,抗氧化和抗炎治療將可能有效遏制疾病的進展,為患者提供一條新的治療途徑。褪黑素是一種主要由松果體分泌的激素,視網膜也有少量分泌[6]。據報道,它是目前發(fā)現的體內抗氧化應激能力最強的分子,可通過多種途徑抗氧化[7-9]。同時也可以通過調節(jié)各種促炎癥因子,發(fā)揮強大的抗炎效應[10]。因此褪黑素對于糖尿病視網膜病變具有潛在的治療作用。本研究運用生物化學,分子生物學等方法,通過原代培養(yǎng)視網膜Muller細胞,建立糖尿病大鼠模型,從細胞水平和整體水平探討褪黑素對糖尿病視網膜病變的保護作用,并進一步深入研究其作用機制,將為以后進行糖尿病視網膜保護新藥的研發(fā)及藥物的臨床試驗提供理論依據,為糖尿病視網膜病變的治療提供新的途徑。第一部分 褪黑素對高糖所致視網膜Muller細胞損傷的保護作用及其機制研究目的：探討褪黑素是否對高糖所致視網膜Muller細胞損傷具有保護效應,并進一步研究其作用通路。方法：酶消化法分離培養(yǎng)大鼠視網膜Muller細胞,免疫熒光法鑒定細胞性質和純化度。細胞免疫熒光法檢測Muller細胞褪黑素膜受體的分布。細胞分為低糖對照組(5.5mmol/l)、甘露醇對照組(5.5mmol/l+24.5mmol/l甘露醇)、高糖組(30mmol/l)、褪黑素+高糖組,褪黑素+高糖+P13k抑制劑(LY294002)組,褪黑素+高糖+褪黑素膜受體拮抗劑(Luzindole)組,分別用不同濃度的褪黑素干預(0.1umol/L,lumol/L,10umol/L,100umol/L),在干預不同時間(24h,48h,72h)后用Western Blot技術檢測細胞中磷酸化的Akt水平,免疫熒光檢測Nrf2的核轉位,用Real time RT-PCR及Western Blot技術檢測HO-1,GCLc,GCLm,IL-1 β,TNF-α,NF-κB的表達,ELISA法檢測VEGF水平,綜合分析褪黑素的保護作用。結果：酶消化法體外分離培養(yǎng)大鼠視網膜Muller細胞純化度高。視網膜Muller細胞存在褪黑素膜受體MT1,MT2分布,且在高糖條件下受體表達升高。褪黑素可以誘導Akt的磷酸化,提高Nrf2及抗氧化酶H0-1,GSH,GCLc,GCLm的表達水平,促使Nrf2發(fā)生核轉位,同時抑制高糖狀態(tài)下顯著提高的VEGF水平。而這些效應可被褪黑素膜受體拮抗劑Luzindole及P13K抑制劑LY294002部分阻斷。高糖條件下,細胞內炎癥因子IL-1β,TNF-α,NF-κB水平顯著升高,褪黑素可降低其表達。結論：褪黑素對高糖所致視網膜Muller細胞損傷具有抗氧化及抗炎保護作用,主要體現在可以提高抗氧化酶水平以及抑制炎癥因子表達。PI3K/Akt-Nrf2通路及褪黑素膜受體參與其抗氧化效應的實現,NF-K B在其抗炎效應中發(fā)揮重要作用。同時褪黑素可以抑制VEGF的表達。第二部分 褪黑素對糖尿病大鼠視網膜組織損傷的保護作用及其機制研究目的：通過建立糖尿病大鼠模型,探討褪黑素是否對糖尿病所致視網膜損傷具有保護作用,并進一步研究其作用機制。方法：成年雄性SD大鼠隨機分為三組：正常對照組,糖尿病組,糖尿病+褪黑素組。采用STZ (60 mg/kg)一次性腹腔注射法建立糖尿病大鼠模型。三天后,尾靜脈采血測血糖,血糖大于16.7 mol/L者為造模成功。糖尿病+褪黑素組在糖尿病造模成功后,給予褪黑素(10 mg/kg)每日腹腔注射。實驗過程中定期檢測實驗動物的血糖和體重。血糖不符合者及時剔除。正常對照組給予相應溶劑腹腔注射。分別在造模4周,8周,12周后將動物處死,取出視網膜組織。免疫熒光法檢測視網膜冰凍切片上褪黑素膜受體MT1, MT2的分布。Real time RT-PCR 及 Western Blot檢測VEGF, HO-1, iNOS, IL1β, TNF-α及Nrf2水平,Western Blot檢測磷酸化IK B,磷酸化Akt及細胞核內NF-κB水平。同時檢測造模12周后各組大鼠ERG水平。結果：STZ腹腔注射法建立糖尿病大鼠模型方法可靠,成功率高,造模組動物血糖在整個實驗中維持高水平,保持穩(wěn)定。糖尿病大鼠體重與正常大鼠相比進行性降低。褪黑素干預對糖尿病大鼠體重和血糖無明顯影響。免疫熒光檢測顯示MT1和MT2主要分布于視網膜神經節(jié)細胞層,內叢狀層,且在糖尿病狀態(tài)下表達升高。糖尿病大鼠視網膜VEGF水平較正常組顯著提高,而褪黑素處理組可降低其表達。TNF-α,IL-1β等炎癥因子,磷酸化IK B及細胞核NF-κB水平在糖尿病狀態(tài)明顯提高,褪黑素可以抑制其表達。褪黑素可以使磷酸化Akt水平提高,并提高抗氧化相關因子Nrf2, HO-1水平。造模12周后,糖尿病大鼠ERG的a波和b波振幅較正常組顯著降低,而褪黑素干預組ERG與正常組相比無統計學差異。結論：褪黑素對糖尿病視網膜病變具有保護作用,主要通過抑制NF-κB及其下游炎癥因子表達,及提高PI3K/Akt-Nrf2介導的抗氧化因子水平實現。同時褪黑素還可以抑制VEGF的表達,改善視網膜功能。因此,褪黑素對于糖尿病視網膜病變的治療具有潛在價值。
[Abstract]:With the change of the rhythm of life and the way of our country, the incidence of diabetes has increased year by year, up to 9.7%[1], indicating that the disease has become one of the major public health problem in China. Diabetes can cause such as cataracts, glaucoma, optic neuropathy and other ocular complications, particularly in diabetic retinopathy (Diabetic Retinopathy DR) is the most serious, has become one of the main reasons for the blindness. Studies show that the occurrence and development of DR is involved in many cellular signaling pathways, including polyol pathway, activation of protein kinase C, advanced glycation end products [2-3]. accumulation, hexosamine pathway etc. but the research stated that oxidative damage is the the fundamental mechanism of DR pathway leading to [4], [5]. and an important factor of chronic inflammation is involved in the course of DR development so that the progress of antioxidant and anti-inflammatory treatment could effectively prevent disease, To provide a new therapeutic approach for patients. Melatonin is a hormone secreted by the pineal gland, retina also have a small amount of secretion of [6]. according to the report, it is the ability of anti oxidative stress found in the strongest antioxidant molecules, through a variety of ways [7-9]. also can be regulated by various proinflammatory cytokines, play an anti-inflammatory effect of [10]. so powerful melatonin for diabetic retinopathy has a potential therapeutic role. On the basis of Biochemistry, molecular biology methods, retinal Muller cells cultured by primary, establish the model of diabetic rats, to investigate the protective effect of melatonin on diabetic retinopathy at the cellular level and the overall level, and further study the mechanism of action, will to provide a theoretical basis for future clinical trials and research and development of new drugs for diabetic retinal protection, diabetes To provide a new way for the treatment of retinopathy. The melatonin protective effect and mechanism of injury of high glucose induced retinal Muller cells Objective: To investigate whether melatonin damage on retinal Muller cells induced by high glucose has a protective effect, and further study its pathway. Methods: cultured rat retinal Muller cells by enzyme digestion, immune cell identification fluorescence properties and purification of Muller cells was detected. The distribution of melatonin membrane receptor cell immunofluorescence. The cells were divided into low glucose control group (5.5mmol/l), mannitol group (5.5mmol/l+24.5mmol/l mannitol), high glucose group (30mmol/l), melatonin melatonin + + high glucose group, high glucose +P13k inhibitor (LY294002) group, high glucose + + melatonin melatonin membrane receptor antagonist (Luzindole) group, respectively with different concentrations of melatonin intervention (0.1umol/L, lumol/L, 10umol/L, 100umol/L), In the intervention of different time (24h, 48h, 72h) after Western cells were detected with phosphoric acid in the Blot technology of Akt, immunofluorescence detection of Nrf2 nuclear translocation was detected by HO-1 Real, time RT-PCR and Western Blot GCLc, GCLm, IL-1 beta, TNF- alpha, the expression of NF- kappa B, VEGF level detection ELISA by comprehensive analysis of the protective effect of melatonin. Results: the cultured rat retinal Muller cells of high purification enzyme digestion method in vitro. Retinal Muller cells exist melatonin receptors MT1, MT2 distribution, and the elevated expression in high glucose conditions. Melatonin can induce the phosphorylation of Akt, Nrf2 increased and antioxidant enzymes H0-1, GSH. GCLc, the expression level of GCLm, promote Nrf2 nuclear translocation, and inhibit the high glucose condition significantly increased levels of VEGF. These effects can be melatonin membrane receptor antagonist Luzindole and P13K inhibitor LY294002 partially blocked. Under high glucose condition, Intracellular inflammatory factor IL-1 beta, TNF- alpha, NF- kappa B had significantly higher levels of melatonin can reduce the expression. Conclusion: melatonin has antioxidant and anti-inflammatory protective effect on retinal Muller cells induced by high glucose injury, mainly reflected in can improve the levels of antioxidant enzymes and can inhibit the inflammatory cytokines expression of.PI3K/Akt-Nrf2 pathway and melatonin membrane receptor involved in the antioxidant the effect of NF-K, B play an important role in the anti-inflammatory effect. At the same time the expression of melatonin can inhibit the VEGF. The second part objective of melatonin on the protective effect and mechanism of retinal tissue injury in diabetic rats: by establishing the model of diabetic rats, to investigate whether melatonin on diabetic retinal injury has a protective effect, and further study its role the mechanism. Methods: adult male SD rats were randomly divided into three groups: normal control group, diabetes group, sugar The urine sickness + melatonin group. Using STZ (60 mg/kg) to establish the model of diabetic rats by intraperitoneal injection. After three days, blood glucose of tail vein was greater than 16.7 mol/L were successful. Diabetes + melatonin group rats in diabetes mellitus after administration of melatonin (10 mg/kg) intraperitoneal injection and blood glucose. The experimental animal weight detection regularly during the experiment. Blood sugar does not conform to timely removed. The normal control group were given intraperitoneal injection of the corresponding solvent. In 4 weeks, 8 weeks, 12 weeks after the animal is killed, remove the retinal tissue. Detection of retinal immunofluorescence on frozen sections of melatonin membrane receptor MT1,.Real time RT-PCR distribution VEGF MT2 and Western Blot detection, HO-1, iNOS, IL1 beta, TNF- alpha and Nrf2 level Western Blot IK detection of phosphorylated B, phosphorylation of Akt and nuclear NF- kappa B level. At the same time detected 12 weeks after modeling, the rats in ERG water Ping. Results: STZ intraperitoneal injection method to establish the diabetic rat model is reliable, high success rate, make blood sugar throughout the experiment animal model to maintain a high level, remained stable. The body weight of diabetic rats compared with normal rats was decreased. Melatonin intervention had no significant effect on body weight and blood glucose of diabetic rats was detected by immunofluorescence. MT1 and MT2 mainly distributed in retinal ganglion cell layer, inner plexiform layer, and in the condition of diabetes. Elevated expression of retinal VEGF levels in diabetic rats was higher than that in normal group and treatment group, melatonin can reduce the expression of.TNF- alpha, IL-1 beta and other inflammatory factors, the phosphorylation of IK B and nuclear NF- kappa B level significantly increased in the diabetic state, melatonin can inhibit its expression. Melatonin can improve the phosphorylation level of Akt, and improve the antioxidant related factor Nrf2, the level of HO-1. After 12 weeks, diabetic rats ER G A and b wave amplitude is significantly lower than the normal group, melatonin intervention group ERG no statistically significant difference compared with the normal group. Conclusion: melatonin has a protective effect on diabetic retinopathy, mainly inhibit the expression of NF- kappa B and its downstream inflammatory factors, and improve the level of antioxidant factor PI3K/Akt-Nrf2 mediated expression of melatonin and implementation. Also can inhibit VEGF and improve the function of retina. Therefore, melatonin has a potential value for the treatment of diabetic retinopathy.

【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R587.2;R774.1

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