钢铁雄心4加资源作弊码:高糖培养下视网膜Müller细胞改变及牛磺酸的防护效应研究

高糖培养下视网膜Müller细胞改变及牛磺酸的防护效应研究
The Cell Change of Muller Cells in the Retina in High Glucose and the Protection Effect of Taurine to It
论文作者
论文导师 糜漫天,论文学位 硕士,论文专业 营养与食品卫生学
论文单位 第三军医大学,点击次数 9,论文页数 106页File Size2273K
2007-05-01论文网 http://www.lw23.com/lunwen_182604162/
Diabetic Retinopahty;; Taurine;; Müller cell;; Apoptosis;; Glial fibrillary acidic protein;; Glutamine synthetase;; Taurine transporter;; S-100
糖尿病视网膜病变(Diabetic Retinopathy, DR )是糖尿病眼病中的特征性病变,常导致视力减退甚至失明。早期大量的实验都集中在糖尿病视网膜血管病变上。近年来越来越多的研究发现,在视网膜血管病变发生之前即可检测到视网膜功能的改变,表明糖尿病对视网膜神经层有直接的影响,而不是发生在血-视网膜屏障破坏之后。新的观点认为,视网膜神经元和神经胶质的改变发生在血管病变之前,并且神经元和神经胶质的改变可能最终导致视网膜血管病变和黄斑水肿。 糖尿病早期的血-视网膜屏障破坏、视网膜缺氧等可引起视网膜内谷氨酸浓度升高,引起视网膜神经元如视网膜神经节细胞等的兴奋毒性损伤及死亡,从而引起视觉丧失,而谷氨酸的兴奋毒性可进一步增加氧化应激。Müller细胞是清除细胞外过量谷氨酸的主要胶质细胞,其胞膜的谷氨酸转运蛋白(Glutamate transporter, GLAST )、胞内的谷氨酰胺合成酶(Glutamine synthetase, GS )及谷氨酸脱羧酶(Glutamate decarboxylase, GD )是转运并降解谷氨酸的几个主要环节。Müller细胞在糖尿病早期功能受损,与其受到的氧化应激增加有关。但尚不清楚其摄取及降解谷氨酸的功能有何改变及血-视网膜屏障破坏中的确切机制。 牛磺酸(Taurine,又名二氨基乙磺酸)是体内一种β-氨基酸,属于非蛋白质氨基酸,主要分布在兴奋性较高的组织如神经系统、肌肉组织、视网膜及淋巴细胞和血小板中,在人体中具有重要的生理作用。牛磺酸是视网膜中含量最丰富的游离氨基酸,尤其在感光细胞和Müller细胞内含量很高,研究发现牛磺酸与视网膜生长发育、维持正常视功能关系密切。最新的研究表明牛磺酸可在视网膜中发挥抑制性神经调质作用,同时能抑制谷氨酸的兴奋毒作用。 有研究显示,糖尿病视网膜病变早期,在视网膜毛细血管病变发生之前视网膜中神经元和神经胶质细胞功能就发生了障碍,表现为Müller细胞中神经胶质原纤维酸性蛋白表达增加、谷氨酸转运蛋白功能障碍及谷氨酸受体NMDA亚型激活等,而视网膜中牛磺酸含量随糖尿病病程延长呈渐进性下降。基于以上分析,本实验在体外以高葡萄糖环境培养大鼠视网膜Müller细胞作为研究DR时Müller细胞改变的体外模型,给予牛磺酸处理后采用TUNEL、免疫细胞化学荧光染色、Western blotting、Real Time-PCR等技术方法,观察高糖环境及牛磺酸处理对视网膜Müller细胞神经胶质原纤维酸性蛋白(Glial fibrillary acidic protein, GFAP)、谷氨酰胺合成酶(Glutamine synthetase, GS)、牛磺酸转运蛋白(Transport protein, TAUT )、S-100表达的影响。旨在深入研究牛磺酸对糖尿病早期视网膜病变的影响及可能的分子机制。 主要实验结果和结论如下: 1.在原代混养的SD大鼠视网膜细胞中,神经细胞和胶质细胞共生良好,神经细胞对高糖损伤更敏感,存活率下降,牛磺酸可使其存活率明显上升。 2.视网膜Müller细胞在高浓度葡萄糖条件下活性下降且出现大量的凋亡细胞,而给予0.1mmol/L、1mmol/L、5mmol/L、10mmol/L牛磺酸处理后Müller细胞活性上调,并使凋亡细胞数明显下降,表明牛磺酸能够浓度依赖性地促进Müller细胞的增殖,抑制高糖所致的视网膜Müller细胞凋亡。 3.高糖可引起视网膜Müller细胞特征蛋白GFAP mRNA和蛋白表达增强,S-100表达核质比降低。0.1mmol/L、1mmol/L、5mmol/L和10mmol/L的牛磺酸可减弱高糖引起的GFAP mRNA和蛋白的强表达。S-100表达核质比在0.1mmol/L、1mmol/L、5mmol/L和10mmol/L的牛磺酸处理后增强。提示高糖导致的Müller细胞功能改变可被牛磺酸减轻或阻止。 4.高糖可诱导Müller细胞GS表达大部分被抑制,GS mRNA及蛋白表达下降。牛磺酸能明显抑制高糖引起的GS mRNA及蛋白表达下降,不同浓度牛磺酸作用后GS逐步向正常水平恢复,有利于谷氨酸在Müller细胞中的正常代谢,从而减弱兴奋毒性。 5.牛磺酸主要通过细胞膜上TAUT跨膜主动转运以维持细胞内高浓度,通过在体外制备Müller细胞高糖模型,观察到高糖抑制了Müller细胞牛磺酸跨细胞膜转运,补充牛磺酸后TAUT表达上调。 综上所述,高糖损伤导致视网膜Müller细胞形态结构和特征蛋白发生改变,给予牛磺酸处理后,对视网膜Müller细胞高糖损伤有明显的防护作用。本实验结果为牛磺酸用于防护糖尿病早期视网膜损伤提供了实验依据。
Diabetic Retinopahty is the characteristic pathological change in the ophthalmopathy of diabetic, it often lead visual extinction and even blind. In the early stage,most experiments concentrated on the retinal vasculopathy in diabetes. Recent years more and more researchs discovered that it can detected the function change of retina before the retinal vasculopathy, it manifested that diabetes had the direct affection to nervous layer of retina and didn’t occurrenced after the breakdown of blood-retina barrier, vasculopathy was one of the directions to cause the handicapped of retina.The new views think that the alteration of retinal neurons and neuroglia development before the vasculopathy, moreover the alteration of retinal neurons and neuroglia possible induce the retinal vasculopathy and macular edema. The breakdown of blood-retina barrier、hypoxia of retina in the early diabetes may cause the concentration of glutamate in retina increased, may provoke the retinal neurons such as retinal ganglial cells exitotoxicity damage and the death, thus cause deprivation of retina. But the exitotoxicity of glutamate will increase the oxidative stress further. Muller cell is the dominant glial cell to scavenge the excess glutamate out of the cell, the GLAST、GS、GD in Müller cell membrane are the several mainly component element to converying and degrade the glutamate. The function of Müller cell damaged in the early diabetes, which has the relationship with the increase of oxidative stress.But it stil didn’t know how to alteration the function after it intake and degrade the glutamate, the really mechanism to breakdown the barrier of blood-retina. Taurine (diamidoethylsulfonic acid ) is one of theβ-amino acids in vivo, it belongs nonprotein amino acid, it mainly distribution in the tissues which have high excitability such as nervous system、muscle tissue、retina、lymphocyte and platelet, it has the important physiological functions in vivo. Taurine is abundance in photoreceptor cell and Müller cell, it relate to the growth and development of retina and maintenance normal visual function. The latest data demonstrate that taurine can inhibit the function of neuromodulator in retina and inhibit the excite toxic action of glutamate simultaneously. It displayed, in the early stage of diabetic retinopathy, the function of retina neuron and glial cell has disordered before the telangiosis in retina developed. It appearanced that the expression of Glial fibrillary acidic protein in Müller cell increased、the function of glutamate transporter disorderd and the subtype of glutamic acid receptor activated, but the contents of taurine in retina decreased gradually with the course of diabetes prolonged. On account of the above analysis, this experiment by means of culture the muller cell of rat retina in high gloucose as the model in vitro to reasearch the change of muller cell in diabetic Retinopathy.Using the TUNEL、immuocytochemistry、Western blotting、Real Time-PCR and so on technique methods after intervention by taurine, observing the influence of high glucose and taurine intervention to the expression of GFAP、GS、TAUT、S-100. Aim at to further research the influence and the possible molecule mechanism of taurine to retinopathy in early diabetes. Results and conclusions: 1. In the retinal cells from SD rat which mix culture at archae-generation, the syngenesis of neurocyte and glial cell is good, the neurocyte is more sensitiveness to high glucose damage, the survival rate decreased, the survival rate can be incresed obviously by taurine. 2. The cytoactive of muller cell in retina which cultured in high glucose can be descended severity and have many apoptosis cells , the cytoactive of muller cell can increased after intervention by 0.1mmol/L、1mmol/L、5mmol/L、10mmol/L taurine, and the number of apoptosis cells decreased obviously, it indicated that taurine can promoted the proliferation of Muller cell by means of dose-dependent and inhibited the apoptosis of Muller cell from retina which induced by high glucose. 3. The damage of high glucose can cause the mRNA and protein expression of GFAP increased which is the characteristic protein of Muller cell from retina, the nuclear-cytoplasmic ratio expression of S-100 is decreased. 0.1mmol/L、1mmol/L、5mmol/L and 10mmol/L tautine can attenuate the high expression of GFAP which caused by high glucose. The nuclear-cytoplasmic ratio expression of S-100 is enhanced after treated by 0.1mmol/L、1mmol/L、5mmol/L and 10mmol/L taurine. It indicated that the cell function change of Muller cell induced by high glucose can been lessen or forbid by taurine. 4. The expression of GS can been inhibited by high glucose, high glucose can cause the mRNA and protein expression of GS decreased. Taurine can inhibit the mRNA and protein expression of GS decreased which caused by high glucose, the expression of GS recover to normal level gradually after contribution by different concentration taurine. 5. Taurine maintenanced the high concentration in cell by the transmembrane active transport of TAUT in cellular membrane, through the high glucose model of Muller cell which constructed in vitro, we observed that high glucose inhibited the transmembrane transport of Muller cell, the expression of TAUT up-regulated after supplied taurine. As the above review, high glucose damage can cause the change of morphous and chatacteristic protein of Muller cell from retina, after treated by taurine, the protective effection to Muller cell from retina which caused by high glucose damage is visible. This experiment have provide the experiment basis to use taurine protective the retina damage in early diabetes.
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