【作者】 罗怡；

【导师】 卢奕； 莫晓芬； 吴继红；

【作者基本信息】 复旦大学 ， 眼科学， 2008， 博士

【摘要】 第一部分晚期糖基化终末产物、转化生长因子与糖尿病性白内障相关性研究目的探讨晚期糖基化终末产物(AGEs)、转化生长因子-β(TGF-β)水平与糖尿病性白内障的相关性,为深入研究糖尿病性白内障发病的分子生物学机理提供实验依据。方法采用生化方法、酶联免疫吸附测定(ELISA)法对糖尿病性和老年性白内障患者房水中葡萄糖(Glueose)、AGEs、TGF-β2,以及晶状体前囊膜上皮细胞AGEs和TGF-β2进行定量检测,Dot blot方法测定前囊膜晶状体上皮细胞内AGEs和TGF-β2的蛋白表达。并采用实时荧光定量聚合酶链式反应(real-time PCR)定量分析晶状体上皮细胞内AGEs受体(RAGE)和TGF-β1、TGF-β2、TGF-β3的mRNA表达。结果糖尿病性白内障房水中葡萄糖水平和AGEs含量分别为6.83±1.4mmol/L、355.79±195.64pg/ml,显著高于老年性白内障患者(4.74±1.3mmol/L、161.67±86.23pg/ml),两组间的差异具有显著的统计学意义(p＜0.01),同时Real—timePCR结果显示晶状体上皮细胞AGEs受体(RAGE)的mRNA表达也显著高于老年性白内障患者。TGF-β2在糖尿病性白内障患者房水(368±264.23pg/ml)和晶状体上皮细胞中的含量(730±403pg/ml)较老年性患者明显降低(732±394.57pg/ml、1147±402pg/ml),TGF-β2 mRNA表达也降低,而TGF-β1mRNA的表达则明显升高,这种差异在老年性白内障和糖尿病性白内障之间均具有统计学意义(p＜0.01)。TGF-β3 mRNA在老年性白内障和糖尿病性白内障晶状体上皮细胞中的表达没有显著差异。结论晶状体前囊膜的上皮细胞有AGE受体(RAGE)mRNA的表达,AGEs及受体在糖尿病性白内障晶状体上皮细胞中的表达高于老年性白内障,而TGF-β2在糖尿病性白内障的房水和晶状体上皮细胞中的表达低于老年性白内障。AGEs和TGF-β2可能与糖尿病性白内障的发病有关。第二部分晚期糖基化终末产物对晶状体上皮细胞作用及其分子机制目的研究晚期糖基化终末产物(AGEs)对体外培养的晶状体上皮细胞增殖及细胞周期的影响及细胞外基质和TGF—β表达变化;探讨糖尿病性白内障晶状体前囊膜上皮细胞p21、p27和c-myc蛋白的变化;从而探讨AGEs在糖尿病性白内障发病中可能的分子机制。方法采用MTT比色法检测AGEs对体外培养的人晶状体上皮细胞增殖的影响,流式细胞仪分析细胞周期。免疫荧光检测AGEs对晶状体上皮细胞TGF-β与细胞外基质FN、α-SMA和I型胶原蛋白表达水平的影响。Western免疫印迹法检测AGEs作用于晶状体上皮细胞后信号分子MEK和ERK蛋白表达和糖尿病性、老年性白内障前囊膜的上皮细胞细胞周期相关蛋白p21、p27、c-myc表达水平。结果AGEs小于1mg/ml时可以抑制晶状体上皮细胞的增殖,甚至出现明显的细胞凋亡。而AGEs浓度大于5mg/ml时促进细胞的增殖,且以AGEs作用于晶状体上皮细胞后48小时最显著。同时氨基胍(AG)抑制AGEs对晶状体上皮细胞的促增殖作用,表现为G1期阻滞。p21和p27在糖尿病性白内障的表达低于老年性白内障,而c-myc则表达升高。体外培养的人晶状体上皮细胞的培养基中加入晚期糖基化产物,晶状体上皮细胞TGF-β(TGF-β1/2/3)、FN、α-SMA和I型胶原蛋白的表达增加,MEK和ERK蛋白表达上调。结论糖尿病性白内障晶状体上皮细胞的增殖状态较老年性白内障活跃。高浓度AGEs促进晶状体上皮细胞增殖,氨基胍通过G1期阻滞抑制AGEs的促增殖作用。AGEs促进晶状体上皮细胞TGF-β(TGF-β1/2/3)、FN、α-SMA和I型胶原蛋白分泌增加。更多还原

【Abstract】 PartⅠAdvanced glycation end-products and TGF-βwith diabetic cataractPurpose To investigate the effects of advanced glycation end-products (AGEs) and TGF-βon the diabetic cataract.These data should provide molecular basis for further studying the pathogenesis of clinical diabetic cataract.Methods Aqueous and anterior capsule lens epithelial cells from the diabetic cataract and age-related cataract patients were extracted to detect the blood glucose,AGEs,and TGF-β2 using biochemical methods and enzyme-linked immunosorbent assay(Elisa).For the anterior capsule lens epithelial cells,dot blot was used to detect the protein expression of AGEs and TGF-β2,and real-time PCR was used to quantitatively detect the mRNA expressions of AGE receptor(RAGE),TGF-β1,TGF-β2,and TGF-β3.Results Blood glucose and AGEs of the aqueous significantly increased in the diabetic cataract compared to the age-related cataract(p＜0.01). RAGE mRNA expression of anterior capsule lens epithelial cells in the diabetic cataract were much higher than that of in the age-related cataract.TGF-βmRNA expression Either TGF-β2 in aqueous and lens epithelial cells or TGF-β2 mRNA expression in the lens epithelial cells of diabetic cataract obviously decreased compared to age-related cataract. In contrast with TGF-β2,TGF-β1 mRNA in the lens epithelial cells of diabetic cataract obviously increased in comparison with age-related cataract(p＜0.01).TGF-β3 mRNA expression in the age-related cataract was slightly higher than that in the diabetic cataract.Conclusion AGEs,RAGE,and TGF-β1 expression increased in the lens epithelial cells of diabetic cataract in comparison with age-related cataract.However,TGF-β2 in the aqueous and lens epithelial cells of diabetic cataract obviously decreased.These results indicated that AGEs and TGF-βwere related with the pathogenesis of diabetic cataract. PartⅡMolecular mechanisms of advanced glycation end-products on lens epithelial cellsPurpose To investigate the effects of advanced glycation end-products on the proliferation and cell cycle of lens epithelial cells,p21、p27 and c-myc protein expressions of lens epithelial cells both in diabetic and age-related cataract were studied as well.Methods The effect of AGEs on the proliferation of lens epithelial cells was detected by MTT methods,and cell cycle was analyzed by flow cytometry. FN,α-SMA and collagenⅠexpressions of cultured lens epithelial cells after adding AGEs by using immunofluorescence.Cell signal molecular expressions of MAPK and ERK in cultured lens epithelial cells with or without AGEs and p21,p27,c-myc protein in anterior capsule lens epithelial cells were determined by western blot.Results Lower concentration of AGEs inhibited the proliferation of lens epithelial cells,even made cell apoptosis.Higher concentration of AGEs accelerated cell proliferation and the effect was most obviously at 48hrs after adding AGEs.Aminoguanidine inhibited the proliferative effect of AGEs and cell cycle was blocked in G1 phase,p21 and p27 protein expression decreased and c-myc increased in diabetic cataract compared to age-related cataract.Expression of FN,α-SMA,TGF-βand collagenⅠof cultured lens epithelial cells increased after adding AGEs.Cell signal moleculars MAKP and ERK expression also increased in the action of AGEs. Conclusion:The proliferation of lens epithelial cells in diabetic cataract was more active compared to age-related cataract.Higher concentration of AGEs promoted the proliferation of lens epithelial cells, and aminoguanidine inhibited this effect by blocking G1 phase of cell cycle.Expression of FN,α-SMA,TGF-βand collagenⅠof cultured lens epithelial cells increased after the action of AGEs.This effects induced from AGEs might be correlated to the MAPK signal transduction pathway.更多还原