【作者】 李琳娜；

【导师】 刘誉；

【作者基本信息】 暨南大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 【目的】研究晚期糖基化终产物(advanced glycation end products,AGEs)对人肾小管上皮细胞株HKC的细胞毒性作用,并探讨其可能的作用机制。【方法】1.葡萄糖与牛血清白蛋白(BSA)共同孵育制备成AGEs,用荧光分光光度计测定AGEs的荧光强度。2.以体外培养的HKC细胞为研究对象,采用MTT法测定不同浓度不同时间AGEs对HKC生长的抑制作用;应用流式细胞仪和DNA琼脂糖凝胶电泳分析细胞的凋亡情况。3.RT-PCR法分析不同浓度AGEs处理HKC后其IL-1β,TNF-α,HMGB1 mRNA表达水平的变化。4.荧光分光光度计检测不同浓度AGEs处理HKC后细胞内活性氧(ROS)的含量,紫外分光光度计测定超氧化物歧化酶(SOD)的活性。【结果】1.MTT结果显示,与对照组相比,AGEs均能显著抑制HKC细胞的增殖,且抑制作用具有时间和剂量依赖性。2.流式细胞仪分析发现,AGEs能促使HKC细胞发生凋亡,凋亡率亦随AGEs浓度的增高而增加。DNA琼脂糖凝胶电泳观察到AGEs作用HKC细胞后出现典型的DNA梯行条带。3.RT-PCR方法检测显示,AGEs使HKC细胞IL-1β,TNF-α,HMGB1 mRNA的表达上调。4.AGEs作用HKC细胞后,ROS含量增高,SOD活性代偿性增高。【结论】1.AGEs对HKC细胞的增殖具有显著的抑制作用,且呈现出良好的时效与量效关系。2.AGEs能促进HKC细胞的凋亡。3.AGEs对HKC细胞的毒性损害可能与上调IL-1β,TNF-α,HMGB1 mRNA表达有关。4.随着AGEs浓度的增高,ROS含量增高,SOD活性代偿性增高,提示AGEs对HKC细胞的毒性作用机制可能与细胞氧化应激有关。更多还原

【Abstract】 Objective:To investigate the cytotoxicity of human renal tubular epithelial cells(HKC) induced by advanced glycation end products(AGEs) and explore the possible mechanisms.Methods:1.AGEs were prepared in vitro by incubating glucose with bovine serum albumin(BSA). The fluorescent intensity of AGEs is measured by spectrofluorophotometer.2.The proliferative inhibition of HKC induced by AGEs at different concentrations and different time was accessed by using MTT assay.Cell apoptosis was analyzed by flow cytometry and DNA agarose gel electrophoresis.3.The expression of IL-1β,TNF-αand HMGB1 mRNA in HKC cells induced by AGEs were determined by RT-PCR..4.The generation of reactive oxygen species(ROS) was determined by spectrofluorophoto-meter. The activity of SOD was detected by UV spectrophotometer.Results:1.Compared with control group(nontreated with AGEs),AGEs significantly inhibited proliferation of HKC,which showed good time- and dose-dependent fashions. 2.AGEs were also found to cause apoptosis in HKC.The flow cytometry analysis showed that AGEs increased the proportion of apoptotic cells in a dose-dependent manner.The DNA Ladder can be observed when the HKC was treated by AGEs.3.RT-PCR analysis demonstrated that the expression of IL-1β,TNF-αand HMGB1 mRNA in HKC cells were up-regulated by AGEs.4.The generation of ROS and the activity of SOD was elevated with AGES concentration increasing.Conlusions:1.AGEs can efficiently inhibit HKC proliferation,these growth inhibitions of HKC induced by the AGEs showed good time- and dose- dependent fashions.2.Apoptosis of HKC can be significantly promoted by AGEs.3.The cytotoxicity of HKC induced by AGEs may be related to the up-regulated expression of IL-1β,TNF-αand HMGB1 mRNA.4.The generation of ROS and the activity of SOD increased when the HKC was treated by AGEs.It suggested that the cytotoxicity of HKC induced by AGEs may be connected with oxidative stress.更多还原