抵抗素对幼鼠主动脉内皮细胞一氧化氮系统的损伤作用

发布时间：2018-02-04 19:21

本文关键词： 抵抗素大鼠主动脉内皮细胞一氧化氮一氧化氮合酶　出处：《安徽医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的: 抵抗素是新近发现的一种脂肪因子,在肥胖和超重儿童中血清浓度有不同程度升高,与心脑血管疾病等密切相关。通过原代培养幼鼠主动脉内皮细胞建立体外模型,以不同浓度抵抗素(resistin)干预,模拟正常和肥胖状态下抵抗素水平,从分子、蛋白、酶的活性、基因的表达及转录、翻译后调节等多个水平研究幼鼠主动脉内皮细胞一氧化氮(NO)系统的调控机制。方法: 取幼鼠胸主动脉,用植环贴壁法原代培养大鼠主动脉内皮细胞,消化、传代,观察细胞形态,VIII因子相关抗原免疫细胞化学染色进行细胞鉴定,建立体外细胞模型。取传至3-5代的大鼠主动脉内皮细胞,以不同浓度鼠抵抗素(10-100 ng/ml)干预24小时。MTT法检测内皮细胞活性,化学比色法检测NO及NOS酶活性,蛋白免疫印迹法检测细胞内eNOS(内皮型一氧化氮合酶)蛋白和P-eNOS(磷酸化一氧化氮合酶)蛋白的表达,RT-PCR检测eNOSmRNA表达的变化。结果: 幼年大鼠主动脉内皮细胞经不同浓度鼠重组抵抗素处理24小时后: 1.光镜下观察细胞形态均未发生明显改变。 2.与对照组和10 ng/ml相比,50 ng/ml和100 ng/ml组细胞活性下降。 3.各组NO产物及eNOS活性均未发生改变。 4.与对照组相比,50 ng/ml和100 ng/ml组细胞eNOS蛋白和mRNA表达减少。 5.各组P-eNOS表达未见改变。结论: 抵抗素可减低幼年大鼠主动脉内皮细胞的细胞活性,调控NO系统,使eNOS蛋白和基因表达水平降低,其但未见通过调控磷酸化作用影响酶的活性从而降低NO产物的合成。
[Abstract]:Objective: Resistin (resistin) is a newly found fatty factor, and serum levels in obese and overweight children have increased to varying degrees. In vitro model was established by primary culture of aortic endothelial cells of young rats and treated with different concentrations of resistin. Mimic normal and obese levels of resistin from molecular, protein, enzyme activity, gene expression and transcription. The regulation mechanism of nitric oxide (no) system in aortic endothelial cells of young rats was studied at several levels such as posttranslational regulation. Methods: The primary cultured rat aortic endothelial cells were isolated from the thoracic aorta of young rats. The endothelial cells of rat aorta were digested and subcultured. The morphology of the cells was observed and the immunocytochemistry staining of factor VIII related antigen was carried out for cell identification. To establish cell model in vitro. The endothelial cells of rat aorta were collected for 3 to 5 passage. The endothelial cell activity was detected by MTT assay with different concentrations of resistin 10-100 ng / ml for 24 hours. The activity of no and NOS was detected by chemical colorimetry, and the expression of Enos (endothelial nitric oxide synthase) protein and P-eNOS- (phosphorylated nitric oxide synthase) protein were detected by Western blot. The expression of eNOSmRNA was detected by RT-PCR. Results: Young rat aortic endothelial cells were treated with different concentrations of recombinant resistin for 24 hours. 1. There was no obvious change in cell morphology under light microscope. 2.Compared with the control group and 10 ng/ml group, the cell activity of 50 ng/ml and 100 ng/ml groups decreased. 3. No products and eNOS activity did not change in each group. 4. Compared with the control group, the expression of eNOS protein and mRNA was decreased in 50 ng/ml and 100 ng/ml groups. 5. There was no change in the expression of P-Enos in each group. Conclusion: Resistin can reduce the cellular activity of aortic endothelial cells, regulate the no system, and decrease the expression of eNOS protein and gene. However, it was not found that the activity of the enzyme was affected by phosphorylation and the synthesis of no products was decreased.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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