AOPP对MRC-5细胞增殖及FN、Collagen I表达的影响
本文选题：晚期氧化蛋白产物 + 人胚肺成纤维细胞 ； 参考：《遵义医学院》2017年硕士论文
【摘要】：目的:研究晚期氧化蛋白产物(7)advanced oxidation protein products,AOPP(8)对人胚肺成纤维细胞(human embryonic lung fibroblasts MRC-5,MRC-5)活性氧自由基(7)reactive oxygen species,ROS(8)及细胞外基质纤维连接蛋白(fibronectin,FN)及I型胶原(collagen I,Col I)的作用。方法:1.将MRC-5细胞分为人血清白蛋白(human serum albium,HSA)组,AO-PP时间效应组(7)0、12、24、48及72小时(8)和浓度效应组(7)0、50、100、200及400μg/ml(8),NADPH氧化酶抑制剂夹竹桃麻素(7)apocynin,APO(8)组(7)AOPP+APO(8)。2.用上述不同浓度的AOPP与MRC-5细胞共同培养不同的时间,MTT法检测细胞的活力。3.采用ELISA法检测FN、Col I及转化生长因子(7)transforming growth factor-β1,TGF-β1(8)在细胞上清中的浓度,通过RT-PCR检测各组细胞上述3个指标m RNA的表达。4.根据MTT实验结果选取AOPP作用最佳的时间点和浓度点刺激MRC-5细胞;流式细胞仪和荧光显微镜检测活性氧自由基ROS的表达。5.NADPH氧化酶抑制剂APO与MRC-5细胞预孵育1小时,加入200μg/ml AOPP刺激48小时,观察细胞内ROS的变化,从蛋白和基因水平检测FN、Col I及TGF-β1的表达。结果:1.不同浓度的AOPP(50、100、200、400μg/ml)作用于MRC-5细胞12、24、48及72小时,发现AOPP对MRC-5细胞具有显著的增殖作用(P0.05(8),且在浓度为200μg/ml及时间48h达到峰值。2.FN、Col I及TGF-β1从蛋白分泌程度和基因表达水平均上调(P0.05(8),且在浓度200μg/ml及时间48h时达到峰值;未经修饰的人血清白蛋白对上述相关纤维指标的表达无明显影响(P0.05)。3.MRC-5细胞与200μg/ml AOPP作用48小时,与对照组相比,细胞内ROS水平显著升高(P0.05)。4.MRC-5细胞与APO预孵育1小时,加入200μg/ml AOPP刺激48小时,细胞上清中FN、Col I及TGF-β1蛋白分泌减少且m RNA表达下调P0.05㖞,MRC-5细胞中ROS水平下降(P0.05),未经修饰的人血清白蛋白对ROS的表达无明显影响P0.05㖞。结论:AOPP能够促进体外MRC-5细胞的增殖,引起细胞外基质FN、Col I表达上调及肺纤维化相关生长因子TGF-β1的表达增加,初步推测可能与NADPH氧化酶介导产生的ROS有关。
[Abstract]:Objective: to study the effects of advanced oxidation protein products of advanced oxidative protein (AOPP8) on human embryonic lung fibroblasts MRC-5 MRC-5 (reactive oxygen species-ROS8), extracellular matrix fibronectin (FNN) and collagen I (I) in human embryonic lung fibroblasts. Method 1: 1. MRC-5 cells were divided into two groups: human serum albumin (HSA) group (serum albium) group (AO-PP time effect group) and concentration response group (HSA group), and the concentration response group (HSA group) was divided into two groups: AO-PP group (n = 7) and concentration response group (n = 7) (n = 10), 7apocynin in AO-PP group (n = 8) and a concentration response group (n = 7), in which 7apocynin in AO-PP group (n = 8) and a concentration response group (n = 7) were divided into two groups. The activity of MRC-5 cells was detected by MTT assay with different concentrations of AOPP and MRC-5 cells at different time points. ELISA assay was used to detect the concentration of ELISA Col I and transforming growth factor- 尾 1 (TGF- 尾 1 + 8) in the cell supernatant. The expression of m RNA was detected by RT-PCR. According to the results of MTT experiment, the best time point and concentration point of AOPP were selected to stimulate MRC-5 cells, and the expression of free radical ROS was detected by flow cytometry and fluorescence microscope. 5. APO, inhibitor of NADPH oxidase, was preincubated with MRC-5 cells for 1 hour. After stimulation with 200 渭 g/ml AOPP for 48 hours, the changes of ROS were observed, and the expression of FNNCol I and TGF- 尾 1 were detected at the level of protein and gene. The result is 1: 1. Different concentrations of AOPP(50100200400 渭 g / ml were applied to MRC-5 cells for 48 and 72 hours. It was found that AOPP had a significant proliferative effect on MRC-5 cells, and reached a peak value of 200 渭 g/ml and 48h. 2. FNCol I and TGF- 尾 1 up-regulated P0.05Col I and TGF- 尾 1 from the level of protein secretion and gene expression, and reached the peak at the concentration of 200 渭 g/ml and 48h. There was no significant effect of unmodified human serum albumin on the expression of the above mentioned fiber markers. 3. MRC-5 cells were exposed to 200 渭 g/ml AOPP for 48 hours. Compared with the control group, the intracellular ROS level was significantly increased. 4. MRC-5 cells were preincubated with APO for 1 hour. After stimulation with 200 渭 g/ml AOPP for 48 h, the secretion of FNNCol I and TGF- 尾 1 protein in supernatant was decreased, and the expression of m RNA was down-regulated. The level of ROS in P0.05 g/ml AOPP MRC-5 cells was down-regulated. Unmodified human serum albumin had no significant effect on the expression of ROS. ConclusionOPP can promote the proliferation of MRC-5 cells in vitro, induce the up-regulation of extracellular matrix FNCol I expression and increase the expression of pulmonary fibrosis related growth factor TGF- 尾 1, which may be related to the ROS production mediated by NADPH oxidase.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563
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