香烟提取物对肺间质纤维化的影响机制以及还原型谷胱甘肽的干预作用

发布时间：2018-06-04 11:11

本文选题：香烟提取物 + 肺间质纤维化　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：香烟烟雾中含有多种有害化学物质，是多种疾病的主要诱因之一，而且被公认为是引起肺间质纤维化的重要危险因素。香烟烟雾中的氧化剂和自由基可通过多种途径产生氧化应激，大量证据表明氧化应激在肺间质纤维化的发病机制中扮演着重要的角色，无论是在急性发作期还是缓解期，病人均存在氧化／抗氧化失衡，然而其分子机制尚不清楚。在临床上肺间质纤维化尚无特效的治疗方法，近来抗氧化治疗引起许多学者关注，研究显示还原性谷胱甘肽（GSH）是机体内重要的还原剂，具有抗氧化作用，可清除自由基及其他活性氧簇，保护肺组织免受氧化损伤。因此本实验就以肺泡Ⅱ型上皮细胞来源的A549细胞为模型，用不同浓度香烟提取物(cigarette smoke extract，CSE)刺激体外培养的A549细胞，，观察细胞生长的形态学改变及增殖抑制情况。通过检测细胞上清液中各项氧化指标及反应纤维化程度的指标，来探讨吸烟对肺间质纤维化影响的可能机制以及还原型谷胱甘肽对吸烟引起的肺间质纤维化是否有逆转作用。方法：体外培养肺泡Ⅱ型上皮细胞来源的A549细胞，取对数生长期的细胞进行试验。参照Carp and Janoff[1]的方法，通过特殊装置制备香烟提取物。抽吸2支去过滤烟嘴的香烟，把烟雾融入不含血清1640培养基中，调整PH值，并除去细菌和大颗粒，然后稀释至不同的浓度，30min中内用于试验。本实验首先尝试应用0%、2.5%、5%、7.5%、10%、12.5%浓度的香烟提取物培养液培养细胞，采用MTT法测定细胞活力，通过相差显微镜观察CSE对细胞增殖形态的影响。依据其结果选定合适的浓度进行实验分组。实验分为空白对照组、2.5%CSE组、5%CSE组、7.5%CSE组，7.5%CSE+GSH组(GSH1mmol/L).分别收集空白对照组和各干预组细胞培养24h小时后的上清液，应用比色分析法测细胞上清液超氧化物歧化酶(SOD)活性和丙二醛（MDA）含量，ELISA法检测细胞上清液中转化生长因子1(TGF-β1)的水平。结果：1倒置显微镜下观察A549细胞在不同浓度CSE作用下形态学变化倒置显微镜拍摄结果显示CSE作用后A549细胞生长密度减低，细胞之间空隙加大，漂浮细胞增多，贴壁细胞也呈回缩变圆的态势，出现萎缩，与对照组的细胞形态区别明显。2不同浓度CSE对A549细胞的生长抑制作用通过MTT比色法检测，CSE对体外培养的A549细胞有较强的增殖抑制作用，抑制作用呈剂量和时间依赖性。相同浓度香烟提取物不同时间（12、24、36h）作用后，随作用时间的延长其抑制率增大，差异有统计学意义(P0.05)。相同时间不同浓度（0%、2.5%、5%、7.5%、10%、12.5%）香烟提取物作用后，随着浓度的增高其抑制率增大，差异有统计学意义(P0.05)。在10%CSE组作用24h时抑制率达70.30±2.21%，在12.5%CSE组作用24h时抑制率高达90.10±1.01%。3不同浓度CSE对A549细胞上清液TGF-β1、MDA、SOD水平的影响不同浓度CSE组TGF-β1、MDA显著高于对照组，差异有统计学意义（P0.05），且CSE浓度越高以上各因子含量越高，呈浓度依赖性，差异有统计学意义（P0.05）。不同浓度CSE组SOD活性低于于对照组，差异有统计学意义（P0.05），且CSE浓度越高SOD活性越低，差异有统计学意义（P0.05）。4GSH作用后A549细胞上清液TGF-β1、MDA、SOD水平的变化 7.5%CSE+GSH组TGF-β1、MDA含量显著低于7.5%CSE组，差异有统计学意义（P0.05），而SOD活性高于7.5%CSE组，差异有统计学意义（P0.05）。结论： 1香烟提取物可抑制肺泡上皮细胞的生长，抑制作用呈剂量和时间依赖性。 2香烟提取物可诱导肺泡上皮细胞分泌TGF-β1、MDA，使SOD活性下降，从而引起其氧化损伤及损伤后细胞修复变化。 3还原型谷胱甘肽可以使CSE刺激后的A549细胞产生的MDA、TGF-β1减少，SOD增加，对吸烟引起的氧化损伤有一定逆转作用。
[Abstract]:Objective: cigarette smoke contains a variety of harmful chemicals, which is one of the main causes of various diseases and is recognized as an important risk factor for pulmonary fibrosis. Oxidizing agents and free radicals in cigarette smoke can produce oxidative stress through a variety of pathways, and a large number of evidence indicate that oxidative stress occurs in the pathogenesis of pulmonary fibrosis. The mechanism plays an important role, in both acute and remission phase, the patient has oxidation / antioxidant imbalance, but its molecular mechanism is not clear. There is no special therapeutic method for pulmonary interstitial fibrosis in clinical. Recently, many researchers pay more attention to antioxidant therapy. The research shows that reduced glutathione (GSH) is a machine. An important reductant in the body has antioxidant effects, which can remove free radicals and other active oxygen clusters and protect lung tissue from oxidative damage. Therefore, this experiment uses A549 cells derived from alveolar type II epithelial cells as a model to stimulate A549 cells cultured in vitro by different concentrations of cigarette smoke extract (CSE) and observe the cells. The possible mechanism of smoking on pulmonary fibrosis and the reverse effect of glutathione on pulmonary interstitial fibrosis caused by smoking were examined by detecting the indexes of oxidation and the degree of fibrosis in the supernatant of the cell.
Methods: A549 cells derived from alveolar type II epithelial cells were cultured in vitro, and the cells in the logarithmic growth period were tested. Cigarette extracts were prepared through special devices with reference to the method of Carp and Janoff[1]. 2 cigarettes were drawn to filter the cigarette mouth, and the smoke was incorporated into the 1640 culture medium without serum, and the pH value was adjusted and the bacteria and large particles were removed. Then diluted to different concentrations, 30min was used in the experiment. First of all, we tried to use 0%, 2.5%, 5%, 7.5%, 10%, 12.5% concentration of cigarette extract culture medium to determine cell viability by MTT method, and observe the effect of CSE on the cell proliferation by phase contrast microscope. The experiment was divided into blank control group, 2.5%CSE group, 5%CSE group, 7.5%CSE group and 7.5%CSE+GSH group (GSH1mmol/L). The supernatant of cell supernatant superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in cell supernatant were measured by colorimetric analysis and ELISA method was used to detect the transformation of cell supernatant. The level of growth factor 1 (TGF- beta 1).
Results: 1 the morphological changes of A549 cells under different concentrations of CSE were observed under inverted microscope.
The results of inverted microscope showed that the growth density of A549 cells decreased after CSE, the gap between cells increased, floating cells increased, and the adherent cells also appeared to shrink and round and atrophy. The difference between the cells of the control group and the cell morphology of the control group was obviously inhibited by the different concentrations of.2 and CSE on the growth of A549 cells.
The MTT colorimetric assay showed that CSE had a strong inhibitory effect on the proliferation of A549 cells in vitro, and the inhibitory effect was dosed and time dependent. After the effect of the same concentration of cigarette extracts at different time (12,24,36h), the inhibition rate increased with the time of action (P0.05). The same time (0%, 2.5%, 5%, 7). .5%, 10%, 12.5%) after the effect of cigarette extract, the inhibitory rate increased with the increase of concentration (P0.05). The inhibition rate was 70.30 + 2.21% in 10%CSE group 24h, and the inhibition rate of CSE in 12.5%CSE group was up to 90.10 + 1.01%.3 and CSE on A549 cell supernatant TGF- beta 1, MDA, SOD level
Different concentrations of CSE group TGF- beta 1, MDA was significantly higher than the control group, the difference was statistically significant (P0.05), and the higher the concentration of CSE, the higher the content of each factor, the concentration dependence, the difference was statistically significant (P0.05). The activity of SOD in the CSE group of different concentrations was lower than that of the control group, the difference was statistically significant (P0.05), and the higher the CSE concentration was, the lower the SOD activity was, the difference was the difference. The change of TGF- TGF- 1, MDA and SOD levels in A549 cell supernatant after P0.05.4GSH treatment was statistically significant.
The contents of TGF- beta 1 and MDA in group 7.5%CSE+GSH were significantly lower than those in group 7.5%CSE (P0.05), while SOD activity was higher than that in 7.5%CSE group (P0.05).
Conclusion:
1 cigarette extract inhibited the growth of alveolar epithelial cells in a dose and time dependent manner.
2 cigarette extract can induce alveolar epithelial cells to secrete TGF- beta 1 and MDA, resulting in a decrease in SOD activity, resulting in oxidative damage and cell repair after injury.
3 reduced glutathione can reduce the production of MDA and TGF- beta 1 from A549 cells stimulated by CSE, and increase SOD, which may reverse the oxidative injury caused by smoking.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R563

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