脂联素球形结构域对烟草烟雾提取物诱导的血管内皮细胞损伤的作用

发布时间：2018-05-19 02:03

本文选题：脂联素球形结构域(gAd) + 烟草烟雾提取物　；参考：《山西医科大学》2014年硕士论文

【摘要】：研究背景肺血管内皮细胞损伤目前被认为是慢性阻塞性肺疾病(Chronic Cbstructive Pulmonary Disease)重要发病机制之一。吸烟作为COPD最重要的危险因素,可以通过直接作用或通过炎症介质的释放等多种方式损伤血管内皮细胞。脂联素是由脂肪细胞分泌的一种具有多种生物学活性的脂肪因子,循环血中的脂联素以三聚体、六聚体、高等分子量体(high molecular weight HMW)以及球形结构域(globular adiponectin gAd)等多种构型存在。Nakanishi等人的研究表明脂联素敲除大鼠表现出随年龄进展的肺气肿,认为肺血管内皮细胞的凋亡是其可能的发病机制之一。目前关于gAd的研究,在不同的细胞系,有些表现为破坏作用,有些表现为保护作用。本研究通过观察不同浓度gAd对烟草烟雾提取物(cigarette smoke extract CSE)诱导的血管内皮细胞凋亡水平、炎症因子以及氧化物质表达的影响,探索gAd在烟草烟雾提取物诱导的血管内皮细胞损伤中的作用。目的通过研究脂联素球形结构域(globular adiponectin gAd)预处理后烟草烟雾提取物(cigarette smoke extract CSE)诱导的血管内皮细胞凋亡水平、炎症因子以及氧化物质表达水平的变化,探讨gAd在烟草烟雾提取物诱导的血管内皮细胞损伤中的作用。方法选取人脐静脉内皮细胞株(human umbilical vein endothelial cells HUVEC)CRL-1730,完成以下实验。 (1)CCK-8测定CSE对HUVEC的毒性作用：细胞用含10%胎牛血清的高糖DMEM培养基常规培养,待细胞生长至80%融合时,0.25%胰酶-EDTA消化,离心,重悬,调整细胞浓度,以4000个细胞/100μE浓度接种于96孔板,1001μL/孔,用加有等量细胞培养液和CCK-8溶液的孔做空白对照。待细胞生长至70%融合时更换含有不同浓度CSE的培养基,CSE浓度为0%,1%,2.5%,5%,7.5%,10%,继续培养24小时,培养结束后,每孔加入10μL CCK-8溶液,在细胞培养箱内孵育1小时,酶标仪测定450nm处吸光度值。 (2)不同浓度gAd预处理对CSE诱导的HUVEC凋亡水平、炎症因子(白介素-6、肿瘤坏死因子-α)以及氧化物质(4-羟基壬烯醛)表达的影响细胞常规培养于含10%胎牛血清的高糖DMEM培养基中,待细胞生长至80%融合时,0.25%胰酶-EDTA消化,离心,重悬,以1×105/孔接种于12孔板,取对数生长期细胞,更换含有不同浓度gAd (0.1μg/ml,0.5μg/ml,2.5μg/ml,5μg/ml,10μg/ml,20μg/ml)的培养基预孵育4小时,随后加入CSE共同作用24h,使CSE终浓度为5%,收集细胞,用AnnexinV-FITC标记后流式细胞检测的方法观察细胞的凋亡水平变化；收集细胞培养的上清液,用ELISA法检测上清液中炎症因子白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-a)以及氧化物质4-羟基壬烯醛(4-HNE)水平。结果 1.CSE对血管内皮细胞的毒性作用不同浓度CSE干预HUVEC,细胞活性下降,与空白对照组相比,差异有统计学意义(F=180.620,P0.05)；细胞活性与CSE浓度变化呈显著负相关,随着CSE浓度的升高,细胞活性逐渐下降(r值为-0.986,P0.05),使细胞活性降低为空白对照组一半的CSE浓度约为5%。 2.5%CSE对血管内皮细胞凋亡水平以及IL-6、TNF-α以及4-HNE的影响： 5%浓度CSE作用于内皮细胞,细胞凋亡率(43.673±0.900%)增加,IL-6(196.265±19.161pg/ml)、TNF-α(297.410±12.232pg/ml)以及4-HNE(5.756±0.255μg/ml)水平升高,与空白对照相比(3.597±0.586%,23.994±4.509pg/ml,76.378±4.663pg/ml,0.865±0.110μg/ml),差异均有统计学意义(t值分别为-64.638，，-15.158,-29.176,-30.482,P均0.05)。 3.gAd预处理对5%CSE诱导的血管内皮细胞凋亡水平以及IL-6、TNF-α以及4-HNE的影响：给予gAd预处理后,细胞凋亡水平、IL-6、TNF-α以及4-HNE水平均下降,差异均有统计学意义(F值分别为74.295,50.630,64.012,61.822；P均0.05)。 4.相关性分析： gAd浓度10μg/ml范围内,gAd浓度与细胞凋亡率、IL-6、TNF-α水平以及4-HNE水平变化呈显著负相关(r值分别为-0.794,-0.871,-0.859,-0.897,p均0.05),随着gAd浓度的升高,细胞凋亡水平以及上述各因子水平逐渐降低,gAd浓度为20μg/ml时,细胞凋亡水平以及上述各因子水平与5%CSE+10μg/ml gAd组相比升高,差异均有统计学意义(P均0.05)。结论 1.烟草烟雾提取物对血管内皮细胞有损伤作用,且其损伤作用呈剂量依赖性； 2.gAd在10μg/ml范围内对烟草烟雾提取物诱导的血管内皮细胞损伤发挥保护作用,且呈剂量依赖性；但当gAd浓度为20μg/ml时,其保护作用下降。 3.脂联素球形结构域对烟草烟雾提取物诱导的血管内皮细胞损伤具有保护作用,而且与其浓度密切相关。
[Abstract]:Research background
The injury of pulmonary vascular endothelial cells is now considered as one of the important pathogenesis of Chronic Cbstructive Pulmonary Disease. Smoking is the most important risk factor for COPD, which can damage vascular endothelial cells by direct action or through the release of inflammatory mediators. Adiponectin is divided into adipocytes. An adipokine with many biological activities, and the existence of adiponectin in circulating blood with triamomers, six polymer, higher molecular weight body (high molecular weight HMW), and spherical domain (globular adiponectin gAd) in the presence of.Nakanishi et al. Shows that adiponectin knockout rats show the lung of the lung that progresses with age. The apoptosis of the pulmonary vascular endothelial cells is one of the possible mechanisms of the pathogenesis of the pulmonary vascular endothelial cells. At present, the study of gAd, in different cell lines, appears to be destructive, some of which are protective. This study was conducted by observing different concentrations of gAd on tobacco smoke extract (cigarette smoke extract CSE) induced vascular endothelial cell withering. Effects of levels of inflammation, cytokines and expression of oxidant on gAd induced injury of vascular endothelial cells induced by tobacco smoke extract were explored.
objective
The role of gAd in vascular endothelial cell injury induced by tobacco smoke extract was investigated by studying the changes in the level of apoptosis, inflammatory factors and the expression level of oxidizing substances induced by globular adiponectin gAd pretreated tobacco smoke extract (cigarette smoke extract CSE).
Method
The human umbilical vein endothelial cell line (human umbilical vein endothelial cells HUVEC) CRL-1730 was selected to complete the following experiments.
(1) CCK-8 determination of the toxic effect of CSE on HUVEC:
The cells were cultured in a high glucose DMEM medium containing 10% fetal bovine serum. When the cells grew to 80% fusion, 0.25% pancreatin -EDTA was digested, centrifuged, and suspended, and the cell concentration was adjusted. 4000 cells were inoculated to 96 orifice and 1001 mu L/ with the concentration of /100 mu E, and the cells were added with the same amount of cell culture solution and the CCK-8 solution in the blank control. The cell growth was 70% thawing. The medium with different concentrations of CSE was changed in time. The concentration of CSE was 0%, 1%, 2.5%, 5%, 7.5%, 10%, and continued to be cultured for 24 hours. After the culture, 10 mu L CCK-8 solution was added to each hole, and incubated for 1 hours in the cell culture box, and the absorbance value at 450nm was measured by the enzyme labelling instrument.
(2) the effect of gAd pretreatment on CSE induced HUVEC apoptosis, inflammatory factors (IL -6, TNF - alpha) and the expression of 4- hydroxyl nonylaldehyde (4- hydroxy nonylaldehyde)
Cells were routinely cultured in high glucose DMEM medium containing 10% fetal bovine serum. When cells grew to 80% fusion, 0.25% pancreatin -EDTA was digested, centrifuged, and suspended, 1 x 105/ holes were inoculated on 12 orifice plates, and logarithmic growth period cells were taken to replace the incubation medium containing different concentrations of gAd (0.1 Mu, 0.5, g/ml, 2.5, g/ml, 5 u g/ml, 10 u g/ml, 20 g/ml). Then, 24h was subsequently added to CSE, the final concentration of CSE was 5%, cells were collected, and the apoptotic level of cells was observed by AnnexinV-FITC labeled flow cytometry, the supernatant of cell culture was collected, and ELISA method was used to detect the inflammatory factors of interleukin -6 (IL-6), tumor necrosis factor alpha (TNF-a) and oxidation substance (4) in the supernatant. - hydroxyl NONYLIC aldehyde (4-HNE) level.
Result
Toxic effect of 1.CSE on vascular endothelial cells
Different concentrations of CSE intervention HUVEC, cell activity decreased, compared with the blank control group, the difference was statistically significant (F=180.620, P0.05), cell activity was significantly negatively correlated with the concentration of CSE. With the increase of CSE concentration, the cell activity gradually decreased (r value was -0.986, P0.05), and the cell activity reduced to half of the control group was about 5 of the CSE concentration. It is.
Effects of 2.5%CSE on the apoptosis of vascular endothelial cells and IL-6, TNF-, and 4-HNE:
5% concentration of CSE acted on endothelial cells, increased the rate of apoptosis (43.673 + 0.900%), IL-6 (196.265 + 19.161pg/ml), TNF- alpha (297.410 + 12.232pg/ml) and 4-HNE (5.756 + 0.255, g/ml). Compared with the blank control (3.597 + 0.586%, 23.994 + 4.509pg/ml, 76.378 + 4.663pg/ml, 0.865 + 0.110 mu g/ml), the difference was statistically significant (T score). Don't be -64.638, -15.158, -29.176, -30.482, P 0.05).
Effects of 3.gAd pretreatment on 5%CSE induced apoptosis of vascular endothelial cells and IL-6, TNF- alpha and 4-HNE:
After gAd preconditioning, the level of apoptosis, IL-6, TNF- alpha and 4-HNE were all decreased, and the difference was statistically significant (F value was 74.295,50.630,64.012,61.822, P was 0.05).
4. correlation analysis:
There was a significant negative correlation between the concentration of gAd and the rate of apoptosis, IL-6, TNF- alpha and the level of 4-HNE (r value was -0.794, -0.871, -0.859, -0.897, P are 0.05). The level of apoptosis and the level of the above factors gradually decreased with the increase of concentration of gAd, and the level of apoptosis and the level of apoptosis at the concentration of 20 mu. The level of each factor was higher than that of 5%CSE+10 g/ml gAd group, and the difference was statistically significant (P 0.05).
conclusion
1. tobacco smoke extract has a damaging effect on vascular endothelial cells, and its damage is dose-dependent.
2.gAd has a protective effect on the vascular endothelial cell injury induced by tobacco smoke extract in the range of 10 micron g/ml, and is dose-dependent, but the protective effect of gAd is decreased when the concentration is 20 g/ml.
3. the adiponectin spherical domain plays a protective role in the injury of vascular endothelial cells induced by tobacco smoke extract, and is closely related to its concentration.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R563.9

【共引文献】