發(fā)布時間：2018-05-19 02:03

  本文選題：脂聯(lián)素球形結構域(gAd) + 煙草煙霧提取物　； 參考：《山西醫(yī)科大學》2014年碩士論文

【摘要】：研究背景 肺血管內(nèi)皮細胞損傷目前被認為是慢性阻塞性肺疾病(Chronic Cbstructive Pulmonary Disease)重要發(fā)病機制之一。吸煙作為COPD最重要的危險因素,可以通過直接作用或通過炎癥介質的釋放等多種方式損傷血管內(nèi)皮細胞。脂聯(lián)素是由脂肪細胞分泌的一種具有多種生物學活性的脂肪因子,循環(huán)血中的脂聯(lián)素以三聚體、六聚體、高等分子量體(high molecular weight HMW)以及球形結構域(globular adiponectin gAd)等多種構型存在。Nakanishi等人的研究表明脂聯(lián)素敲除大鼠表現(xiàn)出隨年齡進展的肺氣腫,認為肺血管內(nèi)皮細胞的凋亡是其可能的發(fā)病機制之一。目前關于gAd的研究,在不同的細胞系,有些表現(xiàn)為破壞作用,有些表現(xiàn)為保護作用。本研究通過觀察不同濃度gAd對煙草煙霧提取物(cigarette smoke extract CSE)誘導的血管內(nèi)皮細胞凋亡水平、炎癥因子以及氧化物質表達的影響,探索gAd在煙草煙霧提取物誘導的血管內(nèi)皮細胞損傷中的作用。 目的 通過研究脂聯(lián)素球形結構域(globular adiponectin gAd)預處理后煙草煙霧提取物(cigarette smoke extract CSE)誘導的血管內(nèi)皮細胞凋亡水平、炎癥因子以及氧化物質表達水平的變化,探討gAd在煙草煙霧提取物誘導的血管內(nèi)皮細胞損傷中的作用。 方法 選取人臍靜脈內(nèi)皮細胞株(human umbilical vein endothelial cells HUVEC)CRL-1730,完成以下實驗。 (1)CCK-8測定CSE對HUVEC的毒性作用： 細胞用含10%胎牛血清的高糖DMEM培養(yǎng)基常規(guī)培養(yǎng),待細胞生長至80%融合時,0.25%胰酶-EDTA消化,離心,重懸,調(diào)整細胞濃度,以4000個細胞/100μE濃度接種于96孔板,1001μL/孔,用加有等量細胞培養(yǎng)液和CCK-8溶液的孔做空白對照。待細胞生長至70%融合時更換含有不同濃度CSE的培養(yǎng)基,CSE濃度為0%,1%,2.5%,5%,7.5%,10%,繼續(xù)培養(yǎng)24小時,培養(yǎng)結束后,每孔加入10μL CCK-8溶液,在細胞培養(yǎng)箱內(nèi)孵育1小時,酶標儀測定450nm處吸光度值。 (2)不同濃度gAd預處理對CSE誘導的HUVEC凋亡水平、炎癥因子(白介素-6、腫瘤壞死因子-α)以及氧化物質(4-羥基壬烯醛)表達的影響 細胞常規(guī)培養(yǎng)于含10%胎牛血清的高糖DMEM培養(yǎng)基中,待細胞生長至80%融合時,0.25%胰酶-EDTA消化,離心,重懸,以1×105/孔接種于12孔板,取對數(shù)生長期細胞,更換含有不同濃度gAd (0.1μg/ml,0.5μg/ml,2.5μg/ml,5μg/ml,10μg/ml,20μg/ml)的培養(yǎng)基預孵育4小時,隨后加入CSE共同作用24h,使CSE終濃度為5%,收集細胞,用AnnexinV-FITC標記后流式細胞檢測的方法觀察細胞的凋亡水平變化；收集細胞培養(yǎng)的上清液,用ELISA法檢測上清液中炎癥因子白細胞介素-6(IL-6)、腫瘤壞死因子α(TNF-a)以及氧化物質4-羥基壬烯醛(4-HNE)水平。 結果 1.CSE對血管內(nèi)皮細胞的毒性作用 不同濃度CSE干預HUVEC,細胞活性下降,與空白對照組相比,差異有統(tǒng)計學意義(F=180.620,P0.05)；細胞活性與CSE濃度變化呈顯著負相關,隨著CSE濃度的升高,細胞活性逐漸下降(r值為-0.986,P0.05),使細胞活性降低為空白對照組一半的CSE濃度約為5%。 2.5%CSE對血管內(nèi)皮細胞凋亡水平以及IL-6、TNF-α以及4-HNE的影響： 5%濃度CSE作用于內(nèi)皮細胞,細胞凋亡率(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,與空白對照相比(3.597±0.586%,23.994±4.509pg/ml,76.378±4.663pg/ml,0.865±0.110μg/ml),差異均有統(tǒng)計學意義(t值分別為-64.638，，-15.158,-29.176,-30.482,P均0.05)。 3.gAd預處理對5%CSE誘導的血管內(nèi)皮細胞凋亡水平以及IL-6、TNF-α以及4-HNE的影響： 給予gAd預處理后,細胞凋亡水平、IL-6、TNF-α以及4-HNE水平均下降,差異均有統(tǒng)計學意義(F值分別為74.295,50.630,64.012,61.822；P均0.05)。 4.相關性分析： gAd濃度10μg/ml范圍內(nèi),gAd濃度與細胞凋亡率、IL-6、TNF-α水平以及4-HNE水平變化呈顯著負相關(r值分別為-0.794,-0.871,-0.859,-0.897,p均0.05),隨著gAd濃度的升高,細胞凋亡水平以及上述各因子水平逐漸降低,gAd濃度為20μg/ml時,細胞凋亡水平以及上述各因子水平與5%CSE+10μg/ml gAd組相比升高,差異均有統(tǒng)計學意義(P均0.05)。 結論 1.煙草煙霧提取物對血管內(nèi)皮細胞有損傷作用,且其損傷作用呈劑量依賴性； 2.gAd在10μg/ml范圍內(nèi)對煙草煙霧提取物誘導的血管內(nèi)皮細胞損傷發(fā)揮保護作用,且呈劑量依賴性；但當gAd濃度為20μg/ml時,其保護作用下降。 3.脂聯(lián)素球形結構域對煙草煙霧提取物誘導的血管內(nèi)皮細胞損傷具有保護作用,而且與其濃度密切相關。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R563.9

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