烟草烟雾对人巨噬细胞超微结构的影响及红霉素的作用

发布时间：2017-12-28 16:11

本文关键词：烟草烟雾对人巨噬细胞超微结构的影响及红霉素的作用　出处：《广西医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的探讨烟草烟雾对人巨噬细胞超微结构的影响及红霉素(Erythromycin, EM)的作用。方法体外培养人类单核细胞系U937细胞,用佛波脂(Phorbol 12-myristate 13-acetate,PMA,10ng/ml)诱导分化人巨噬细胞,用烟草烟雾提取物(Cigarette Smoke Extract,CSE)处理人巨噬细胞或EM预处理后用CSE刺激人巨噬细胞,(一)将细胞分组：①空白对照组；②CSE组(加入1%CSE孵育24小时)；③CSE+EM24h组(1μg/ml红霉素预孵育24h后,加入1%CSE继续孵育24小时);④CSE+EM48h组(1μg/ml红霉素预孵育48h后,加入1%CSE继续孵育24小时)。(二)取孵育后各组细胞经预包埋、固定、漂洗、脱水、渗透、聚合、修块、切片后,经醋酸铀及柠檬酸铅染色后,用电子显微镜观察各处理组细胞超微结构。(三)取各组上清液分别离心去除沉淀后,避光加入2’,7’_二氯二氢荧光素二乙酯(2’,7'-dichlorofluorescin-diacetat, DCFH-DA),使其浓度为10uM,37℃下避光孵育30分钟后,立即予荧光酶标仪检测各组荧光强度。结果(一)各组人巨噬细胞超微结构的观察空白对照组：细胞圆或椭圆形,细胞胞质内可见少许线粒体、粗面内质网及溶酶体。绝大多数表面有许多微绒毛样突起,核偏心位,呈圆形或椭圆形,少数呈不规则形,染色质聚积于核膜下。CSE组：与空白对照组比较,烟草烟雾组中细胞胞质内不仅可见溶酶体、线粒体、内质网数量明显增多,亦出现线粒体嵴排列紊乱,嵴断裂逐渐崩解形成空泡(线粒体空泡化改变)、线粒体基质电子密度增高浓聚,次级溶酶体与初级溶酶体同时存在,甚至可见多个溶酶体聚集融合、粗面内质网轻度扩张的、细胞内基质较浓聚,即粗面内质网脱颗粒现象较明显。CSE+EM组：与CSE组比较,线粒体数量减少,未见空泡化改变,溶酶体数量减少,未见次级溶酶体,内质网数量减少、无扩张,粗面内质网脱颗粒现象较CSE组减少。(二)CSE刺激及EM干预对人巨噬细胞活性氧(Reactive Oxygen Species,ROS)释放量的影响：CSE组ROS释放量明显高于空白对照组(P0.05),CSE+EM24h组ROS释放量较CSE组减少(P0.05),CSE+EM48h组ROS释放量明显减少(P0.05)。结论1、烟草烟雾刺激人巨噬细胞引起细胞超微结构的改变,表现为线粒体的肿胀及空泡化改变,溶酶体数量的增多,出现多个溶酶体聚集融合,粗面内质网扩张及脱颗粒现象；而红霉素预处理后可改善线粒体肿胀及空泡化改变,溶酶体数量减少,粗面内质网未见扩张,粗面内质网脱颗粒现象减少。2、烟草烟雾刺激人巨噬细胞释放活性氧明显增多,红霉素预处理后可降低活性氧释放量,提示可能是通过保护线粒体功能而抑制ROS的释放,这可能是红霉素抗氧化应激作用的机理。
[Abstract]:Objective to investigate the effect of tobacco smoke on the ultrastructure of human macrophages and the effect of Erythromycin (EM). The method of in vitro human monocytic U937 cells cultured with phorbol ester (Phorbol 12-myristate, 13-acetate, PMA, 10ng/ml) induced differentiation of human macrophages with cigarette smoke extract (Cigarette Smoke, Extract, CSE) treatment of human macrophages or EM after pretreatment with CSE stimulated macrophages, (a) were divided into four groups: blank control group II; group CSE (adding 1%CSE incubated for 24 hours); group CSE+EM24h (1 g/ml erythromycin pre incubated 24h, add 1%CSE to 24 h of incubation); the CSE+EM48h group (1 g/ml erythromycin pre incubated 48h, add 1%CSE to incubated for 24 hours). (two) after incubation, the cells in each group were pre embedded, fixed, rinsed, dehydrated, permeated, polymerized, repaired and sliced, then stained with uranyl and lead citrate, and the ultrastructure of cells in each treatment group was observed by electron microscope. (three) from the supernatant were removed by centrifugation after precipitation, light with 2 ', 7' _ two chloro two ethyl two hydrogen fluorescence (2, 7'-dichlorofluorescin-diacetat, DCFH-DA), the concentration of 10uM, 37 DEG C under light after 30 min of incubation, immediately to the fluorescence microplate fluorescence intensity detected. Results (1) the ultrastructure of macrophages in each group was observed in blank control group: round or oval cells, and a few mitochondria, rough endoplasmic reticulum and lysosome in cytoplasm. Most of the surface there are many microvilli, nuclear eccentric, round or oval, a few irregular chromatin accumulation in the nuclear membrane. CSE group: compared with the control group, the cell group in tobacco smoke matter not only visible lysosomes, mitochondria and endoplasmic reticulum also significantly increased the number of mitochondrial cristae disordered cristae gradually disintegrated vacuoles (change mitochondrial vacuolation), mitochondrial matrix electron density increased concentration, there are secondary lysosomes and primary lysosomes at the same time, even more than the visible lysosomes and endoplasmic reticulum aggregation and fusion of mild dilatation and intracellular matrix concentration, i.e. the degranulation of rough endoplasmic reticulum is evident. Group CSE+EM: compared with group CSE, the number of mitochondria decreased, no vacuolation changed, the number of lysosomes decreased, no secondary lysosomes were found, the number of endoplasmic reticulum decreased, no expansion was found, and rough granular endoplasmic reticulum degranulation was reduced compared with group CSE. (two) the effect of CSE stimulation and EM intervention on the release of Reactive Oxygen Species (ROS) in human macrophages: the ROS release in CSE group was significantly higher than that in the blank control group (P0.05), the release of CSE+EM24h in CSE+EM24h group was less than that in the control group, and the release rate of group B was significantly reduced. Due to the change of ultrastructure of human macrophage stimulating conclusion 1, tobacco smoke, showed altered mitochondrial swelling and vacuolization, increase the number of lysosomes, multiple aggregation lysosome fusion, rough endoplasmic reticulum expansion and degranulation; while erythromycin preconditioning may improve mitochondrial swelling and vacuolization, reduce the number of lysosomes, no rough endoplasmic reticulum expansion, reduced rough endoplasmic reticulum degranulation. 2, tobacco smoke stimulates the release of reactive oxygen species from human macrophages. Erythromycin can decrease the release of reactive oxygen species after pretreatment. It suggests that the release of ROS may be inhibited by the protection of mitochondrial function. This may be the mechanism of erythromycin anti oxidative stress.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563.9

【相似文献】