氧化石墨烯对大鼠角膜上皮细胞致损伤作用的初步研究

发布时间：2018-05-06 16:26

本文选题：氧化石墨烯 + 角膜上皮细胞　；参考：《兰州大学》2017年硕士论文

【摘要】：石墨烯是目前人们已知的最薄材料,它是由单层的碳原子以sp2杂化轨道组成的蜂窝状二维碳纳米材料,氧化石墨烯作为众多石墨烯衍生物中的一种,也受到相当大的关注。近年来随着石墨烯及其衍生物的大规模生产和应用,它的生物安全问题也备受关注。本实验选用氧化石墨烯作为干扰材料,模式生物为标准体重的Wistar雌鼠,通过培养大鼠角膜上皮组织,获得原代大鼠角膜上皮细胞;通过MTT法检测正常细胞的细胞活性并进行曲线拟合;当细胞培养至对数生长期,分别加入浓度为0μg/mL、5μg/mL、10μg/m L、20μg/m L、50μg/m L、100μg/m L的氧化石墨烯悬浊液与大鼠角膜上皮细胞进行共培养,之后选取适宜浓度的氧化石墨烯干预细胞培养24 h、48 h、72 h,通过MTT法检测不同条件下细胞的活性变化;通过Hoechst33258、HE染色对细胞的外观形态以及核质变化进行观察,观察正常细胞组及干预组细胞的形态变化;流式细胞术检测细胞的凋亡/死亡分布;通过DAPI/PI双染以及台盼兰染色确定正常组和干预组死亡细胞的比例;流式细胞术检测正常细胞组及干预组的细胞周期变化。结果显示:正常的大鼠角膜上皮细胞贴壁后呈翼状分布,密度增大后细胞主要以梭形排列,核质均匀无明显分界;Hoechst33258染色后细胞核呈现圆形或椭圆形,边缘光滑,大小均一;正常细胞曲线呈“S”型,铺板后26 h进入对数生长期,43 h后细胞密度可达到80%;加入氧化石墨烯干预后,细胞延展性降低,形态出现明显皱缩,伪足部分呈现纤维状;染色后发现细胞核的大小差异明显,核边缘部分有类似破损状,核内容物溢出;经流式细胞术检测,正常细胞的比例明显降低,而破损、死亡细胞的数目明显增多;通过DAPI/PI双染以及台盼兰染色,初步推测细胞活性的降低主要由细胞死亡导致而非凋亡引起;HE染色发现,氧化石墨烯能够穿透细胞膜进入细胞并残留,细胞表面也有氧化石墨烯附着;检测细胞周期后,发现氧化石墨烯干预后的角膜上皮细胞会产生S期及G2/M期阻滞,细胞碎片大量增多。以上结果表明:浓度在5μg/m L—100μg/m L范围内的氧化石墨烯对大鼠角膜上皮细胞具有明显的致损伤作用,提示氧化石墨烯对大鼠角膜上皮细胞的生物安全性存在隐患。
[Abstract]:Graphene is the thinnest material known at present. It is a honeycomb two-dimensional carbon nanomaterial composed of monolayer carbon atoms with sp2 hybrid orbitals. Graphene oxide as one of many graphene derivatives has also received considerable attention. In recent years, with the large-scale production and application of graphene and its derivatives, the biosafety of graphene has attracted much attention. In this experiment, graphene oxide was used as interference material and model organism was used as standard weight Wistar female rat. Primary rat corneal epithelial cells were obtained by cultured rat corneal epithelium. The cell activity of normal cells was measured by MTT method and the curve fitting was carried out. When the cells were cultured to logarithmic growth stage, 10 渭 g / mL 10 渭 g / mL of graphene oxide suspension solution with 100 渭 g / mL of graphene oxide was co-cultured with the corneal epithelial cells of rats when the cells were cultured to logarithmic growth stage, and the concentration of 10 渭 g / mL was 20 渭 g / m L ~ (-1) or 50 渭 g 路m ~ (L) ~ (-1). Then the suitable concentration of graphene oxide was selected to interfere with cell culture for 24 h or 48 h or 72 h, and the changes of cell activity under different conditions were detected by MTT assay, and the morphology and nuclear and cytoplasmic changes of cells were observed by Hoechst33258 he staining. The morphologic changes of normal cells and intervention groups were observed, apoptosis / death distribution was detected by flow cytometry, the proportion of dead cells in normal and intervention groups was determined by DAPI/PI double staining and Trypan blue staining. The changes of cell cycle in normal cell group and intervention group were detected by flow cytometry. The results showed that the corneal epithelial cells of normal rats showed winglike distribution after adhering to the wall, the cells were mainly arranged in fusiform shape after the density increased, and the nuclei were round or elliptical after the homogeneous and homogeneous Hoechst33258 staining, the edges were smooth and the size was uniform. The normal cell curve was "S", and the cell density could reach 80 after 26 hours of logarithmic growth and 43 hours after the addition of graphene oxide, the cell ductility was decreased, the shape of the pseudopodid was obviously crumpled, and the pseudopodid part was fibrous. It was found that the size of the nucleus varied significantly after staining, with similar damage to the edge of the nucleus and overflow of the nuclear contents, and the percentage of normal cells was significantly decreased, but the number of damaged and dead cells was increased significantly by flow cytometry (FCM). By means of DAPI/PI double staining and Trypan blue staining, it was preliminarily assumed that the decrease of cell activity was mainly caused by cell death rather than apoptosis. It was found that graphene oxide could penetrate the cell membrane and remain. The cell surface was also attached to graphene oxide, and after cell cycle detection, it was found that the corneal epithelial cells treated with graphene oxide could produce S phase and G 2 / M phase arrest, and a large number of cell fragments would be increased. The above results showed that graphene oxide at the concentration of 5 渭 g / mL -100 渭 g / mL had a significant damage effect on rat corneal epithelial cells, suggesting that graphene oxide had a hidden danger to the biological safety of rat corneal epithelial cells.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O613.71;R772.2

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