發(fā)布時(shí)間：2018-05-06 16:26

  本文選題：氧化石墨烯 + 角膜上皮細(xì)胞�。� 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：石墨烯是目前人們已知的最薄材料,它是由單層的碳原子以sp2雜化軌道組成的蜂窩狀二維碳納米材料,氧化石墨烯作為眾多石墨烯衍生物中的一種,也受到相當(dāng)大的關(guān)注。近年來隨著石墨烯及其衍生物的大規(guī)模生產(chǎn)和應(yīng)用,它的生物安全問題也備受關(guān)注。本實(shí)驗(yàn)選用氧化石墨烯作為干擾材料,模式生物為標(biāo)準(zhǔn)體重的Wistar雌鼠,通過培養(yǎng)大鼠角膜上皮組織,獲得原代大鼠角膜上皮細(xì)胞;通過MTT法檢測正常細(xì)胞的細(xì)胞活性并進(jìn)行曲線擬合;當(dāng)細(xì)胞培養(yǎng)至對數(shù)生長期,分別加入濃度為0μg/mL、5μg/mL、10μg/m L、20μg/m L、50μg/m L、100μg/m L的氧化石墨烯懸濁液與大鼠角膜上皮細(xì)胞進(jìn)行共培養(yǎng),之后選取適宜濃度的氧化石墨烯干預(yù)細(xì)胞培養(yǎng)24 h、48 h、72 h,通過MTT法檢測不同條件下細(xì)胞的活性變化;通過Hoechst33258、HE染色對細(xì)胞的外觀形態(tài)以及核質(zhì)變化進(jìn)行觀察,觀察正常細(xì)胞組及干預(yù)組細(xì)胞的形態(tài)變化;流式細(xì)胞術(shù)檢測細(xì)胞的凋亡/死亡分布;通過DAPI/PI雙染以及臺盼蘭染色確定正常組和干預(yù)組死亡細(xì)胞的比例;流式細(xì)胞術(shù)檢測正常細(xì)胞組及干預(yù)組的細(xì)胞周期變化。結(jié)果顯示:正常的大鼠角膜上皮細(xì)胞貼壁后呈翼狀分布,密度增大后細(xì)胞主要以梭形排列,核質(zhì)均勻無明顯分界;Hoechst33258染色后細(xì)胞核呈現(xiàn)圓形或橢圓形,邊緣光滑,大小均一;正常細(xì)胞曲線呈“S”型,鋪板后26 h進(jìn)入對數(shù)生長期,43 h后細(xì)胞密度可達(dá)到80%;加入氧化石墨烯干預(yù)后,細(xì)胞延展性降低,形態(tài)出現(xiàn)明顯皺縮,偽足部分呈現(xiàn)纖維狀;染色后發(fā)現(xiàn)細(xì)胞核的大小差異明顯,核邊緣部分有類似破損狀,核內(nèi)容物溢出;經(jīng)流式細(xì)胞術(shù)檢測,正常細(xì)胞的比例明顯降低,而破損、死亡細(xì)胞的數(shù)目明顯增多;通過DAPI/PI雙染以及臺盼蘭染色,初步推測細(xì)胞活性的降低主要由細(xì)胞死亡導(dǎo)致而非凋亡引起;HE染色發(fā)現(xiàn),氧化石墨烯能夠穿透細(xì)胞膜進(jìn)入細(xì)胞并殘留,細(xì)胞表面也有氧化石墨烯附著;檢測細(xì)胞周期后,發(fā)現(xiàn)氧化石墨烯干預(yù)后的角膜上皮細(xì)胞會產(chǎn)生S期及G2/M期阻滯,細(xì)胞碎片大量增多。以上結(jié)果表明:濃度在5μg/m L—100μg/m L范圍內(nèi)的氧化石墨烯對大鼠角膜上皮細(xì)胞具有明顯的致?lián)p傷作用,提示氧化石墨烯對大鼠角膜上皮細(xì)胞的生物安全性存在隱患。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O613.71;R772.2

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