单壁碳纳米角抑制人胶质瘤细胞增殖与促进其凋亡的作用机制研究

发布时间：2018-04-17 08:42

本文选题：单壁碳纳米角 + 胶质瘤细胞　；参考：《中国人民解放军医学院》2015年博士论文

【摘要】：目的：研究单壁碳纳米角(SWNHs)是通过何种途径来对人胶质瘤细胞的增殖过程进行抑制,并诱导其凋亡过程。方法：1.利用铺有不同浓度SWNHs的6孔板或培养皿接种人胶质瘤细胞系U251细胞,培养一定时间后,通过普通电子倒置显微镜和共聚焦激光显微镜来对细胞的形态和数目进行观察。2. BrdU检测SWNHs对细胞增殖和有丝分裂的影响；SWNHs对U251细胞增殖和活力的影响分别用XTT法和CCK8法来定量地评价。3. SWNHs对胶质瘤细胞凋亡及细胞周期的影响采用流式细胞术进行评价；Western Blot法分析SWNHs材料导致胶质瘤细胞凋亡相关蛋白.4.使用Seahorse XF24分析仪测量不同浓度SWNHS对U251细胞氧气消耗量的影响,采用生物发光法进行ATP生产分析,及通过发光法检测不同浓度SWNHS培养后的U251细胞的产生NAD的影响,全面评估SWNHS对U251细胞的线粒体功能影响。结果：1.人胶质瘤U251细胞分别和具有浓度梯度的SWNHs材料共同培后,在设定的时间用倒置显微镜对细胞进行观察,结果提示随着SWNHs浓度的增加,U251细胞数量呈逐渐减少,细胞不能完全正常贴壁生长,细胞变圆固缩,甚至有些细胞死亡后漂浮在培养液中；共聚焦激光显微镜观察结果示U251细胞的核发生固缩,且生成大量的核小体碎片。2.根据XTT法和CCK8法的实验结果,证明了SWNHs抑制U251细胞增殖存在时间和剂量依赖关系。3.流式细胞术检测提示SWNHs显著诱导U251细胞凋亡,并且凋亡相关蛋白Bad、 Bax的浓度与SWNHS的浓度呈正相关,而凋亡抑制蛋白Bcl-2随着SWNHS的浓度增加而减少,呈负相关。4. SWNHS影响U251细胞的线粒体功能：细胞氧消耗量、ATP产生和NAD的功能都以剂量依赖的方式受到阻碍。结论：SWNHs抑制人胶质瘤细胞生长和增殖,并促进胶质瘤细胞凋亡,同时存在剂量依赖关系。其具体机制可能通过Bcl-2信号系统,引起了U251细胞线粒体功能的障碍。
[Abstract]:Aim: to investigate how SWNHs can inhibit the proliferation and induce apoptosis of human glioma cells.Method 1: 1.Human glioma cell line U251 cells were inoculated with 6-well plate or culture dish with different concentrations of SWNHs for a certain time. The morphology and number of U251 cells were observed by conventional electron inverted microscope and confocal laser microscope.The effects of BrdU on cell proliferation and mitosis were evaluated quantitatively by XTT and CCK8.The effect of SWNHs on apoptosis and cell cycle of glioma cells was evaluated by flow cytometry. Western Blot assay was used to analyze apoptosis-related protein .4.The effects of different concentrations of SWNHS on the oxygen consumption of U251 cells were measured by Seahorse XF24 analyzer. The production of ATP was analyzed by bioluminescence assay, and the effect of NAD production on U251 cells cultured with different concentrations of SWNHS was detected by luminescence assay.To evaluate the effects of SWNHS on mitochondrial function of U251 cells.The result is 1: 1.Human glioma U251 cells were cultured with SWNHs material with concentration gradient, and the cells were observed by inverted microscope at a fixed time. The results showed that the number of U251 cells decreased gradually with the increase of SWNHs concentration.The cells could not completely adhere to the wall, the cells became round and pyknotic, and some cells died and floated in the culture medium. Confocal laser microscopy showed that the nuclei of U251 cells had pyknosis and produced a large number of nucleosome fragments. 2.According to the experimental results of XTT and CCK8 methods, it was proved that SWNHs inhibited the proliferation of U251 cells in a time-and dose-dependent manner.Flow cytometry showed that SWNHs significantly induced apoptosis of U251 cells, and the concentration of apoptosis-related protein Badand Bax was positively correlated with the concentration of SWNHS, while the inhibitor of apoptosis protein Bcl-2 decreased with the increase of SWNHS concentration, which was negatively correlated with the concentration of SWNHS.SWNHS affects the mitochondrial function of U251 cells. The production of ATP and the function of NAD in U251 cells are obstructed in a dose-dependent manner.Conclusion the growth and proliferation of human glioma cells were inhibited and the apoptosis of glioma cells was promoted by WSWNHs in a dose-dependent manner.The mechanism may cause dysfunction of mitochondria function in U251 cells through Bcl-2 signaling system.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.41

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