羟基芫花素配伍芹菜素体外协同抗胶质瘤作用及其机制研究

发布时间：2018-03-01 12:12

本文关键词： 羟基芫花素芹菜素协同配伍胶质瘤凋亡　出处：《大连医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：初步探索羟基芫花素与芹菜配伍协同抗胶质瘤作用及其分子机制方法：MTT法检测经不同浓度的芹菜素、羟基芫花素、羟基芫花素+芹菜素（配伍组）处理不同时间后的C6胶质瘤细胞增殖情况，初步判定各组药物抑制C6胶质瘤增殖的时间依赖、浓度依赖特点。用AO/EB（acridine orange/ethidiumbromide，，吖啶橙/溴化乙锭）和DAPI（4,6-diamidino-2-phenylindole，4,6-二脒基-2-苯基吲哚）荧光染色的方法检测核固缩等细胞凋亡形态变化；应用扫描电镜（transmission electron microscope，TEM）和JC-1荧光探针的方法从形态和功能的角度检测各组药物对线粒体结构和膜电位的损伤情况；单细胞凝胶电泳（single cell gel eletrophoresis，SCGE)实验检测细胞DNA损伤；流式细胞仪检测各组药物对C6细胞的周期阻滞作用。在分子机制研究方面，采用Western blot考察了各组药物对线粒体凋亡通路相关抗凋亡蛋白（Bcl-2、Bcl-xl）和凋亡诱导蛋白（Bax、Bak、Bid、Bad）表达的影响；利用放免试剂盒测定了经各组药物处理后C6胶质瘤细胞上清液中TNF-的浓度；Caspase活性测定试剂盒检测各组药物对肿瘤细胞Caspase-3和8酶活性的影响。结果：羟基芫花素和芹菜素在总药物浓度为25μM时对C6胶质瘤细胞均有一定的抗增殖效应，其中羟基芫花素的抗胶质瘤效应稍强于芹菜素；当二者配伍联合应用后，联合用药组的抗胶质瘤效应明显强于各单用药组；AO/EB染色、DAPI染色结果表明羟基芫花素和芹菜素是通过诱导肿瘤细胞凋亡的方式达到抗增殖效应；进一步机制研究表明，羟基芫花素对线粒体结构和功能（线粒体膜电位）造成明显的损伤，联合用药组表现出明显的协同效应；SCGE检测到各处理组中肿瘤细胞DNA损伤。细胞周期分析表明，羟基芫花素具有明显的S期阻滞效应，而芹菜素对肿瘤细胞周期无明显的阻滞效应；进一步分子机制研究表明，相对于对照组，羟基芫花素和芹菜素能明显增强肿瘤细胞TNF-的表达、增强Caspase活化，当联合应用后二者表现出明显的协同效应。但是Western blot结果分析表明，羟基芫花素和芹菜素对线粒体凋亡通路某些关键蛋白表达的影响具有无明显的协同效应；药物毒性初步评估结果表明，在一定浓度下，羟基芫花素对高分化的PC12细胞系无明显毒性效应（IC50㧐120μM）。结论：羟基芫花素具有一定的抗胶质瘤作用，当羟基芫花素与芹菜素联合应用后，二者表现出明显的协同增效抗胶质瘤效应；羟基芫花素和芹菜素通过线粒体损伤、DNA损伤、细胞周期S期阻滞，启动线粒体凋亡通路等机制达到抗胶质瘤作用。本研究首次证明了羟基芫花素联合芹菜素的巨大抗胶质瘤潜在价值并为进一步动物体内研究提供了一定的理论依据。
[Abstract]:Objective: to explore the synergistic effect of hydroxygenkwa with celery on glioma and its molecular mechanism. Methods the proliferation of C6 glioma cells treated with different concentrations of apigenin, hydroxygenkapenin and hydroxygenkapenin (compatibility group) for different time was detected by MTT assay. The time and concentration dependence of drug inhibition on the proliferation of C6 gliomas was preliminarily determined. The morphological changes of nuclear pyknosis were detected by fluorescence staining of AO/EB(acridine orange / ethidium rodede (acridine orange / ethidium bromide) and DAPIX 46-diamidino-2-phenylindoledol (46-diamidino-2-phenylindoledol). The morphology and function of mitochondrial structure and membrane potential of each group were examined by scanning electron microscope transmission electron microscopetem and JC-1 fluorescence probe, and single cell gel eletrophoresis-SCGE assay was used to detect cell DNA damage. The cell cycle arrest of C6 cells was detected by flow cytometry. In the study of molecular mechanism, Western blot was used to investigate the effects of each group on the expression of Bcl-2Bcl-xl) and Bax-induced protein Baxy BakBidadad. The concentration of TNF- in the supernatant of C6 glioma cells was determined by radioimmunoassay kit. The effects of various drugs on the activities of Caspase-3 and 8 enzymes in the supernatant of C6 glioma cells were measured. Results: both hydroxygenkaphnin and apigenin could inhibit the proliferation of C6 glioma cells at a concentration of 25 渭 M, and the anti-glioma effect of hydroxygenkwa was slightly stronger than that of apigenin. The anti-glioma effect of the combined treatment group was significantly stronger than that of each single drug group by AO-EB staining and DAPI staining. The results showed that hydroxygenkaphnin and apigenin could induce the apoptosis of tumor cells to achieve the anti-proliferation effect. Hydroxygenkwa caused obvious damage to mitochondrial structure and function (mitochondrial membrane potential), and the combined treatment group showed obvious synergistic effect. Cell cycle analysis showed that the tumor cell DNA damage was detected in each treatment group. Hydroxy Daphne genkwa has obvious S phase arrest effect, while apigenin has no obvious blocking effect on tumor cell cycle. Hydroxygenkwa and apigenin could significantly enhance the expression of TNF- and the activation of Caspase in tumor cells, but the results of Western blot analysis showed that the expression of TNF- and the activation of TNF- in tumor cells were significantly increased after combined treatment. There was no significant synergistic effect of hydroxygenkwa and apigenin on the expression of some key proteins in mitochondrial apoptotic pathway. Hydroxygenkwanin has no toxic effect on highly differentiated PC12 cell line. 120 渭 m. Conclusion: hydroxygenkwa has a certain anti-glioma effect. When the combination of hydroxygenkwa and apigenin is used, both of them have synergistic effect on glioma, and hydroxygenkaphnin and apigenin damage DNA through mitochondria. Cell cycle S phase arrest, This study demonstrated the potential value of hydroxygenkaphnin combined with apigenin in anti-glioma and provided a theoretical basis for further study in vivo.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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